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Regulation of NuA4 Histone Acetyltransferase Activity in Transcription and DNA Repair by Phosphorylation of Histone H4
Utley, Rhea T.; Lacoste, Nicolas; Jobin-Robitaille, Olivier; Allard, Stéphane; Côté, Jacques
The NuA4 complex is a histone H4/H2A acetyltransferase involved in transcription and DNA repair. While histone acetylation is important in many processes, it has become increasingly clear that additional histone modifications also play a crucial interrelated role. To understand how NuA4 action is regulated, we tested various H4 tail peptides harboring known modifications in HAT assays. While dimethylation at arginine 3 (R3M) had little effect on NuA4 activity, phosphorylation of serine 1 (S1P...
Enhanced MYC association with the NuA4 histone acetyltransferase complex mediated by the adenovirus E1A N-terminal domain activates a subset of MYC target genes highly expressed in cancer cells.
Zhao, Ling-Jun; Loewenstein, Paul M; Green, Maurice
The proto-oncogene MYC is a transcription factor over-expressed in many cancers and required for cell survival. Its function is regulated by histone acetyltransferase (HAT) complexes, such as the GCN5 complex and the NuA4/Tip60 complex. However, the roles of the HAT complexes during MYC function in cancer have not been well characterized. We recently showed that adenovirus E1A and its N-terminal 80 aa region, E1A 1-80, interact with the NuA4 complex, through the E1A TRRAP-targeting (ET) domain, and enhance MYC association with the NuA4 complex. We show here that the ET domain mainly targets the MYC-NuA4 complex. By global gene expression analysis using E1A 1-80 and deletion mutants, we have identified a panel of genes activated by targeting the MYC-NuA4 complex and notably enriched for genes involved in ribosome biogenesis and gene expression. A second panel of genes is activated by E1A 1-80 targeting of both the MYC-NuA4 complex and p300, and is enriched for genes involved in DNA replication and cell cycle processes. Both panels of genes are highly expressed in cancer cells. Since the ET domain is essential for E1A-mediated cellular transformation, our results suggest that MYC and the NuA4 complex function cooperatively in cell transformation and cancer.
NuA4 Lysine Acetyltransferase Complex Contributes to Phospholipid Homeostasis in Saccharomyces cerevisiae
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Louis Dacquay
Full Text Available Actively proliferating cells constantly monitor and readjust their metabolic pathways to ensure the replenishment of phospholipids necessary for membrane biogenesis and intracellular trafficking. In Saccharomyces cerevisiae, multiple studies have suggested that the lysine acetyltransferase complex NuA4 plays a role in phospholipid homeostasis. For one, NuA4 mutants induce the expression of the inositol-3-phosphate synthase gene, INO1, which leads to excessive accumulation of inositol, a key metabolite used for phospholipid biosynthesis. Additionally, NuA4 mutants also display negative genetic interactions with sec14-1ts, a mutant of a lipid-binding gene responsible for phospholipid remodeling of the Golgi. Here, using a combination of genetics and transcriptional profiling, we explore the connections between NuA4, inositol, and Sec14. Surprisingly, we found that NuA4 mutants did not suppress but rather exacerbated the growth defects of sec14-1ts under inositol-depleted conditions. Transcriptome studies reveal that while loss of the NuA4 subunit EAF1 in sec14-1ts does derepress INO1 expression, it does not derepress all inositol/choline-responsive phospholipid genes, suggesting that the impact of Eaf1 on phospholipid homeostasis extends beyond inositol biosynthesis. In fact, we find that NuA4 mutants have impaired lipid droplet levels and through genetic and chemical approaches, we determine that the genetic interaction between sec14-1ts and NuA4 mutants potentially reflects a role for NuA4 in fatty acid biosynthesis. Altogether, our work identifies a new role for NuA4 in phospholipid homeostasis.
Roles for the Histone Modifying and Exchange Complex NuA4 in Cell Cycle Progression in Drosophila melanogaster.
Flegel, Kerry; Grushko, Olga; Bolin, Kelsey; Griggs, Ellen; Buttitta, Laura
Robust and synchronous repression of E2F-dependent gene expression is critical to the proper timing of cell cycle exit when cells transition to a postmitotic state. Previously NuA4 was suggested to act as a barrier to proliferation in Drosophila by repressing E2F-dependent gene expression. Here we show that NuA4 activity is required for proper cell cycle exit and the repression of cell cycle genes during the transition to a postmitotic state in vivo However, the delay of cell cycle exit caused by compromising NuA4 is not due to additional proliferation or effects on E2F activity. Instead NuA4 inhibition results in slowed cell cycle progression through late S and G2 phases due to aberrant activation of an intrinsic p53-independent DNA damage response. A reduction in NuA4 function ultimately produces a paradoxical cell cycle gene expression program, where certain cell cycle genes become derepressed in cells that are delayed during the G2 phase of the final cell cycle. Bypassing the G2 delay when NuA4 is inhibited leads to abnormal mitoses and results in severe tissue defects. NuA4 physically and genetically interacts with components of the E2F complex termed D: rosophila, R: bf, E: 2F A: nd M: yb/ M: ulti-vulva class B: (DREAM/MMB), and modulates a DREAM/MMB-dependent ectopic neuron phenotype in the posterior wing margin. However, this effect is also likely due to the cell cycle delay, as simply reducing Cdk1 is sufficient to generate a similar phenotype. Our work reveals that the major requirement for NuA4 in the cell cycle in vivo is to suppress an endogenous DNA damage response, which is required to coordinate proper S and G2 cell cycle progression with differentiation and cell cycle gene expression. Copyright © 2016 by the Genetics Society of America.
Recruitment of the NuA4 complex poises the PHO5 promoter for chromatin remodeling and activation
Nourani, Amine; Utley, Rhea T; Allard, Stéphane; Côté, Jacques
The remodeling of the promoter chromatin structure is a key event for the induction of the PHO5 gene. Two DNA-binding proteins Pho2 and Pho4 are critical for this step. We found that the NuA4 histone acetyltransferase complex is essential for PHO5 transcriptional induction without affecting Pho4 translocation upon phosphate starvation. Our data also indicate that NuA4 is critical for the chromatin remodeling event that occurs over the PHO5 promoter prior to activation. Using Chromatin IP anal...
Histone acetyltransferases : challenges in targeting bi-substrate enzymes
Wapenaar, Hannah; Dekker, Frank J
Histone acetyltransferases (HATs) are epigenetic enzymes that install acetyl groups onto lysine residues of cellular proteins such as histones, transcription factors, nuclear receptors, and enzymes. HATs have been shown to play a role in diseases ranging from cancer and inflammatory diseases to
Small molecule inhibitors of histone deacetylases and acetyltransferases as potential therapeutics in oncology
van den Bosch, Thea; Leus, Niek; Timmerman, Tirza; Dekker, Frank J
Uncontrolled cell proliferation and resistance to apoptosis in cancer are, among others, regulated by post-translational modifications of histone proteins. The most investigated type of histone modification is lysine acetylation. Histone acetyltransferases (HATs), acetylate histone lysine residues,
Dysregulation of Histone Acetyltransferases and Deacetylases in Cardiovascular Diseases
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Yonggang Wang
Full Text Available Cardiovascular disease (CVD remains a leading cause of mortality worldwide despite advances in its prevention and management. A comprehensive understanding of factors which contribute to CVD is required in order to develop more effective treatment options. Dysregulation of epigenetic posttranscriptional modifications of histones in chromatin is thought to be associated with the pathology of many disease models, including CVD. Histone acetyltransferases (HATs and deacetylases (HDACs are regulators of histone lysine acetylation. Recent studies have implicated a fundamental role of reversible protein acetylation in the regulation of CVDs such as hypertension, pulmonary hypertension, diabetic cardiomyopathy, coronary artery disease, arrhythmia, and heart failure. This reversible acetylation is governed by enzymes that HATs add or HDACs remove acetyl groups respectively. New evidence has revealed that histone acetylation regulators blunt cardiovascular and related disease states in certain cellular processes including myocyte hypertrophy, apoptosis, fibrosis, oxidative stress, and inflammation. The accumulating evidence of the detrimental role of histone acetylation in cardiac disease combined with the cardioprotective role of histone acetylation regulators suggests that the use of histone acetylation regulators may serve as a novel approach to treating the millions of patients afflicted by cardiac diseases worldwide.
The Functional Analysis of Histone Acetyltransferase MOF in Tumorigenesis
Su, Jiaming; Wang, Fei; Cai, Yong; Jin, Jingji
Changes in chromatin structure and heritably regulating the gene expression by epigenetic mechanisms, such as histone post-translational modification, are involved in most cellular biological processes. Thus, abnormal regulation of epigenetics is implicated in the occurrence of various diseases, including cancer. Human MOF (males absent on the first) is a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of histone acetyltransferases (HATs). As a catalytic subunit, MOF can form at least two distinct multiprotein complexes (MSL and NSL) in human cells. Both complexes can acetylate histone H4 at lysine 16 (H4K16); however, the NSL complex possesses broader substrate specificity and can also acetylate histone H4 at lysines 5 and 8 (H4K5 and H4K8), suggesting the complexity of the intracellular functions of MOF. Silencing of MOF in cells leads to genomic instability, inactivation of gene transcription, defective DNA damage repair and early embryonic lethality. Unbalanced MOF expression and its corresponding acetylation of H4K16 have been found in certain primary cancer tissues, including breast cancer, medulloblastoma, ovarian cancer, renal cell carcinoma, colorectal carcinoma, gastric cancer, as well as non-small cell lung cancer. In this review, we provide a brief overview of MOF and its corresponding histone acetylation, introduce recent research findings that link MOF functions to tumorigenesis and speculate on the potential role that may be relevant to tumorigenic pathways. PMID:26784169
Histone acetyltransferases: challenges in targeting bi-substrate enzymes.
Wapenaar, Hannah; Dekker, Frank J
Histone acetyltransferases (HATs) are epigenetic enzymes that install acetyl groups onto lysine residues of cellular proteins such as histones, transcription factors, nuclear receptors, and enzymes. HATs have been shown to play a role in diseases ranging from cancer and inflammatory diseases to neurological disorders, both through acetylations of histone proteins and non-histone proteins. Several HAT inhibitors, like bi-substrate inhibitors, natural product derivatives, small molecules, and protein-protein interaction inhibitors, have been developed. Despite their potential, a large gap remains between the biological activity of inhibitors in in vitro studies and their potential use as therapeutic agents. To bridge this gap, new potent HAT inhibitors with improved properties need to be developed. However, several challenges have been encountered in the investigation of HATs and HAT inhibitors that hinder the development of new HAT inhibitors. HATs have been shown to function in complexes consisting of many proteins. These complexes play a role in the activity and target specificity of HATs, which limits the translation of in vitro to in vivo experiments. The current HAT inhibitors suffer from undesired properties like anti-oxidant activity, reactivity, instability, low potency, or lack of selectivity between HAT subtypes and other enzymes. A characteristic feature of HATs is that they are bi-substrate enzymes that catalyze reactions between two substrates: the cofactor acetyl coenzyme A (Ac-CoA) and a lysine-containing substrate. This has important-but frequently overlooked-consequences for the determination of the inhibitory potency of small molecule HAT inhibitors and the reproducibility of enzyme inhibition experiments. We envision that a careful characterization of molecular aspects of HATs and HAT inhibitors, such as the HAT catalytic mechanism and the enzyme kinetics of small molecule HAT inhibitors, will greatly improve the development of potent and
Adenovirus E1A TRRAP-targeting domain-mediated enhancement of MYC association with the NuA4 complex activates a panel of MYC target genes enriched for gene expression and ribosome biogenesis.
Zhao, Ling-Jun; Loewenstein, Paul M; Green, Maurice
Cellular transformation by adenovirus E1A requires targeting TRRAP, a scaffold protein which helps assemble histone acetyltransferase complexes, including the NuA4 complex. We recently reported that E1A and E1A 1-80 (N-terminal 80 aa) promote association of the proto-oncogene product MYC with the NuA4 complex. The E1A N-terminal TRRAP-targeting (ET) domain is required for E1A 1-80 to interact with the NuA4 complex. We demonstrate that an ET-MYC fusion associates with the NuA4 complex more efficiently than does MYC alone. Because MYC regulates genes for multiple cellular pathways, we performed global RNA-sequence analysis of cells expressing MYC or ET-MYC, and identified a panel of genes (262) preferentially activated by ET-MYC and significantly enriched in genes involved in gene expression and ribosome biogenesis, suggesting that E1A enhances MYC association with the NuA4 complex to activate a set of MYC target genes likely involved in cellular proliferation and cellular transformation by E1A and by MYC. Copyright © 2017 Elsevier Inc. All rights reserved.
The histone acetyltransferase inhibitor Nir regulates epidermis development.
Duteil, Delphine; Tourrette, Yves; Eberlin, Adrien; Willmann, Dominica; Patel, Dharmeshkumar; Friedrichs, Nicolaus; Müller, Judith M; Schüle, Roland
In addition to its function as an inhibitor of histone acetyltransferases, Nir (Noc2l) binds to p53 and TAp63 to regulate their activity. Here, we show that epidermis-specific ablation of Nir impairs epidermal stratification and barrier function, resulting in perinatal lethality. Nir-deficient epidermis lacks appendages and remains single layered during embryogenesis. Cell proliferation is inhibited, whereas apoptosis and p53 acetylation are increased, indicating that Nir is controlling cell proliferation by limiting p53 acetylation. Transcriptome analysis revealed that Nir regulates the expression of essential factors in epidermis development, such as keratins, integrins and laminins. Furthermore, Nir binds to and controls the expression of p63 and limits H3K18ac at the p63 promoter. Corroborating the stratification defects, asymmetric cell divisions were virtually absent in Nir-deficient mice, suggesting that Nir is required for correct mitotic spindle orientation. In summary, our data define Nir as a key regulator of skin development. © 2018. Published by The Company of Biologists Ltd.
CAT in the HAT: catabolic inhibition by the histone acetyltransferase GCN5.
Liu, Yi; Montminy, Marc
The nuclear hormone receptor coactivator PGC-1alpha is a key regulator of gluconeogenic genes during fasting. In this issue of Cell Metabolism, Puigserver and colleagues (Lerin et al., 2006) report that the histone acetyltransferase GCN5 inhibits gluconeogenesis by acetylating and sequestering PGC-1alpha in nuclear foci.
Diversification of the Histone Acetyltransferase GCN5 through Alternative Splicing in Brachypodium distachyon
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Alexandre Martel
Full Text Available The epigenetic modulatory SAGA complex is involved in various developmental and stress responsive pathways in plants. Alternative transcripts of the SAGA complex's enzymatic subunit GCN5 have been identified in Brachypodium distachyon. These splice variants differ based on the presence and integrity of their conserved domain sequences: the histone acetyltransferase domain, responsible for catalytic activity, and the bromodomain, involved in acetyl-lysine binding and genomic loci targeting. GCN5 is the wild-type transcript, while alternative splice sites result in the following transcriptional variants: L-GCN5, which is missing the bromodomain and S-GCN5, which lacks the bromodomain as well as certain motifs of the histone acetyltransferase domain. Absolute mRNA quantification revealed that, across eight B. distachyon accessions, GCN5 was the dominant transcript isoform, accounting for up to 90% of the entire transcript pool, followed by L-GCN5 and S-GCN5. A cycloheximide treatment further revealed that the S-GCN5 splice variant was degraded through the nonsense-mediated decay pathway. All alternative BdGCN5 transcripts displayed similar transcript profiles, being induced during early exposure to heat and displaying higher levels of accumulation in the crown, compared to aerial tissues. All predicted protein isoforms localize to the nucleus, which lends weight to their purported epigenetic functions. S-GCN5 was incapable of forming an in vivo protein interaction with ADA2, the transcriptional adaptor that links the histone acetyltransferase subunit to the SAGA complex, while both GCN5 and L-GCN5 interacted with ADA2, which suggests that a complete histone acetyltransferase domain is required for BdGCN5-BdADA2 interaction in vivo. Thus, there has been a diversification in BdGCN5 through alternative splicing that has resulted in differences in conserved domain composition, transcript fate and in vivo protein interaction partners. Furthermore, our
Histone acetyltransferase PCAF is required for Hedgehog-Gli-dependent transcription and cancer cell proliferation
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Malatesta, Martina; Steinhauer, Cornelia; Mohammad, Faizaan
as in several cancers, including brain tumors like medulloblastoma and glioblastoma. Inhibition of aberrant Hh-Gli signaling has, thus, emerged as an attractive approach for anticancer therapy; however, the mechanisms that mediate Hh-Gli signaling in vertebrates remain poorly understood. Here, we show...... that the histone acetyltransferase PCAF/KAT2B is an important factor of the Hh pathway. Specifically, we show that PCAF depletion impairs Hh activity and reduces expression of Hh target genes. Consequently, PCAF downregulation in medulloblastoma and glioblastoma cells leads to decreased proliferation and increased...... in vivo. In summary, our study identified the acetyltransferase PCAF as a positive cofactor of the Hh-Gli signaling pathway, leading us to propose PCAF as a candidate therapeutic target for the treatment of patients with medulloblastoma and glioblastoma....
Histone acetyltransferase p300 mediates histone acetylation of PS1 and BACE1 in a cellular model of Alzheimer's disease.
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Xi Lu
Full Text Available Epigenetic modifications, particularly histone acetylation, have been implicated in Alzheimer's disease (AD. While previous studies have suggested that histone hypoacetylation may regulate the expression of genes associated with memory and learning in AD, little is known about histone regulation of AD-related genes such as Presenilin 1(PS1 and beta-site amyloid precursor protein cleaving enzyme 1(BACE1. By utilizing neuroblastoma N2a cells transfected with Swedish mutated human amyloid precursor protein (APP (N2a/APPswe and wild-type APP (N2a/APPwt as cellular models of AD, we examined the alterations of histone acetylation at the promoter regions of PS1 and BACE1 in these cells. Our results revealed that histone H3 acetylation in PS1 and BACE1 promoters is markedly increased in N2a/APPswe cells when compared to N2a/APPwt cells and control cells (vector-transfected, respectively, causing the elevated expression of PS1 and BACE1. In addition, expression of histone acetyltransferase (HAT adenoviral E1A-associated 300-kDa protein (p300 is dramatically enhanced in N2a/APPswe cells compared to N2a/APPwt and control cells. We have further demonstrated the direct binding of p300 protein to the PS1 and BACE1 promoters in N2a/APPswe cells. The expression levels of H3 acetylation of the PS1 and BACE1 promoters and p300 protein, however, were found to be not significantly different in N2a/APPwt cells when compared to controls in our studies. Furthermore, curcumin, a natural selective inhibitor of p300 in HATs, significantly suppressed the expression of PS1 and BACE1 through inhibition of H3 acetylation in their promoter regions in N2a/APPswe cells. These findings indicated that histone acetyltransferase p300 plays a critical role in controlling the expression of AD-related genes through regulating the acetylation of their promoter regions, suggesting that p300 may represent a novel potential therapeutic target for AD.
Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p
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Greenblatt Jack F
Full Text Available Abstract Background Histone modifications have been implicated in the regulation of transcription and, more recently, in DNA replication and repair. In yeast, a major conserved histone acetyltransferase, Hat1p, preferentially acetylates lysine residues 5 and 12 on histone H4. Results Here, we report that a nuclear sub-complex consisting of Hat1p and its partner Hat2p interacts physically and functionally with the origin recognition complex (ORC. While mutational inactivation of the histone acetyltransferase (HAT gene HAT1 alone does not compromise origin firing or initiation of DNA replication, a deletion in HAT1 (or HAT2 exacerbates the growth defects of conditional orc-ts mutants. Thus, the ORC-associated Hat1p-dependent histone acetyltransferase activity suggests a novel linkage between histone modification and DNA replication. Additional genetic and biochemical evidence points to the existence of partly overlapping histone H3 acetyltransferase activities in addition to Hat1p/Hat2p for proper DNA replication efficiency. Furthermore, we demonstrated a dynamic association of Hat1p with chromatin during S-phase that suggests a role of this enzyme at the replication fork. Conclusion We have found an intriguing new association of the Hat1p-dependent histone acetyltransferase in addition to its previously known role in nuclear chromatin assembly (Hat1p/Hat2p-Hif1p. The participation of a distinct Hat1p/Hat2p sub-complex suggests a linkage of histone H4 modification with ORC-dependent DNA replication.
Cinnamoyl compounds as simple molecules that inhibit p300 histone acetyltransferase.
Costi, Roberta; Di Santo, Roberto; Artico, Marino; Miele, Gaetano; Valentini, Paola; Novellino, Ettore; Cereseto, Anna
Cinnamoly compounds 1a-c and 2a-d were designed, synthesized, and in vitro tested as p300 inhibitors. At different degrees, all tested compounds were proven to inactivate p300, particularly, derivative 2c was the most active inhibitor, also showing high specificity for p300 as compared to other histone acetyltransferases. Most notably, 2c showed anti-acetylase activity in mammalian cells. These compounds represent a new class of synthetic inhibitors of p300, characterized by simple chemical structures.
EPC1/TIP60-mediated histone acetylation facilitates spermiogenesis in mice
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Dong, Yixin; Isono, Kyo Ichi; Ohbo, Kazuyuki
Global histone hyperacetylation is suggested to play a critical role for replacement of histones by transition proteins and protamines to compact the genome during spermiogenesis. However, the underlying mechanisms for hyperacetylation- mediated histone replacement remains poorly understood. Here......, we report that EPC1 and TIP60, two critical components of the mammalian nucleosome acetyltransferase of H4 (NuA4) complexes, are coexpressed in male germ cells. Strikingly, genetic ablation of either Epc1 or Tip60 disrupts hyperacetylation and impairs histone replacement, in turn causing aberrant...... spermatid development. Taking these observations together, we reveal an essential role of the NuA4 complexes for histone hyperacetylation and subsequent compaction of the spermatid genome....
Histone acetyltransferase inhibitors antagonize AMP-activated protein kinase in postmortem glycolysis
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Qiong Li
Full Text Available Objective The purpose of this study was to investigate the influence of AMP-activated protein kinase (AMPK activation on protein acetylation and glycolysis in postmortem muscle to better understand the mechanism by which AMPK regulates postmortem glycolysis and meat quality. Methods A total of 32 mice were randomly assigned to four groups and intraperitoneally injected with 5-Aminoimidazole-4-carboxamide1-β-D-ribofuranoside (AICAR, a specific activator of AMPK, AICAR and histone acetyltransferase inhibitor II, or AICAR, Trichostatin A (TSA, an inhibitor of histone deacetylase I and II and Nicotinamide (NAM, an inhibitor of the Sirt family deacetylases. After mice were euthanized, the Longissimus dorsi muscle was collected at 0 h, 45 min, and 24 h postmortem. AMPK activity, protein acetylation and glycolysis in postmortem muscle were measured. Results Activation of AMPK by AICAR significantly increased glycolysis in postmortem muscle. At the same time, it increased the total acetylated proteins in muscle 45 min postmortem. Inhibition of protein acetylation by histone acetyltransferase inhibitors reduced AMPK activation induced increase in the total acetylated proteins and glycolytic rate in muscle early postmortem, while histone deacetylase inhibitors further promoted protein acetylation and glycolysis. Several bands of proteins were detected to be differentially acetylated in muscle with different glycolytic rates. Conclusion Protein acetylation plays an important regulatory role in postmortem glycolysis. As AMPK mediates the effects of pre-slaughter stress on postmortem glycolysis, protein acetylation is likely a mechanism by which antemortem stress influenced postmortem metabolism and meat quality though the exact mechanism is to be elucidated.
Quinoline Derivative MC1626, a Putative GCN5 Histone Acetyltransferase (HAT) Inhibitor, Exhibits HAT-Independent Activity against Toxoplasma gondii▿
Smith, Aaron T.; Livingston, Meredith R.; Mai, Antonello; Filetici, Patrizia; Queener, Sherry F.; Sullivan, William J.
We report that quinoline derivative MC1626, first described as an inhibitor of the histone acetyltransferase (HAT) GCN5, is active against the protozoan parasite Toxoplasma gondii in vitro. However, MC1626 does not inhibit Toxoplasma GCN5 HATs or reduce HAT-mediated activity; rather, this quinoline may target the plastid organelle called the apicoplast. PMID:17178801
The histone acetyltransferase GCN5 affects the inflorescence meristem and stamen development in Arabidopsis.
Cohen, Ross; Schocken, John; Kaldis, Athanasios; Vlachonasios, Konstantinos E; Hark, Amy T; McCain, Elizabeth R
A central question in biology is to understand how gene expression is precisely regulated to give rise to a variety of forms during the process of development. Epigenetic effects such as DNA methylation or histone modification have been increasingly shown to play a critical role in regulation of genome function. GCN5 is a prototypical histone acetyltransferase that participates in regulating developmental gene expression in several metazoan species. In Arabidopsis thaliana, plants with T-DNA insertions in GCN5 (also known as HAG1) display a variety of pleiotropic effects including dwarfism, loss of apical dominance, and floral defects affecting fertility. We sought to determine when during early development floral abnormalities first arise. Using scanning electron microscopy, we demonstrate that gcn5-1/hag1-1 and gcn5-5/hag1-5 mutants display overproliferation of young buds and development of abnormal structures around the inflorescence meristem. gcn5 mutants also display defects in stamen number and arrangement at later stages. This analysis provides temporal and spatial information to aid in the identification of GCN5 target genes in the developing flower. Preliminary studies of putative targets using reverse transcriptase PCR suggest that the floral meristem identity gene LEAFY is among factors upregulated in gcn5-1 mutants.
Histone acetyltransferase mediated regulation of FOXP3 acetylation and Treg function.
Xiao, Yan; Li, Bin; Zhou, Zhaocai; Hancock, Wayne W; Zhang, Hongtao; Greene, Mark I
Regulatory T cells (Tregs) are required for the maintenance of immune homeostasis as first clearly described by Herman Waldmann's laboratory. Dysfunction of Treg cells also leads to fatal autoimmunity in humans and mice. Conversely, the activation of different classes of Tregs operative systemically and within the cancer microenvironment can suppress host anti-tumor immune responses and promote tumor progression. Therefore, the development of new therapeutic approaches to regulate the activity of Treg cells may have considerable clinical potential. FOXP3 is the key transcriptional regulator of Treg development and function. The activity of FOXP3 is regulated by acetylation, a process catalyzed by distinct types of histone/protein acetyltransferases (HATs) that regulate the functions of many transcription factors, independently of FOXP3, as well as non-histone proteins, in addition to their effects on chromatin accessibility. Interactions between FOXP3 and these enzymes determine the suppressive function of FOXP3. Clearly, small molecules targeting these enzymes are candidates for the regulation of Treg function in vaccines and tumor therapies. Published by Elsevier Ltd.
Metabolic Regulation of Histone Acetyltransferases by Endogenous Acyl-CoA Cofactors.
Montgomery, David C; Sorum, Alexander W; Guasch, Laura; Nicklaus, Marc C; Meier, Jordan L
The finding that chromatin modifications are sensitive to changes in cellular cofactor levels potentially links altered tumor cell metabolism and gene expression. However, the specific enzymes and metabolites that connect these two processes remain obscure. Characterizing these metabolic-epigenetic axes is critical to understanding how metabolism supports signaling in cancer, and developing therapeutic strategies to disrupt this process. Here, we describe a chemical approach to define the metabolic regulation of lysine acetyltransferase (KAT) enzymes. Using a novel chemoproteomic probe, we identify a previously unreported interaction between palmitoyl coenzyme A (palmitoyl-CoA) and KAT enzymes. Further analysis reveals that palmitoyl-CoA is a potent inhibitor of KAT activity and that fatty acyl-CoA precursors reduce cellular histone acetylation levels. These studies implicate fatty acyl-CoAs as endogenous regulators of histone acetylation, and suggest novel strategies for the investigation and metabolic modulation of epigenetic signaling. Copyright © 2015 Elsevier Ltd. All rights reserved.
Disposition, Metabolism and Histone Deacetylase and Acetyltransferase Inhibition Activity of Tetrahydrocurcumin and Other Curcuminoids
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Júlia T. Novaes
Full Text Available Tetrahydrocurcumin (THC, curcumin and calebin-A are curcuminoids found in turmeric (Curcuma longa. Curcuminoids have been established to have a variety of pharmacological activities and are used as natural health supplements. The purpose of this study was to identify the metabolism, excretion, antioxidant, anti-inflammatory and anticancer properties of these curcuminoids and to determine disposition of THC in rats after oral administration. We developed a UHPLC–MS/MS assay for THC in rat serum and urine. THC shows multiple redistribution phases with corresponding increases in urinary excretion rate. In-vitro antioxidant activity, histone deacetylase (HDAC activity, histone acetyltransferase (HAT activity and anti-inflammatory inhibitory activity were examined using commercial assay kits. Anticancer activity was determined in Sup-T1 lymphoma cells. Our results indicate THC was poorly absorbed after oral administration and primarily excreted via non-renal routes. All curcuminoids exhibited multiple pharmacological effects in vitro, including potent antioxidant activity as well as inhibition of CYP2C9, CYP3A4 and lipoxygenase activity without affecting the release of TNF-α. Unlike curcumin and calebin-A, THC did not inhibit HDAC1 and PCAF and displayed a weaker growth inhibition activity against Sup-T1 cells. We show evidence for the first time that curcumin and calebin-A inhibit HAT and PCAF, possibly through a Michael-addition mechanism.
Garcinol, a Histone Acetyltransferase Inhibitor, Radiosensitizes Cancer Cells by Inhibiting Non-Homologous End Joining
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Oike, Takahiro [Division of Multistep Carcinogenesis, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi, Gunma (Japan); Ogiwara, Hideaki [Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Torikai, Kohta [Gunma University Heavy Ion Medical Center, Maebashi, Gunma (Japan); Nakano, Takashi [Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi, Gunma (Japan); Yokota, Jun [Division of Multistep Carcinogenesis, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Kohno, Takashi, E-mail: email@example.com [Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan)
Purpose: Non-homologous end joining (NHEJ), a major pathway used to repair DNA double-strand breaks (DSBs) generated by ionizing radiation (IR), requires chromatin remodeling at DSB sites through the acetylation of histones by histone acetyltransferases (HATs). However, the effect of compounds with HAT inhibitory activities on the DNA damage response (DDR), including the NHEJ and cell cycle checkpoint, as well as on the radiosensitivity of cancer cells, remains largely unclear. Here, we investigated whether garcinol, a HAT inhibitor found in the rinds of Garcinia indica fruit (called mangosteens), has effects on DDR, and whether it can be used for radiosensitization. Methods and Materials: The following assays were used to examine the effect of garcinol on the inhibition of DSB repair, including the following: a conventional neutral comet assay; a cell-based assay recently developed by us, in which NHEJ repair of DSBs on chromosomal DNA was evaluated; the micrococcal nuclease sensitivity assay; and immunoblotting for autophosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs). We assessed the effect of garcinol on the cell cycle checkpoint after IR treatment by analyzing the phosphorylation levels of checkpoint kinases CHK1 and CHK2 and histone H3, and by cell cycle profile analysis using flow cytometry. The radiosensitizing effect of garcinol was assessed by a clonogenic survival assay, whereas its effects on apoptosis and senescence were examined by annexin V and senescence-associated {beta}-galactosidase (SA-{beta}-Gal) staining, respectively. Results: We found that garcinol inhibits DSB repair, including NHEJ, without affecting cell cycle checkpoint. Garcinol radiosensitized A549 lung and HeLa cervical carcinoma cells with dose enhancement ratios (at 10% surviving fraction) of 1.6 and 1.5, respectively. Cellular senescence induced by IR was enhanced by garcinol. Conclusion: These results suggest that garcinol is a radiosensitizer that
The MYST family histone acetyltransferase complex regulates stress resistance and longevity through transcriptional control of DAF-16/FOXO transcription factors.
Ikeda, Takako; Uno, Masaharu; Honjoh, Sakiko; Nishida, Eisuke
The well-known link between longevity and the Sir2 histone deacetylase family suggests that histone deacetylation, a modification associated with repressed chromatin, is beneficial to longevity. However, the molecular links between histone acetylation and longevity remain unclear. Here, we report an unexpected finding that the MYST family histone acetyltransferase complex (MYS-1/TRR-1 complex) promotes rather than inhibits stress resistance and longevity in Caenorhabditis elegans Our results show that these beneficial effects are largely mediated through transcriptional up-regulation of the FOXO transcription factor DAF-16. MYS-1 and TRR-1 are recruited to the promoter regions of the daf-16 gene, where they play a role in histone acetylation, including H4K16 acetylation. Remarkably, we also find that the human MYST family Tip60/TRRAP complex promotes oxidative stress resistance by up-regulating the expression of FOXO transcription factors in human cells. Tip60 is recruited to the promoter regions of the foxo1 gene, where it increases H4K16 acetylation levels. Our results thus identify the evolutionarily conserved role of the MYST family acetyltransferase as a key epigenetic regulator of DAF-16/FOXO transcription factors. © 2017 The Authors.
The transcriptional cofactor MIER1-beta negatively regulates histone acetyltransferase activity of the CREB-binding protein
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Gillespie Laura L
Full Text Available Abstract Background Mier1 encodes a novel transcriptional regulator and was originally isolated as a fibroblast growth factor early response gene. Two major protein isoforms have been identified, MIER1α and β, which differ in their C-terminal sequence. Previously, we demonstrated that both isoforms recruit histone deacetylase 1 (HDAC1 to repress transcription. To further explore the role of MIER1 in chromatin remodeling, we investigated the functional interaction of MIER1 with the histone acetyltransferase (HAT, Creb-binding protein (CBP. Findings Using GST pull-down assays, we demonstrate that MIER1 interacts with CBP and that this interaction involves the N-terminal half (amino acids 1–283 of MIER1, which includes the acidic activation and ELM2 domains and the C-terminal half (amino acids 1094–2441 of CBP, which includes the bromo-, HAT, C/H3 and glutamine-rich domains. Functional analysis, using HEK293 cells, shows that the CBP bound to MIER1 in vivo has no detectable HAT activity. Histone 4 peptide binding assays demonstrate that this inhibition of HAT activity is not the result of interference with histone binding. Conclusion Our data indicate that an additional mechanism by which MIER1 could repress transcription involves the inhibition of histone acetyltransferase activity.
The histone acetyltransferase PsGcn5 mediates oxidative stress responses and is required for full virulence of Phytophthora sojae.
Zhao, Wei; Wang, Tao; Liu, Shusen; Chen, Qingqing; Qi, Rende
In eukaryotic organisms, histone acetyltransferase complexes are coactivators that are important for transcriptional activation by modifying chromatin. In this study, a gene (PsGcn5) from Phytophthora sojae encoding a histone acetyltransferase was identified as a homolog of one component of the histone acetyltransferase complex from yeasts to mammals. PsGcn5 was constitutively expressed in each stage tested, but had a slightly higher expression in sporulating hyphae and 3 h after infection. PsGcn5-silenced mutants were generated using polyethylene glycol-mediated protoplast stable transformation. These mutants had normal development, but compared to wild type strains they had higher sensitivity to hydrogen peroxide (H2O2) and significantly reduced virulence in soybean. Diaminobenzidine staining revealed an accumulation of H2O2 around the infection sites of PsGcn5-silenced mutants but not for wild type strains. Inhibition of the plant NADPH oxidase by diphenyleneiodonium prevented host-derived H2O2 accumulation in soybean cells and restored infectious hyphal growth of the mutants. Thus, we concluded that PsGcn5 is important for growth under conditions of oxidative stress and contributes to the full virulence of P. sojae by suppressing the host-derived reactive oxygen species. Copyright © 2015 Elsevier Ltd. All rights reserved.
Interactions of Histone Acetyltransferase p300 with the Nuclear Proteins Histone and HMGB1, As Revealed by Single Molecule Atomic Force Spectroscopy.
Banerjee, S; Rakshit, T; Sett, S; Mukhopadhyay, R
One of the important properties of the transcriptional coactivator p300 is histone acetyltransferase (HAT) activity that enables p300 to influence chromatin action via histone modulation. p300 can exert its HAT action upon the other nuclear proteins too--one notable example being the transcription-factor-like protein HMGB1, which functions also as a cytokine, and whose accumulation in the cytoplasm, as a response to tissue damage, is triggered by its acetylation. Hitherto, no information on the structure and stability of the complexes between full-length p300 (p300FL) (300 kDa) and the histone/HMGB1 proteins are available, probably due to the presence of unstructured regions within p300FL that makes it difficult to be crystallized. Herein, we have adopted the high-resolution atomic force microscopy (AFM) approach, which allows molecularly resolved three-dimensional contour mapping of a protein molecule of any size and structure. From the off-rate and activation barrier values, obtained using single molecule dynamic force spectroscopy, the biochemical proposition of preferential binding of p300FL to histone H3, compared to the octameric histone, can be validated. Importantly, from the energy landscape of the dissociation events, a model for the p300-histone and the p300-HMGB1 dynamic complexes that HAT forms, can be proposed. The lower unbinding forces of the complexes observed in acetylating conditions, compared to those observed in non-acetylating conditions, indicate that upon acetylation, p300 tends to weakly associate, probably as an outcome of charge alterations on the histone/HMGB1 surface and/or acetylation-induced conformational changes. To our knowledge, for the first time, a single molecule level treatment of the interactions of HAT, where the full-length protein is considered, is being reported.
Early adipogenesis is regulated through USP7-mediated deubiquitination of the histone acetyltransferase TIP60
Gao, Yuan; Koppen, Arjen; Rakhshandehroo, Maryam; Tasdelen, Ismayil; van de Graaf, Stan F.; van Loosdregt, Jorg; van Beekum, Olivier; Hamers, Nicole; van Leenen, Dik; Berkers, Celia R.; Berger, Ruud; Holstege, Frank C. P.; Coffer, Paul J.; Brenkman, Arjan B.; Ovaa, Huib; Kalkhoven, Eric
Transcriptional coregulators, including the acetyltransferase Tip60, have a key role in complex cellular processes such as differentiation. Whereas post-translational modifications have emerged as an important mechanism to regulate transcriptional coregulator activity, the identification of the
Metabolic regulation of histone acetyltransferases by endogenous Acyl-CoA cofactors | Center for Cancer Research
Unraveling the metabolic regulation of lysine acetyltransferases (KATs). Montgomery et al. detail the application of a competitive chemoproteomic strategy to quantitatively characterize the interactions of acyl-CoA metabolites with cellular KAT enzymes.
Arsenic Trioxide Reduces Global Histone H4 Acetylation at Lysine 16 through Direct Binding to Histone Acetyltransferase hMOF in Human Cells
Liu, Da; Wu, Donglu; Zhao, Linhong; Yang, Yang; Ding, Jian; Dong, Liguo; Hu, Lianghai; Wang, Fei; Zhao, Xiaoming; Cai, Yong; Jin, Jingji
Histone post-translational modification heritably regulates gene expression involved in most cellular biological processes. Experimental studies suggest that alteration of histone modifications affects gene expression by changing chromatin structure, causing various cellular responses to environmental influences. Arsenic (As), a naturally occurring element and environmental pollutant, is an established human carcinogen. Recently, increasing evidence suggests that As-mediated epigenetic mechanisms may be involved in its toxicity and carcinogenicity, but how this occurs is still unclear. Here we present evidence that suggests As-induced global histone H4K16 acetylation (H4K16ac) partly due to the direct physical interaction between As and histone acetyltransferase (HAT) hMOF (human male absent on first) protein, leading to the loss of hMOF HAT activity. Our data show that decreased global H4K16ac and increased deacetyltransferase HDAC4 expression occurred in arsenic trioxide (As2O3)-exposed HeLa or HEK293T cells. However, depletion of HDAC4 did not affect global H4K16ac, and it could not raise H4K16ac in cells exposed to As2O3, suggesting that HDAC4 might not directly be involved in histone H4K16 de-acetylation. Using As-immobilized agarose, we confirmed that As binds directly to hMOF, and that this interaction was competitively inhibited by free As2O3. Also, the direct interaction of As and C2CH zinc finger peptide was verified by MAIDI-TOF mass and UV absorption. In an in vitro HAT assay, As2O3 directly inhibited hMOF activity. hMOF over-expression not only increased resistance to As and caused less toxicity, but also effectively reversed reduced H4K16ac caused by As exposure. These data suggest a theoretical basis for elucidating the mechanism of As toxicity. PMID:26473953
A naturally-occurring histone acetyltransferase inhibitor derived from Garcinia indica impairs newly acquired and reactivated fear memories.
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Stephanie A Maddox
Full Text Available The study of the cellular and molecular mechanisms underlying the consolidation and reconsolidation of traumatic fear memories has progressed rapidly in recent years, yet few compounds have emerged that are readily useful in a clinical setting for the treatment of anxiety disorders such as post-traumatic stress disorder (PTSD. Here, we use a combination of biochemical, behavioral, and neurophysiological methods to systematically investigate the ability of garcinol, a naturally-occurring histone acetyltransferase (HAT inhibitor derived from the rind of the fruit of the Kokum tree (Garcina indica, to disrupt the consolidation and reconsolidation of Pavlovian fear conditioning, a widely studied rodent model of PTSD. We show that local infusion of garcinol into the rat lateral amygdala (LA impairs the training and retrieval-related acetylation of histone H3 in the LA. Further, we show that either intra-LA or systemic administration of garcinol within a narrow window after either fear conditioning or fear memory retrieval significantly impairs the consolidation and reconsolidation of a Pavlovian fear memory and associated neural plasticity in the LA. Our findings suggest that a naturally-occurring compound derived from the diet that regulates chromatin function may be useful in the treatment of newly acquired or recently reactivated traumatic memories.
Absence of Rtt109p, a fungal-specific histone acetyltransferase, results in improved acetic acid tolerance of Saccharomyces cerevisiae.
Cheng, Cheng; Zhao, Xinqing; Zhang, Mingming; Bai, Fengwu
RTT109 is a histone acetyltransferase for the acetylation of histone H3. It is still not clear whether RTT109 plays a role in regulation of gene expression under environmental stresses. In this study, the involvement of RTT109 in acetic acid stress tolerance of Saccharomyces cerevisiae was investigated. It was revealed that the absence of RTT109 enhanced resistance to 5.5 g L(-1) acetic acid, which was indicated by improved growth of RTT109Δ mutant compared with that of the wild-type BY4741 strain. Meanwhile, the lag phase was shortened for 48 h and glucose consumption completed 36 h in advance for RTT109Δ mutant compared to the wild-type strain, with ethanol production rate increased from 0.39 to 0.60 g L(-1) h(-1). Significantly, elevated transcription levels of HSP12, CTT1 and GSH1, as well as increased activities of antioxidant enzymes were observed in RTT109Δ under acetic acid stress. Improved flocculation of RTT109Δ compared to that of the control strain BY4741 under the acetic acid stress was also observed. These results suggest that the absence of RTT109 not only activates transcription of stress responsive genes, but also improves resistance to oxidative stress, which ultimately contributes to improved acetic acid tolerance in S. cerevisiae. © FEMS 2016. All rights reserved. For permissions, please e-mail: firstname.lastname@example.com.
Mutation of the CH1 Domain in the Histone Acetyltransferase CREBBP Results in Autism-Relevant Behaviors in Mice.
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Fei Zheng
Full Text Available Autism spectrum disorders (ASDs are a group of neurodevelopmental afflictions characterized by repetitive behaviors, deficits in social interaction, and impaired communication skills. For most ASD patients, the underlying causes are unknown. Genetic mutations have been identified in about 25 percent of ASD cases, including mutations in epigenetic regulators, suggesting that dysregulated chromatin or DNA function is a critical component of ASD. Mutations in the histone acetyltransferase CREB binding protein (CBP, CREBBP cause Rubinstein-Taybi Syndrome (RTS, a developmental disorder that includes ASD-like symptoms. Recently, genomic studies involving large numbers of ASD patient families have theoretically modeled CBP and its paralog p300 (EP300 as critical hubs in ASD-associated protein and gene interaction networks, and have identified de novo missense mutations in highly conserved residues of the CBP acetyltransferase and CH1 domains. Here we provide animal model evidence that supports this notion that CBP and its CH1 domain are relevant to autism. We show that mice with a deletion mutation in the CBP CH1 (TAZ1 domain (CBPΔCH1/ΔCH1 have an RTS-like phenotype that includes ASD-relevant repetitive behaviors, hyperactivity, social interaction deficits, motor dysfunction, impaired recognition memory, and abnormal synaptic plasticity. Our results therefore indicate that loss of CBP CH1 domain function contributes to RTS, and possibly ASD, and that this domain plays an essential role in normal motor function, cognition and social behavior. Although the key physiological functions affected by ASD-associated mutation of epigenetic regulators have been enigmatic, our findings are consistent with theoretical models involving CBP and p300 in ASD, and with a causative role for recently described ASD-associated CBP mutations.
E2A proteins enhance the histone acetyltransferase activity of the transcriptional co-activators CBP and p300.
Hyndman, Brandy D; Thompson, Patrick; Bayly, Richard; Côté, Graham P; LeBrun, David P
The E2A gene encodes the E-protein transcription factors E12 and E47 that play critical roles in B-lymphopoiesis. A somatic chromosomal translocation detectable in 5% of cases of acute lymphoblastic leukemia (ALL) involves E2A and results in expression of the oncogenic transcription factor E2A-PBX1. CREB binding protein (CBP) and its close paralog p300 are transcriptional co-activators with intrinsic histone acetyltransferase (HAT) activity. We and others have shown that direct binding of an N-terminal transcriptional activation domain present in E12/E47 and E2A-PBX1 to the KIX domain of CBP/p300 contributes to E2A protein function. In the current work we show for the first time that the catalytic HAT activity of CBP/p300 is increased in the presence of residues 1-483 of E2A (i.e., the portion present in E2A-PBX1). The addition of purified, recombinant E2A protein to in vitro assays results in a two-fold augmentation of CBP/p300 HAT activity, whereas in vivo assays show a ten-fold augmentation of HAT-dependent transcriptional induction and a five-fold augmentation of acetylation of reporter plasmid-associated histone by CBP in response to co-transfected E2A. Our results indicate that the HAT-enhancing effect is independent of the well-documented E2A-CBP interaction involving the KIX domain and suggest a role for direct, perhaps low affinity binding of E2A to a portion of CBP that includes the HAT domain and flanking elements. Our findings add to a growing body of literature indicating that interactions between CBP/p300 and transcription factors can function in a specific manner to modulate HAT catalytic activity. Copyright © 2012 Elsevier B.V. All rights reserved.
Delphinidin, a specific inhibitor of histone acetyltransferase, suppresses inflammatory signaling via prevention of NF-κB acetylation in fibroblast-like synoviocyte MH7A cells
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Seong, Ah-Reum; Yoo, Jung-Yoon; Choi, KyungChul; Lee, Mee-Hee; Lee, Yoo-Hyun; Lee, Jeongmin; Jun, Woojin; Kim, Sunoh; Yoon, Ho-Geun
Highlights: → Delphinidin is a novel inhibitor of p300/CBP histone acetyltransferase. → Delphinidin prevents the hyperacetylation of p65 by inhibiting the HAT activity of p300/CBP. → Delphinidin efficiently suppresses the expression of inflammatory cytokines in MH7A cells via hypoacetylation of NF-κB. → Delphinidin inhibits cytokine release in the Jurkat T lymphocyte cell line. -- Abstract: Histone acetyltransferase (HAT) inhibitors (HATi) isolated from dietary compounds have been shown to suppress inflammatory signaling, which contributes to rheumatoid arthritis. Here, we identified a novel HATi in Punica granatum L. known as delphinidin (DP). DP did not affect the activity of other epigenetic enzymes (histone deacetylase, histone methyltransferase, or sirtuin1). DP specifically inhibited the HAT activities of p300/CBP. It also inhibited p65 acetylation in MH7A cells, a human rheumatoid arthritis synovial cell line. DP-induced hypoacetylation was accompanied by cytosolic accumulation of p65 and nuclear localization of IKBα. Accordingly, DP treatment inhibited TNFα-stimulated increases in NF-κB function and expression of NF-κB target genes in these cells. Importantly, DP suppressed lipopolysaccharide-induced pro-inflammatory cytokine expression in Jurkat T lymphocytes, demonstrating that HATi efficiently suppresses cytokine-mediated immune responses. Together, these results show that the HATi activity of DP counters anti-inflammatory signaling by blocking p65 acetylation and that this compound may be useful in preventing inflammatory arthritis.
Delphinidin, a specific inhibitor of histone acetyltransferase, suppresses inflammatory signaling via prevention of NF-{kappa}B acetylation in fibroblast-like synoviocyte MH7A cells
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Seong, Ah-Reum; Yoo, Jung-Yoon; Choi, KyungChul [Department of Biochemistry and Molecular Biology, Center for Chronic Metabolic Disease Research, College of Medicine, Yonsei University, Seoul (Korea, Republic of); Lee, Mee-Hee [Department of Biochemistry and Molecular Biology, Center for Chronic Metabolic Disease Research, College of Medicine, Yonsei University, Seoul (Korea, Republic of); Brain Korea 21 Project for Medical Sciences, Yonsei University, College of Medicine, Seoul (Korea, Republic of); Lee, Yoo-Hyun [Department of Food Science and Nutrition, The University of Suwon, Kyunggi-do (Korea, Republic of); Lee, Jeongmin [Department of Medical Nutrition, Kyung Hee University, Kyunggi-do (Korea, Republic of); Jun, Woojin [Department of Food and Nutrition, Chonnam National University, Gwangju (Korea, Republic of); Kim, Sunoh, E-mail: firstname.lastname@example.com [Jeollanamdo Institute of Natural Resources Research, Jeonnam (Korea, Republic of); Yoon, Ho-Geun, E-mail: email@example.com [Department of Biochemistry and Molecular Biology, Center for Chronic Metabolic Disease Research, College of Medicine, Yonsei University, Seoul (Korea, Republic of); Brain Korea 21 Project for Medical Sciences, Yonsei University, College of Medicine, Seoul (Korea, Republic of)
Highlights: {yields} Delphinidin is a novel inhibitor of p300/CBP histone acetyltransferase. {yields} Delphinidin prevents the hyperacetylation of p65 by inhibiting the HAT activity of p300/CBP. {yields} Delphinidin efficiently suppresses the expression of inflammatory cytokines in MH7A cells via hypoacetylation of NF-{kappa}B. {yields} Delphinidin inhibits cytokine release in the Jurkat T lymphocyte cell line. -- Abstract: Histone acetyltransferase (HAT) inhibitors (HATi) isolated from dietary compounds have been shown to suppress inflammatory signaling, which contributes to rheumatoid arthritis. Here, we identified a novel HATi in Punica granatum L. known as delphinidin (DP). DP did not affect the activity of other epigenetic enzymes (histone deacetylase, histone methyltransferase, or sirtuin1). DP specifically inhibited the HAT activities of p300/CBP. It also inhibited p65 acetylation in MH7A cells, a human rheumatoid arthritis synovial cell line. DP-induced hypoacetylation was accompanied by cytosolic accumulation of p65 and nuclear localization of IKB{alpha}. Accordingly, DP treatment inhibited TNF{alpha}-stimulated increases in NF-{kappa}B function and expression of NF-{kappa}B target genes in these cells. Importantly, DP suppressed lipopolysaccharide-induced pro-inflammatory cytokine expression in Jurkat T lymphocytes, demonstrating that HATi efficiently suppresses cytokine-mediated immune responses. Together, these results show that the HATi activity of DP counters anti-inflammatory signaling by blocking p65 acetylation and that this compound may be useful in preventing inflammatory arthritis.
The enhancing effect of genistein on apoptosis induced by trichostatin A in lung cancer cells with wild type p53 genes is associated with upregulation of histone acetyltransferase
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Wu, Tzu-Chin; Lin, Yi-Chin; Chen, Hsiao-Ling; Huang, Pei-Ru; Liu, Shang-Yu; Yeh, Shu-Lan
Genistein has been shown to enhance the antitumor activity of trichostatin A (TSA) in human lung carcinoma A549 cells. However, whether the combined treatment exerts the same effect in other lung cancer cells is unclear. In the present study we first compared the enhancing effect of genistein on the antitumor effect of TSA in ABC-1, NCI-H460 (H460) and A549 cells. Second, we investigated whether the effects of genistein are associated with increased histone/non-histone protein acetylation. We found that the enhancing effect of genistein on cell-growth-arrest in ABC-1 cells (p53 mutant) was less than in A549 and H460 cells. Genistein enhanced TSA induced apoptosis in A549 and H460 cells rather than in ABC-1 cells. After silencing p53 expression in A549 and H460 cells, the enhancing effect of genistein was diminished. In addition, genistein increased TSA-induced histone H3/H4 acetylation in A549 and H460 cells. Genistein also increased p53 acetylation in H460 cells. The inhibitor of acetyltransferase, anacardic acid, diminished the enhancing effect of genistein on all TSA-induced histone/p53 acetylation and apoptosis. Genistein in combination with TSA increased the expression of p300 protein, an acetyltransferase, in A549 and NCI-H460 cells. Furthermore, we demonstrated that genistein also enhanced the antitumor effect of genistein in A549-tumor-bearing mice. Taken together, these results suggest that the enhancing effects of genistein on TSA-induced apoptosis in lung cancer cells were p53-dependent and were associated with histone/non-histone protein acetylation. - Highlights: • Genistein enhances the antitumor effect of TSA through p53-associated pathways. • Genistein enhances TSA-induced histone acetylation commonly. • An acetyltransferase inhibitor diminishes the antitumor effect of genistein + TSA. • TSA in combination with genistein increases the expression of p300. • Genistein given by i.p. injection increases the antitumor effect of TSA in vivo.
3D structure prediction of histone acetyltransferase (HAC proteins of the p300/CBP family and their interactome in Arabidopsis thaliana
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Amar Cemanovic
Full Text Available Histone acetylation is an important posttranslational modification correlated with gene activation. In Arabidopsis thaliana the histone acetyltransferase (HAC proteins of the CBP family are homologous to animal p300/CREB (cAMP-responsive element-binding proteins, which are important histone acetyltransferases participating in many physiological processes, including proliferation, differentiation, and apoptosis. In this study the 3-D structure of all HAC protein subunits in Arabidopsis thaliana: HAC1, HAC2, HAC4, HAC5 and HAC12 is predicted by homology modeling and confirmed by Ramachandran plot analysis. The amino acid sequences HAC family members are highly similar to the sequences of the homologous human p300/CREB protein. Conservation of p300/CBP domains among the HAC proteins was examined further by sequence alignment and pattern search. The domains of p300/CBP required for the HAC function, such as PHD, TAZ and ZZ domains, are conserved in all HAC proteins. Interactome analysis revealed that HAC1, HAC5 and HAC12 proteins interact with S-adenosylmethionine-dependent methyltransferase domaincontaining protein that shows methyltransferase activity, suggesting an additional function of the HAC proteins. Additionally, HAC5 has a strong interaction value for the putative c-myb-like transcription factor MYB3R-4, which suggests that it also may have a function in regulation of DNA replication.
Three-dimensional collagen I promotes gemcitabine resistance in vitro in pancreatic cancer cells through HMGA2-dependent histone acetyltransferase expression.
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Surabhi Dangi-Garimella
Full Text Available Pancreatic ductal adenocarcinoma (PDAC is associated with a pronounced collagen-rich stromal reaction that has been shown to contribute to chemo-resistance. We have previously shown that PDAC cells are resistant to gemcitabine chemotherapy in the collagen microenvironment because of increased expression of the chromatin remodeling protein high mobility group A2 (HMGA2. We have now found that human PDAC tumors display higher levels of histone H3K9 and H3K27 acetylation in fibrotic regions. We show that relative to cells grown on tissue culture plastic, PDAC cells grown in three-dimensional collagen gels demonstrate increased histone H3K9 and H3K27 acetylation, along with increased expression of p300, PCAF and GCN5 histone acetyltransferases (HATs. Knocking down HMGA2 attenuates the effect of collagen on histone H3K9 and H3K27 acetylation and on collagen-induced p300, PCAF and GCN5 expression. We also show that human PDAC tumors with HMGA2 demonstrate increased histone H3K9 and H3K27 acetylation. Additionally, we show that cells in three-dimensional collagen gels demonstrate increased protection against gemcitabine. Significantly, down-regulation of HMGA2 or p300, PCAF and GCN5 HATs sensitizes the cells to gemcitabine in three-dimensional collagen. Overall, our results increase our understanding of how the collagen microenvironment contributes to chemo-resistance in vitro and identify HATs as potential therapeutic targets against this deadly cancer.
Inhibition of Different Histone Acetyltransferases (HATs) Uncovers Transcription-Dependent and -Independent Acetylation-Mediated Mechanisms in Memory Formation
Merschbaecher, Katja; Hatko, Lucyna; Folz, Jennifer; Mueller, Uli
Acetylation of histones changes the efficiency of the transcription processes and thus contributes to the formation of long-term memory (LTM). In our comparative study, we used two inhibitors to characterize the contribution of different histone acetyl transferases (HATs) to appetitive associative learning in the honeybee. For one we applied…
Depletion of histone N-terminal-acetyltransferase Naa40 induces p53-independent apoptosis in colorectal cancer cells via the mitochondrial pathway.
Pavlou, Demetria; Kirmizis, Antonis
Protein N-terminal acetylation is an abundant post-translational modification in eukaryotes implicated in various fundamental cellular and biochemical processes. This modification is catalysed by evolutionarily conserved N-terminal acetyltransferases (NATs) whose deregulation has been linked to cancer development and thus, are emerging as useful diagnostic and therapeutic targets. Naa40 is a highly selective NAT that acetylates the amino-termini of histones H4 and H2A and acts as a sensor of cell growth in yeast. In the present study, we examine the role of Naa40 in cancer cell survival. We demonstrate that depletion of Naa40 in HCT116 and HT-29 colorectal cancer cells decreases cell survival by enhancing apoptosis, whereas Naa40 reduction in non-cancerous mouse embryonic fibroblasts has no effect on cell viability. Specifically, Naa40 knockdown in colon cancer cells activates the mitochondrial caspase-9-mediated apoptotic cascade. Consistent with this, we show that caspase-9 activation is required for the induced apoptosis because treatment of cells with an irreversible caspase-9 inhibitor impedes apoptosis when Naa40 is depleted. Furthermore, the effect of Naa40-depletion on cell-death is mediated through a p53-independent mechanism since p53-null HCT116 cells still undergo apoptosis upon reduction of the acetyltransferase. Altogether, these findings reveal an anti-apoptotic role for Naa40 and exhibit its potential as a therapeutic target in colorectal cancers.
The Histone Acetyltransferase Gcn5 Regulates ncRNA-ICR1 and FLO11 Expression during Pseudohyphal Development in Saccharomyces cerevisiae
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Long-Chi Wang
Full Text Available Filamentous growth is one of the key features of pathogenic fungi during the early infectious phase. The pseudohyphal development of yeast Saccharomyces cerevisiae shares similar characteristics with hyphae elongation in pathogenic fungi. The expression of FLO11 is essential for adhesive growth and filament formation in yeast and is governed by a multilayered transcriptional network. Here we discovered a role for the histone acetyltransferase general control nonderepressible 5 (Gcn5 in regulating FLO11-mediated pseudohyphal growth. The expression patterns of FLO11 were distinct in haploid and diploid yeast under amino acid starvation induced by 3-amino-1,2,4-triazole (3AT. In diploids, FLO11 expression was substantially induced at a very early stage of pseudohyphal development and decreased quickly, but in haploids, it was gradually induced. Furthermore, the transcription factor Gcn4 was recruited to the Sfl1-Flo8 toggle sites at the FLO11 promoter under 3AT treatment. Moreover, the histone acetylase activity of Gcn5 was required for FLO11 induction. Finally, Gcn5 functioned as a negative regulator of the noncoding RNA ICR1, which is known to suppress FLO11 expression. Gcn5 plays an important role in the regulatory network of FLO11 expression via Gcn4 by downregulating ICR1 expression, which derepresses FLO11 for promoting pseudohyphal development.
Histone acetyltransferase (HAT) activity of p300 modulates human T lymphotropic virus type 1 p30II-mediated repression of LTR transcriptional activity
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Michael, Bindhu; Nair, Amrithraj M.; Datta, Antara; Hiraragi, Hajime; Ratner, Lee; Lairmore, Michael D.
Human T-lymphotropic virus type-1 (HTLV-1) is a deltaretrovirus that causes adult T cell leukemia/lymphoma, and is implicated in a variety of lymphocyte-mediated inflammatory disorders. HTLV-1 provirus has regulatory and accessory genes in four pX open reading frames. HTLV-1 pX ORF-II encodes two proteins, p13 II and p30 II , which are incompletely defined in virus replication or pathogenesis. We have demonstrated that pX ORF-II mutations block virus replication in vivo and that ORF-II encoded p30 II , a nuclear-localizing protein that binds with CREB-binding protein (CBP)/p300, represses CREB and Tax responsive element (TRE)-mediated transcription. Herein, we have identified p30 II motifs important for p300 binding and in regulating TRE-mediated transcription in the absence and presence of HTLV-1 provirus. Within amino acids 100-179 of p30 II , a region important for repression of LTR-mediated transcription, we identified a single lysine residue at amino acid 106 (K3) that significantly modulates the ability of p30 II to repress TRE-mediated transcription. Exogenous p300, in a dose-responsive manner, reverses p30 II -dependent repression of TRE-mediated transcription, in the absence or presence of the provirus, In contrast to wild type p300, p300 HAT mutants (defective in histone acetyltransferase activity) only partially rescued p30 II -mediated LTR repression. Deacetylation by histone deacetylase-1 (HDAC-1) enhanced p30 II -mediated LTR repression, while inhibition of deacetylation by trichostatin A decreases p30 II -mediated LTR repression. Collectively, our data indicate that HTLV-1 p30 II modulates viral gene expression in a cooperative manner with p300-mediated acetylation
Whole-exome-sequencing identifies mutations in histone acetyltransferase gene KAT6B in individuals with the Say-Barber-Biesecker variant of Ohdo syndrome.
Clayton-Smith, Jill; O'Sullivan, James; Daly, Sarah; Bhaskar, Sanjeev; Day, Ruth; Anderson, Beverley; Voss, Anne K; Thomas, Tim; Biesecker, Leslie G; Smith, Philip; Fryer, Alan; Chandler, Kate E; Kerr, Bronwyn; Tassabehji, May; Lynch, Sally-Ann; Krajewska-Walasek, Malgorzata; McKee, Shane; Smith, Janine; Sweeney, Elizabeth; Mansour, Sahar; Mohammed, Shehla; Donnai, Dian; Black, Graeme
Say-Barber-Biesecker-Young-Simpson syndrome (SBBYSS or Ohdo syndrome) is a multiple anomaly syndrome characterized by severe intellectual disability, blepharophimosis, and a mask-like facial appearance. A number of individuals with SBBYSS also have thyroid abnormalities and cleft palate. The condition usually occurs sporadically and is therefore presumed to be due in most cases to new dominant mutations. In individuals with SBBYSS, a whole-exome sequencing approach was used to demonstrate de novo protein-truncating mutations in the highly conserved histone acetyltransferase gene KAT6B (MYST4/MORF)) in three out of four individuals sequenced. Sanger sequencing was used to confirm truncating mutations of KAT6B, clustering in the final exon of the gene in all four individuals and in a further nine persons with typical SBBYSS. Where parental samples were available, the mutations were shown to have occurred de novo. During mammalian development KAT6B is upregulated specifically in the developing central nervous system, facial structures, and limb buds. The phenotypic features seen in the Qkf mouse, a hypomorphic Kat6b mutant, include small eyes, ventrally placed ears and long first digits that mirror the human phenotype. This is a further example of how perturbation of a protein involved in chromatin modification might give rise to a multisystem developmental disorder. Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.
Directional Migration in Esophageal Squamous Cell Carcinoma (ESCC is Epigenetically Regulated by SET Nuclear Oncogene, a Member of the Inhibitor of Histone Acetyltransferase Complex
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Xiang Yuan
Full Text Available Directional cell migration is of fundamental importance to a variety of biological events, including metastasis of malignant cells. Herein, we specifically investigated SET oncoprotein, a subunit of the recently identified inhibitor of acetyltransferases (INHAT complex and identified its role in the establishment of front–rear cell polarity and directional migration in Esophageal Squamous Cell Carcinoma (ESCC. We further define the molecular circuits that govern these processes by showing that SET modulated DOCK7/RAC1 and cofilin signaling events. Moreover, a detailed analysis of the spatial distribution of RAC1 and cofilin allowed us to decipher the synergistical contributions of the two in coordinating the advancing dynamics by measuring architectures, polarities, and cytoskeletal organizations of the lamellipodia leading edges. In further investigations in vivo, we identified their unique role at multiple levels of the invasive cascade for SET cell and indicate the necessity for their functional balance to enable efficient invasion as well. Additionally, SET epigenetically repressed miR-30c expression by deacetylating histones H2B and H4 on its promoter, which was functionally important for the biological effects of SET in our cell-context. Finally, we corroborated our findings in vivo by evaluating the clinical relevance of SET signaling in the metastatic burden in mice and a large series of patients with ESCC at diagnosis, observing it's significance in predicting metastasis formation. Our findings uncovered a novel signaling network initiated by SET that epigenetically modulated ESCC properties and suggest that targeting the regulatory axis might be a promising strategy to inhibit migration and metastasis.
Directional Migration in Esophageal Squamous Cell Carcinoma (ESCC) is Epigenetically Regulated by SET Nuclear Oncogene, a Member of the Inhibitor of Histone Acetyltransferase Complex.
Yuan, Xiang; Wang, Xinshuai; Gu, Bianli; Ma, Yingjian; Liu, Yiwen; Sun, Man; Kong, Jinyu; Sun, Wei; Wang, Huizhi; Zhou, Fuyou; Gao, Shegan
Directional cell migration is of fundamental importance to a variety of biological events, including metastasis of malignant cells. Herein, we specifically investigated SET oncoprotein, a subunit of the recently identified inhibitor of acetyltransferases (INHAT) complex and identified its role in the establishment of front-rear cell polarity and directional migration in Esophageal Squamous Cell Carcinoma (ESCC). We further define the molecular circuits that govern these processes by showing that SET modulated DOCK7/RAC1 and cofilin signaling events. Moreover, a detailed analysis of the spatial distribution of RAC1 and cofilin allowed us to decipher the synergistical contributions of the two in coordinating the advancing dynamics by measuring architectures, polarities, and cytoskeletal organizations of the lamellipodia leading edges. In further investigations in vivo, we identified their unique role at multiple levels of the invasive cascade for SET cell and indicate the necessity for their functional balance to enable efficient invasion as well. Additionally, SET epigenetically repressed miR-30c expression by deacetylating histones H2B and H4 on its promoter, which was functionally important for the biological effects of SET in our cell-context. Finally, we corroborated our findings in vivo by evaluating the clinical relevance of SET signaling in the metastatic burden in mice and a large series of patients with ESCC at diagnosis, observing it's significance in predicting metastasis formation. Our findings uncovered a novel signaling network initiated by SET that epigenetically modulated ESCC properties and suggest that targeting the regulatory axis might be a promising strategy to inhibit migration and metastasis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.
No germline mutations in the histone acetyltransferase gene EP300 in BRCA1 and BRCA2 negative families with breast cancer and gastric, pancreatic, or colorectal cancer
International Nuclear Information System (INIS)
Campbell, Ian G; Choong, David; Chenevix-Trench, Georgia
Mutations in BRCA1, BRCA2, ATM, TP53, CHK2 and PTEN account for many, but not all, multiple-case breast and ovarian cancer families. The histone acetyltransferase gene EP300 may function as a tumour suppressor gene because it is sometimes somatically mutated in breast, colorectal, gastric and pancreatic cancers, and is located on a region of chromosome 22 that frequently undergoes loss of heterozygosity in many cancer types. We hypothesized that germline mutations in EP300 may account for some breast cancer families that include cases of gastric, pancreatic and/or colorectal cancer. We screened the entire coding region of EP300 for mutations in the youngest affected members of 23 non-BRCA1/BRCA2 breast cancer families with at least one confirmed case of gastric, pancreatic and/or colorectal cancer. These families were ascertained in Australia through the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer. Denaturing HPLC analysis identified a heterozygous alteration at codon 211, specifically a GGC to AGC (glycine to serine) alteration, in two individuals. This conservative amino acid change was not within any known functional domains of EP300. The frequency of the Ser211 variant did not differ significanlty between a series of 352 breast cancer patients (4.0%) and 254 control individuals (2.8%; P = 0.5). The present study does not support a major role for EP300 mutations in breast and ovarian cancer families with a history of gastric, pancreatic and/or colorectal cancer
Plant Responses to Abiotic Stress Regulated by Histone Deacetylases
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Ming Luo
Full Text Available In eukaryotic cells, histone acetylation and deacetylation play an important role in the regulation of gene expression. Histone acetylation levels are modulated by histone acetyltransferases and histone deacetylases (HDACs. Recent studies indicate that HDACs play essential roles in the regulation of gene expression in plant response to environmental stress. In this review, we discussed the recent advance regarding the plant HDACs and their functions in the regulation of abiotic stress responses. The role of HDACs in autophagy was also discussed.
Histone deacetylase inhibitors induced differentiation and accelerated mineralization of pulp-derived cells.
LENUS (Irish Health Repository)
Duncan, Henry F
Histone deacetylase inhibitors (HDACis) alter the homeostatic balance between 2 groups of cellular enzymes, histone deacetylases (HDACs) and histone acetyltransferases (HATs), increasing transcription and influencing cell behavior. This study investigated the potential of 2 HDACis, valproic acid (VPA) and trichostatin A (TSA), to promote reparative processes in pulp cells as assayed by viability, cell cycle, and mineralization analyses.
Expression profiling of S. pombe acetyltransferase mutants identifies redundant pathways of gene regulation
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Wright Anthony PH
Full Text Available Abstract Background Histone acetyltransferase enzymes (HATs are implicated in regulation of transcription. HATs from different families may overlap in target and substrate specificity. Results We isolated the elp3+ gene encoding the histone acetyltransferase subunit of the Elongator complex in fission yeast and characterized the phenotype of an Δelp3 mutant. We examined genetic interactions between Δelp3 and two other HAT mutants, Δmst2 and Δgcn5 and used whole genome microarray analysis to analyze their effects on gene expression. Conclusions Comparison of phenotypes and expression profiles in single, double and triple mutants indicate that these HAT enzymes have overlapping functions. Consistent with this, overlapping specificity in histone H3 acetylation is observed. However, there is no evidence for overlap with another HAT enzyme, encoded by the essential mst1+ gene.
A Non-Isotopic In Vitro Assay for Histone Acetylation
Kuninger, David; Lundblad, James; Semirale, Anthony; Rotwein, Peter
We describe a simple, robust, and relatively inexpensive non-radioactive in vitro assay for measuring histone acetyl-transferase activity. The assay takes advantage of easy to purify recombinant E. coli-derived fusion proteins containing the NH2-terminal tails of histones H3 and H4 linked to epitope-tagged maltose binding protein (MBP), and immunoblotting with antibodies specific to acetylated H3 and H4. Here we show the specificity and dynamic range of this assay for the histone acetyl-transferases, p300 and PCAF. This assay may be adapted readily for other substrates by simply generating new fusion proteins and for other acetyl-transferases by modifying reaction conditions. PMID:17698235
A Role for Histone Deacetylases in the Cellular and Behavioral Mechanisms Underlying Learning and Memory
Mahgoub, Melissa; Monteggia, Lisa M.
Histone deacetylases (HDACs) are a family of chromatin remodeling enzymes that restrict access of transcription factors to the DNA, thereby repressing gene expression. In contrast, histone acetyltransferases (HATs) relax the chromatin structure allowing for an active chromatin state and promoting gene transcription. Accumulating data have…
Snail acetylation by histone acetyltransferase p300 in lung cancer
Chang, Rui; Zhang, Yinjie; Zhang, Peng; Zhou, Qinghua
Background Epithelial to mesenchymal transition (EMT) is a complex and dynamic molecular event in lung cancer metastasis that has not yet been thoroughly investigated. EMT transcriptional factors, such as Snail, play a central role in regulation of the EMT process. In this study, we sought to identify an association between p300 and Snail in lung cancer, as well as the engagement of p300 in Snail acetylation. Methods We transfected p300 small interfering RNA into lung cancer cells to detect S...
Histone deacetylases and their roles in mineralized tissue regeneration
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Nam Cong-Nhat Huynh
Full Text Available Histone acetylation is an important epigenetic mechanism that controls expression of certain genes. It includes non-sequence-based changes of chromosomal regional structure that can alter the expression of genes. Acetylation of histones is controlled by the activity of two groups of enzymes: the histone acetyltransferases (HATs and histone deacetylases (HDACs. HDACs remove acetyl groups from the histone tail, which alters its charge and thus promotes compaction of DNA in the nucleosome. HDACs render the chromatin structure into a more compact form of heterochromatin, which makes the genes inaccessible for transcription. By altering the transcriptional activity of bone-associated genes, HDACs control both osteogenesis and osteoclastogenesis. This review presents an overview of the function of HDACs in the modulation of bone formation. Special attention is paid to the use of HDAC inhibitors in mineralized tissue regeneration from cells of dental origin.
Basic nuclear processes affected by histone acetyltransferases and histone deacetylase inhibitors
Czech Academy of Sciences Publication Activity Database
Legartová, Soňa; Stixová, Lenka; Strnad, Hynek; Kozubek, Stanislav; Martinet, N.; Dekker, F.J.; Franěk, Michal; Bártová, Eva
Roč. 5, č. 4 (2013), s. 379-396 ISSN 1750-1911 R&D Projects: GA MŠk(CZ) LD11020; GA ČR(CZ) GAP302/10/1022; GA ČR(CZ) GBP302/12/G157; GA ČR(CZ) GA13-07822S; GA ČR(CZ) EE2.3.30.0030 Institutional support: RVO:68081707 ; RVO:68378050 Keywords : cDNA microarray * DNA repair * epi-drug Subject RIV: BO - Biophysics; EB - Genetics ; Molecular Biology (UMG-J) Impact factor: 5.215, year: 2013
Neisseria meningitidis serogroup A capsular polysaccharide acetyltransferase, methods and compositions
Energy Technology Data Exchange (ETDEWEB)
Stephens, David S [Stone Mountain, GA; Gudlavalleti, Seshu K [Kensington, MD; Tzeng, Yih-Ling [Atlanta, GA; Datta, Anup K [San Diego, CA; Carlson, Russell W [Athens, GA
Provided are methods for recombinant production of an O-acetyltransferase and methods for acetylating capsular polysaccharides, especially those of a Serogroup A Neisseria meningitidis using the recombinant O-acetyltransferase, and immunogenic compositions comprising the acetylated capsular polysaccharide.
Histone deacetylase inhibitors enhance phosphorylation of histone H2AX after ionizing radiation
International Nuclear Information System (INIS)
Zhang Yubin; Adachi, Masaaki; Zou Huichao; Hareyama, Masato; Imai, Kohzoh; Shinomura, Yasuhisa
Purpose Histone deacetylase (HDAC) inhibitors are believed to be promising radiosensitizers. To explore their effects on ionizing radiation (IR), we examined whether the HDAC inhibitors m-carboxycinnamic acid bis-hydroxamide (CBHA) and depsipeptide FK228 affect H2AX phosphorylation (γ-H2AX), a landmark of DNA double-strand breaks after IR exposure. Methods and Materials We evaluated the effects of the HDAC inhibitors on clonogenic assay in human lung carcinoma A549 cells and progression of A549 xenograft tumors. IR-induced DNA damage was evaluated by histone γ-H2AX. Histone hyperacetylation was induced by overexpression of histone acetyltransferase p300 and evaluated by Western blots. Results M-carboxycinnamic acid bishydroxyamide pretreatment radiosensitized A549 cells and strongly inhibited A549 xenograft tumor progression. CBHA and FK228, but not 5-fluorouracil, enhanced IR-induced γ-H2AX in A549 and other cancer cell lines. Overexpression of p300 similarly augmented IR-induced γ-H2AX. Conclusion The results of this study suggest that HDAC inhibitors enhance IR-induced γ-H2AX, most likely through histone hyperacetylation, and radiosensitize various cancers
Histone deacetylases in memory and cognition.
Penney, Jay; Tsai, Li-Huei
Over the past 30 years, lysine acetylation of histone and nonhistone proteins has become established as a key modulator of gene expression regulating numerous aspects of cell biology. Neuronal growth and plasticity are no exception; roles for lysine acetylation and deacetylation in brain function and dysfunction continue to be uncovered. Transcriptional programs coupling synaptic activity to changes in gene expression are critical to the plasticity mechanisms underlying higher brain functions. These transcriptional programs can be modulated by changes in histone acetylation, and in many cases, transcription factors and histone-modifying enzymes are recruited together to plasticity-associated genes. Lysine acetylation, catalyzed by lysine acetyltransferases (KATs), generally promotes cognitive performance, whereas the opposing process, catalyzed by histone lysine deacetylases (HDACs), appears to negatively regulate cognition in multiple brain regions. Consistently, mutation or deregulation of different KATs or HDACs contributes to neurological dysfunction and neurodegeneration. HDAC inhibitors have shown promise as a treatment to combat the cognitive decline associated with aging and neurodegenerative disease, as well as to ameliorate the symptoms of depression and posttraumatic stress disorder, among others. In this review, we discuss the evidence for the roles of HDACs in cognitive function as well as in neurological disorders and disease. In particular, we focus on HDAC2, which plays a central role in coupling lysine acetylation to synaptic plasticity and mediates many of the effects of HDAC inhibition in cognition and disease. Copyright © 2014, American Association for the Advancement of Science.
Inhibition of histone deacetylases prevents cytokine-induced toxicity in beta cells
DEFF Research Database (Denmark)
Larsen, L; Tonnesen, M; Ronn, S G
B (NFkappaB) is a critical signalling molecule in inflammation and is required for expression of the gene encoding inducible NO synthase (iNOS) and of pro-apoptotic genes. NFkappaB has recently been shown to associate with chromatin-modifying enzymes histone acetyltransferases and histone...... by immunoblotting and by immunoblotting combined with electrophoretic mobility shift assay, respectively. Viability was analysed by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and apoptosis by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and histone...
PTEN Interacts with Histone H1 and Controls Chromatin Condensation
Chen, Zhu Hong; Zhu, Minglu; Yang, Jingyi; Liang, Hui; He, Jinxue; He, Shiming; Wang, Pan; Kang, Xi; McNutt, Michael A.; Yin, Yuxin; Shen, Wen H.
SUMMARY Chromatin organization and dynamics are integral to global gene transcription. Histone modification influences chromatin status and gene expression. PTEN plays multiple roles in tumor suppression, development and metabolism. Here we report on the interplay of PTEN, histone H1 and chromatin. We show that loss of PTEN leads to dissociation of histone H1 from chromatin and decondensation of chromatin. PTEN deletion also results in elevation of histone H4 acetylation at lysine 16, an epigenetic marker for chromatin activation. We found that PTEN and histone H1 physically interact through their C-terminal domains. Disruption of the PTEN C-terminus promotes the chromatin association of MOF acetyltransferase and induces H4K16 acetylation. Hyperacetylation of H4K16 impairs the association of PTEN with histone H1, which constitutes regulatory feedback that may deteriorate chromatin stability. Our results demonstrate that PTEN controls chromatin condensation, thus influencing gene expression. We propose that PTEN regulates global gene transcription profiling through histones and chromatin remodeling. PMID:25199838
Synthesis and bioactivity of novel histone acetylation inhibitors : potential new drugs for treatment of cancer and inflammation
Ghizzoni, Massimo
There is an increasing interest in histone acetyltransferases (HATs) as new therapeutic targets for treatment of diseases like, for example, inflammation and cancer. However, only few small and cell-permeable molecule inhibitors of HATs are currently available. The work described in this thesis
Histone Deacetylase Inhibitors as Anticancer Drugs
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Tomas Eckschlager
Full Text Available Carcinogenesis cannot be explained only by genetic alterations, but also involves epigenetic processes. Modification of histones by acetylation plays a key role in epigenetic regulation of gene expression and is controlled by the balance between histone deacetylases (HDAC and histone acetyltransferases (HAT. HDAC inhibitors induce cancer cell cycle arrest, differentiation and cell death, reduce angiogenesis and modulate immune response. Mechanisms of anticancer effects of HDAC inhibitors are not uniform; they may be different and depend on the cancer type, HDAC inhibitors, doses, etc. HDAC inhibitors seem to be promising anti-cancer drugs particularly in the combination with other anti-cancer drugs and/or radiotherapy. HDAC inhibitors vorinostat, romidepsin and belinostat have been approved for some T-cell lymphoma and panobinostat for multiple myeloma. Other HDAC inhibitors are in clinical trials for the treatment of hematological and solid malignancies. The results of such studies are promising but further larger studies are needed. Because of the reversibility of epigenetic changes during cancer development, the potency of epigenetic therapies seems to be of great importance. Here, we summarize the data on different classes of HDAC inhibitors, mechanisms of their actions and discuss novel results of preclinical and clinical studies, including the combination with other therapeutic modalities.
Ubiquitylation of the acetyltransferase MOF in Drosophila melanogaster.
Schunter, Sarah; Villa, Raffaella; Flynn, Victoria; Heidelberger, Jan B; Classen, Anne-Kathrin; Beli, Petra; Becker, Peter B
The nuclear acetyltransferase MOF (KAT8 in mammals) is a subunit of at least two multi-component complexes involved in transcription regulation. In the context of complexes of the 'Non-Specific-Lethal' (NSL) type it controls transcription initiation of many nuclear housekeeping genes and of mitochondrial genes. While this function is conserved in metazoans, MOF has an additional, specific function in Drosophila in the context of dosage compensation. As a subunit of the male-specific-lethal dosage compensation complex (MSL-DCC) it contributes to the doubling of transcription output from the single male X chromosome by acetylating histone H4. Proper dosage compensation requires finely tuned levels of MSL-DCC and an appropriate distribution of MOF between the regulatory complexes. The amounts of DCC formed depends directly on the levels of the male-specific MSL2, which orchestrates the assembly of the DCC, including MOF recruitment. We found earlier that MSL2 is an E3 ligase that ubiquitylates most MSL proteins, including MOF, suggesting that ubiquitylation may contribute to a quality control of MOF's overall levels and folding state as well as its partitioning between the complex entities. We now used mass spectrometry to map the lysines in MOF that are ubiquitylated by MSL2 in vitro and identified in vivo ubiquitylation sites of MOF in male and female cells. MSL2-specific ubiquitylation in vivo could not be traced due to the dominance of other, sex-independent ubiquitylation events and conceivably may be rare or transient. Expressing appropriately mutated MOF derivatives we assessed the importance of the ubiquitylated lysines for dosage compensation by monitoring DCC formation and X chromosome targeting in cultured cells, and by genetic complementation of the male-specific-lethal mof2 allele in flies. Our study provides a comprehensive analysis of MOF ubiquitylation as a reference for future studies.
Ubiquitylation of the acetyltransferase MOF in Drosophila melanogaster.
Directory of Open Access Journals (Sweden)
Sarah Schunter
Full Text Available The nuclear acetyltransferase MOF (KAT8 in mammals is a subunit of at least two multi-component complexes involved in transcription regulation. In the context of complexes of the 'Non-Specific-Lethal' (NSL type it controls transcription initiation of many nuclear housekeeping genes and of mitochondrial genes. While this function is conserved in metazoans, MOF has an additional, specific function in Drosophila in the context of dosage compensation. As a subunit of the male-specific-lethal dosage compensation complex (MSL-DCC it contributes to the doubling of transcription output from the single male X chromosome by acetylating histone H4. Proper dosage compensation requires finely tuned levels of MSL-DCC and an appropriate distribution of MOF between the regulatory complexes. The amounts of DCC formed depends directly on the levels of the male-specific MSL2, which orchestrates the assembly of the DCC, including MOF recruitment. We found earlier that MSL2 is an E3 ligase that ubiquitylates most MSL proteins, including MOF, suggesting that ubiquitylation may contribute to a quality control of MOF's overall levels and folding state as well as its partitioning between the complex entities. We now used mass spectrometry to map the lysines in MOF that are ubiquitylated by MSL2 in vitro and identified in vivo ubiquitylation sites of MOF in male and female cells. MSL2-specific ubiquitylation in vivo could not be traced due to the dominance of other, sex-independent ubiquitylation events and conceivably may be rare or transient. Expressing appropriately mutated MOF derivatives we assessed the importance of the ubiquitylated lysines for dosage compensation by monitoring DCC formation and X chromosome targeting in cultured cells, and by genetic complementation of the male-specific-lethal mof2 allele in flies. Our study provides a comprehensive analysis of MOF ubiquitylation as a reference for future studies.
Histone deacetylases and their role in motor neuron degeneration
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Rafael eLazo-Gómez
Full Text Available Amyotrophic lateral sclerosis (ALS is a fatal neurodegenerative disease, characterized by the progressive loss of motor neurons. The cause of this selective neuronal death is unknown, but transcriptional dysregulation is recently emerging as an important factor. The physical substrate for the regulation of the transcriptional process is chromatin, a complex assembly of histones and DNA. Histones are subject to several post-translational modifications, like acetylation, that are a component of the transcriptional regulation process. Histone acetylation and deacetylation is performed by a group of enzymes (histone acetyltransferases and deacetylases, respectively whose modulation can alter the transcriptional state of many regions of the genome, and thus may be an important target in diseases that share this pathogenic process, as is the case for ALS. This review will discuss the present evidence of transcriptional dysregulation in ALS, the role of histone deacetylases in disease pathogenesis, and the novel pharmacologic strategies that are being comprehensively studied to prevent motor neuron death, with focus on sirtuins and their effectors.
Histone variants and lipid metabolism
Borghesan, Michela; Mazzoccoli, Gianluigi; Sheedfar, Fareeba; Oben, Jude; Pazienza, Valerio; Vinciguerra, Manlio
Within nucleosomes, canonical histones package the genome, but they can be opportunely replaced with histone variants. The incorporation of histone variants into the nucleosome is a chief cellular strategy to regulate transcription and cellular metabolism. In pathological terms, cellular steatosis
The Histone Database: an integrated resource for histones and histone fold-containing proteins
Mariño-Ramírez, Leonardo; Levine, Kevin M.; Morales, Mario; Zhang, Suiyuan; Moreland, R. Travis; Baxevanis, Andreas D.; Landsman, David
Eukaryotic chromatin is composed of DNA and protein components—core histones—that act to compactly pack the DNA into nucleosomes, the fundamental building blocks of chromatin. These nucleosomes are connected to adjacent nucleosomes by linker histones. Nucleosomes are highly dynamic and, through various core histone post-translational modifications and incorporation of diverse histone variants, can serve as epigenetic marks to control processes such as gene expression and recombination. The Histone Sequence Database is a curated collection of sequences and structures of histones and non-histone proteins containing histone folds, assembled from major public databases. Here, we report a substantial increase in the number of sequences and taxonomic coverage for histone and histone fold-containing proteins available in the database. Additionally, the database now contains an expanded dataset that includes archaeal histone sequences. The database also provides comprehensive multiple sequence alignments for each of the four core histones (H2A, H2B, H3 and H4), the linker histones (H1/H5) and the archaeal histones. The database also includes current information on solved histone fold-containing structures. The Histone Sequence Database is an inclusive resource for the analysis of chromatin structure and function focused on histones and histone fold-containing proteins. Database URL: The Histone Sequence Database is freely available and can be accessed at http://research.nhgri.nih.gov/histones/. PMID:22025671
The lysine acetyltransferase activator Brpf1 governs dentate gyrus development through neural stem cells and progenitors.
Directory of Open Access Journals (Sweden)
Linya You
Full Text Available Lysine acetylation has recently emerged as an important post-translational modification in diverse organisms, but relatively little is known about its roles in mammalian development and stem cells. Bromodomain- and PHD finger-containing protein 1 (BRPF1 is a multidomain histone binder and a master activator of three lysine acetyltransferases, MOZ, MORF and HBO1, which are also known as KAT6A, KAT6B and KAT7, respectively. While the MOZ and MORF genes are rearranged in leukemia, the MORF gene is also mutated in prostate and other cancers and in four genetic disorders with intellectual disability. Here we show that forebrain-specific inactivation of the mouse Brpf1 gene causes hypoplasia in the dentate gyrus, including underdevelopment of the suprapyramidal blade and complete loss of the infrapyramidal blade. We trace the developmental origin to compromised Sox2+ neural stem cells and Tbr2+ intermediate neuronal progenitors. We further demonstrate that Brpf1 loss deregulates neuronal migration, cell cycle progression and transcriptional control, thereby causing abnormal morphogenesis of the hippocampus. These results link histone binding and acetylation control to hippocampus development and identify an important epigenetic regulator for patterning the dentate gyrus, a brain structure critical for learning, memory and adult neurogenesis.
Histone deacetylase regulation of immune gene expression in tumor cells
Tomasi, Thomas B.
Epigenetic modifications of chromatin, such as histone acetylation, are involved in repression of tumor antigens and multiple immune genes that are thought to facilitate tumor escape. The status of acetylation in a cell is determined by the balance of the activities of histone acetyltransferases and histone deacetylases. Inhibitors of histone deacetylase (HDACi) can enhance the expression of immunologically important molecules in tumor cells and HDACi treated tumor cells are able to induce immune responses in vitro and in vivo. Systemic HDACi are in clinical trails in cancer and also being used in several autoimmune disease models. To date, 18 HDACs have been reported in human cells and more than thirty HDACi identified, although only a few immune targets of these inhibitors have been identified. Here, we discuss the molecular pathways employed by HDACi and their potential role in inducing immune responses against tumors. We review data suggesting that selection of target specific HDACi and combinations with other agents and modalities, including those that activate stress pathways, may further enhance the efficacy of epigenetic therapies. PMID:18213528
Solar Simulated Ultraviolet Radiation Induces Global Histone Hypoacetylation in Human Keratinocytes.
Zhang, Xiaoru; Kluz, Thomas; Gesumaria, Lisa; Matsui, Mary S; Costa, Max; Sun, Hong
Ultraviolet radiation (UVR) from sunlight is the primary effector of skin DNA damage. Chromatin remodeling and histone post-translational modification (PTM) are critical factors in repairing DNA damage and maintaining genomic integrity, however, the dynamic changes of histone marks in response to solar UVR are not well characterized. Here we report global changes in histone PTMs induced by solar simulated UVR (ssUVR). A decrease in lysine acetylation of histones H3 and H4, particularly at positions of H3 lysine 9, lysine 56, H4 lysine 5, and lysine 16, was found in human keratinocytes exposed to ssUVR. These acetylation changes were highly associated with ssUVR in a dose-dependent and time-specific manner. Interestingly, H4K16ac, a mark that is crucial for higher order chromatin structure, exhibited a persistent reduction by ssUVR that was transmitted through multiple cell divisions. In addition, the enzymatic activities of histone acetyltransferases were significantly reduced in irradiated cells, which may account for decreased global acetylation. Moreover, depletion of histone deacetylase SIRT1 in keratinocytes rescued ssUVR-induced H4K16 hypoacetylation. These results indicate that ssUVR affects both HDAC and HAT activities, leading to reduced histone acetylation.
Histone acetylation and CREB binding protein are required for neuronal resistance against ischemic injury.
Directory of Open Access Journals (Sweden)
Ferah Yildirim
Full Text Available Epigenetic transcriptional regulation by histone acetylation depends on the balance between histone acetyltransferase (HAT and deacetylase activities (HDAC. Inhibition of HDAC activity provides neuroprotection, indicating that the outcome of cerebral ischemia depends crucially on the acetylation status of histones. In the present study, we characterized the changes in histone acetylation levels in ischemia models of focal cerebral ischemia and identified cAMP-response element binding protein (CREB-binding protein (CBP as a crucial factor in the susceptibility of neurons to ischemic stress. Both neuron-specific RNA interference and neurons derived from CBP heterozygous knockout mice showed increased damage after oxygen-glucose deprivation (OGD in vitro. Furthermore, we demonstrated that ischemic preconditioning by a short (5 min subthreshold occlusion of the middle cerebral artery (MCA, followed 24 h afterwards by a 30 min occlusion of the MCA, increased histone acetylation levels in vivo. Ischemic preconditioning enhanced CBP recruitment and histone acetylation at the promoter of the neuroprotective gene gelsolin leading to increased gelsolin expression in neurons. Inhibition of CBP's HAT activity attenuated neuronal ischemic preconditioning. Taken together, our findings suggest that the levels of CBP and histone acetylation determine stroke outcome and are crucially associated with the induction of an ischemia-resistant state in neurons.
Metabolic Regulation of Histone Acetyltransferases by Endogenous Acyl-CoA Cofactors
Montgomery, David C.; Sorum, Alexander W.; Guasch, Laura; Nicklaus, Marc C.; Meier, Jordan L.
The finding that chromatin modifications are sensitive to changes in cellular cofactor levels potentially links altered tumor cell metabolism and gene expression. However, the specific enzymes and metabolites that connect these two processes remain obscure. Characterizing these metabolic-epigenetic axes is critical to understanding how metabolism supports signaling in cancer, and developing therapeutic strategies to disrupt this process. Here, we describe a chemical approach to define the met...
International Nuclear Information System (INIS)
Mee, L.K.; Adelstein, S.J.
The damage to histones irradiated in isolation is being elaborated to aid the identification of the crosslinking sites in radiation-induced DNA-histone adducts. Histones are being examined by amino acid analysis to determine the destruction of residues and by polyacrylamide gel electrophoresis to delineate changes in conformation. For the slightly lysine-rich histone, H2A, a specific attack on selective residues has been established, the aromatic residues, tyrosine and phenylalanine, and the heterocyclic residue, histidine, being significantly destroyed. In addition, a significant increase in aspartic acid was found; this may represent a radiation product from scission of the ring in the histidine residues. The similarity of the effects on residues in nitrous oxide-saturated and nitrogen-saturated solutions suggests that OH . and e/sub aq//sup -/ are equally efficient and selective in their attack. On gel electrophoresis degradation of the histone H2A was found to be greatest for irradiations in nitrous oxide-saturated solutions, suggesting CH . is the most effective radical for producing changes in conformation; O/sub 2//sup -/ was essentially ineffective. Other histones are being examined for changes in amino acid composition, conformation, and for the formation of radiation products
Comparative effects of histone deacetylases inhibitors and resveratrol on Trypanosoma cruzi replication, differentiation, infectivity and gene expression
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Vanina A. Campo
Full Text Available Histone post-translational modification, mediated by histone acetyltransferases and deacetylases, is one of the most studied factors affecting gene expression. Recent data showing differential histone acetylation states during the Trypanosoma cruzi cell cycle suggest a role for epigenetics in the control of this process. As a starting point to study the role of histone deacetylases in the control of gene expression and the consequences of their inhibition and activation in the biology of T. cruzi, two inhibitors for different histone deacetylases: trichostatin A for class I/II and sirtinol for class III and the activator resveratrol for class III, were tested on proliferative and infective forms of this parasite. The two inhibitors tested caused histone hyperacetylation whereas resveratrol showed the opposite effect on both parasite forms, indicating that a biologically active in vivo level of these compounds was achieved. Histone deacetylase inhibitors caused life stage-specific effects, increasing trypomastigotes infectivity and blocking metacyclogenesis. Moreover, these inhibitors affected specific transcript levels, with sirtinol causing the most pronounced change. On the other hand, resveratrol showed strong anti-parasitic effects. This compound diminished epimastigotes growth, promoted metacyclogenesis, reduced in vitro infection and blocked differentiation and/or replication of intracellular amastigotes. In conclusion, the data presented here supports the notion that these compounds can modulate T. cruzi gene expression, differentiation, infection and histones deacetylase activity. Furthermore, among the compounds tested in this study, the results point to Resveratrol as promising trypanocidal drug candidate.
Ethanol Exposure RegulatesGabra1Expression via Histone Deacetylation at the Promoter in Cultured Cortical Neurons.
Bohnsack, John Peyton; Patel, Vraj K; Morrow, A Leslie
γ -Aminobutyric acid A receptors (GABA A -Rs) mediate the majority of inhibitory neurotransmission in the adult brain. The α 1-containing GABA A -Rs are the most prominent subtype in the adult brain and are important in both homeostatic function and several disease pathologies including alcohol dependence, epilepsy, and stress. Ethanol exposure causes a decrease of α 1 transcription and peptide expression both in vivo and in vitro, but the mechanism that controls the transcriptional regulation is unknown. Because ethanol is known to activate epigenetic regulation of gene expression, we tested the hypothesis that ethanol regulates α 1 expression through histone modifications in cerebral cortical cultured neurons. We found that class I histone deacetylases (HDACs) regulate ethanol-induced changes in α 1 gene and protein expression as pharmacologic inhibition or knockdown of HDAC1-3 prevents the effects of ethanol exposure. Targeted histone acetylation associated with the Gabra1 promoter using CRISPR (clustered regularly interspaced palindromic repeat) dCas9-P300 (a nuclease-null Cas9 fused with a histone acetyltransferase) increases histone acetylation and prevents the decrease of Gabra1 expression. In contrast, there was no effect of a mutant histone acetyltransferase or generic transcriptional activator or targeting P300 to a distant exon. Conversely, using a dCas9-KRAB construct that increases repressive methylation (H3K9me3) does not interfere with ethanol-induced histone deacetylation. Overall our results indicate that ethanol deacetylates histones associated with the Gabra1 promoter through class I HDACs and that pharmacologic, genetic, or epigenetic intervention prevents decreases in α 1 expression in cultured cortical neurons. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.
Analysis of Myc-induced histone modifications on target chromatin.
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Francesca Martinato
Full Text Available The c-myc proto-oncogene is induced by mitogens and is a central regulator of cell growth and differentiation. The c-myc product, Myc, is a transcription factor that binds a multitude of genomic sites, estimated to be over 10-15% of all promoter regions. Target promoters generally pre-exist in an active or poised chromatin state that is further modified by Myc, contributing to fine transcriptional regulation (activation or repression of the afferent gene. Among other mechanisms, Myc recruits histone acetyl-transferases to target chromatin and locally promotes hyper-acetylation of multiple lysines on histones H3 and H4, although the identity and combination of the modified lysines is unknown. Whether Myc dynamically regulates other histone modifications (or marks at its binding sites also remains to be addressed. Here, we used quantitative chromatin immunoprecipitation (qChIP to profile a total of 24 lysine-acetylation and -methylation marks modulated by Myc at target promoters in a human B-cell line with a regulatable c-myc transgene. Myc binding promoted acetylation of multiple lysines, primarily of H3K9, H3K14, H3K18, H4K5 and H4K12, but significantly also of H4K8, H4K91 and H2AK5. Dimethylation of H3K79 was also selectively induced at target promoters. A majority of target promoters showed co-induction of multiple marks - in various combinations - correlating with recruitment of the two HATs tested (Tip60 and HBO1, incorporation of the histone variant H2A.Z and transcriptional activation. Based on this and previous findings, we surmise that Myc recruits the Tip60/p400 complex to achieve a coordinated histone acetylation/exchange reaction at activated promoters. Our data are also consistent with the additive and redundant role of multiple acetylation events in transcriptional activation.
Histone Acetylation Regulation in Sleep Deprivation-Induced Spatial Memory Impairment.
Duan, Ruifeng; Liu, Xiaohua; Wang, Tianhui; Wu, Lei; Gao, Xiujie; Zhang, Zhiqing
Sleep disorders negatively affect cognition and health. Recent evidence has indicated that chromatin remodeling via histone acetylation regulates cognitive function. This study aimed to investigate the possible roles of histone acetylation in sleep deprivation (SD)-induced cognitive impairment. Results of the Morris water maze test showed that 3 days of SD can cause spatial memory impairment in Wistar rats. SD can also decrease histone acetylation levels, increase histone deacetylase 2 (HDAC2) expression, and decrease histone acetyltransferase (CBP) expression. Furthermore, SD can reduce H3 and H4 acetylation levels in the promoters of the brain-derived neurotrophic factor (Bdnf) gene and thus significantly downregulate BDNF expression and impair the activity of key BDNF signaling pathways (pCaMKII, pErk2, and pCREB). However, treatment with the HDAC inhibitor trichostatin A attenuated all the negative effects induced by SD. Therefore, BDNF and its histone acetylation regulation may play important roles in SD-induced spatial memory impairment, whereas HDAC inhibition possibly confers protection against SD-induced impairment in spatial memory and hippocampal functions.
Chemical mechanisms of histone lysine and arginine modifications.
Smith, Brian C; Denu, John M
Histone lysine and arginine residues are subject to a wide array of post-translational modifications including methylation, citrullination, acetylation, ubiquitination, and sumoylation. The combinatorial action of these modifications regulates critical DNA processes including replication, repair, and transcription. In addition, enzymes that modify histone lysine and arginine residues have been correlated with a variety of human diseases including arthritis, cancer, heart disease, diabetes, and neurodegenerative disorders. Thus, it is important to fully understand the detailed kinetic and chemical mechanisms of these enzymes. Here, we review recent progress towards determining the mechanisms of histone lysine and arginine modifying enzymes. In particular, the mechanisms of S-adenosyl-methionine (AdoMet) dependent methyltransferases, FAD-dependent demethylases, iron dependent demethylases, acetyl-CoA dependent acetyltransferases, zinc dependent deacetylases, NAD(+) dependent deacetylases, and protein arginine deiminases are covered. Particular attention is paid to the conserved active-site residues necessary for catalysis and the individual chemical steps along the catalytic pathway. When appropriate, areas requiring further work are discussed.
The yeast SAS (something about silencing) protein complex contains a MYST-type putative acetyltransferase and functions with chromatin assembly factor ASF1
Osada, Shigehiro; Sutton, Ann; Muster, Nemone; Brown, Christine E.; Yates, John R.; Sternglanz, Rolf; Workman, Jerry L.
It is well established that acetylation of histone and nonhistone proteins is intimately linked to transcriptional activation. However, loss of acetyltransferase activity has also been shown to cause silencing defects, implicating acetylation in gene silencing. The something about silencing (Sas) 2 protein of Saccharomyces cerevisiae, a member of the MYST (MOZ, Ybf2/Sas3, Sas2, and TIP60) acetyltransferase family, promotes silencing at HML and telomeres. Here we identify a ∼450-kD SAS complex containing Sas2p, Sas4p, and the tf2f-related Sas5 protein. Mutations in the conserved acetyl-CoA binding motif of Sas2p are shown to disrupt the ability of Sas2p to mediate the silencing at HML and telomeres, providing evidence for an important role for the acetyltransferase activity of the SAS complex in silencing. Furthermore, the SAS complex is found to interact with chromatin assembly factor Asf1p, and asf1 mutants show silencing defects similar to mutants in the SAS complex. Thus, ASF1-dependent chromatin assembly may mediate the role of the SAS complex in silencing. PMID:11731479
Histone Chaperone Jun Dimerization Protein 2 (JDP2: Role in Cellular Senescence and Aging
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Yu-Chang Huang
Full Text Available Transcription factor Jun dimerization protein 2 (JDP2 binds directly to histones and DNA, and inhibits p300-mediated acetylation of core histones and reconstituted nucleosomes that contain JDP2-recognition DNA sequences. The region of JDP2 that encompasses its histone-binding domain and DNA-binding region is essential to inhibit histone acetylation by histone acetyltransferases. Moreover, assays of nucleosome assembly in vitro demonstrate that JDP2 also has histone-chaperone activity. The mutation of the region responsible for inhibition of histone acetyltransferase activity within JDP2 eliminates repression of transcription from the c-jun promoter by JDP2, as well as JDP2-mediated inhibition of retinoic-acid-induced differentiation. Thus JDP2 plays a key role as a repressor of cell differentiation by regulating the expression of genes with an activator protein 1 (AP-1 site via inhibition of histone acetylation and/or assembly and disassembly of nucleosomes. Senescent cells show a series of alterations, including flatten and enlarged morphology, increase in nonspecific acidic β-galactosidase activity, chromatin condensation, and changes in gene expression patterns. The onset and maintenance of senescence are regulated by two tumor suppressors, p53 and retinoblastoma proteins. The expression of p53 and retinoblastoma proteins is regulated by two distinct proteins, p16Ink4a and Arf, respectively, which are encoded by cdkn2a. JDP2 inhibits recruitment of the polycomb repressive complexes 1 and 2 (PRC-1 and PRC-2 to the promoter of the gene that encodes p16Ink4a and inhibits the methylation of lysine 27 of histone H3 (H3K27. The PRCs associate with the p16Ink4a/Arf locus in young proliferating cells and dissociate from it in senescent cells. Therefore, it seems that chromatin-remodeling factors that regulate association and dissociation of PRCs, and are controlled by JDP2, might play an important role in the senescence program. The molecular
Genome-wide integration on transcription factors, histone acetylation and gene expression reveals genes co-regulated by histone modification patterns.
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Yayoi Natsume-Kitatani
Full Text Available N-terminal tails of H2A, H2B, H3 and H4 histone families are subjected to posttranslational modifications that take part in transcriptional regulation mechanisms, such as transcription factor binding and gene expression. Regulation mechanisms under control of histone modification are important but remain largely unclear, despite of emerging datasets for comprehensive analysis of histone modification. In this paper, we focus on what we call genetic harmonious units (GHUs, which are co-occurring patterns among transcription factor binding, gene expression and histone modification. We present the first genome-wide approach that captures GHUs by combining ChIP-chip with microarray datasets from Saccharomyces cerevisiae. Our approach employs noise-robust soft clustering to select patterns which share the same preferences in transcription factor-binding, histone modification and gene expression, which are all currently implied to be closely correlated. The detected patterns are a well-studied acetylation of lysine 16 of H4 in glucose depletion as well as co-acetylation of five lysine residues of H3 with H4 Lys12 and H2A Lys7 responsible for ribosome biogenesis. Furthermore, our method further suggested the recognition of acetylated H4 Lys16 being crucial to histone acetyltransferase ESA1, whose essential role is still under controversy, from a microarray dataset on ESA1 and its bypass suppressor mutants. These results demonstrate that our approach allows us to provide clearer principles behind gene regulation mechanisms under histone modifications and detect GHUs further by applying to other microarray and ChIP-chip datasets. The source code of our method, which was implemented in MATLAB (http://www.mathworks.com/, is available from the supporting page for this paper: http://www.bic.kyoto-u.ac.jp/pathway/natsume/hm_detector.htm.
N-acetyltransferase in human skin and keratinocytes
Vogel, Tanja; Bonifas, Jutta; Wiegman, Marjon; Pas, Hendrikus; Blömeke, Brunhilde; Coenraads, Pieter Jan; Schuttelaar, Marie-Louise
Background: N-acetyltransferase 1 (NAT1) mediated Nacetylation in human skin and keratinocytes is an important detoxification pathway for aromatic amines including the strong sensitizer para-phenylenediamine (PPD), an important component of oxidative hair dyes. Objectives: Human skin and
Inhibitors of histone deacetylase
DEFF Research Database (Denmark)
The present invention relates to compounds of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, wherein X1, X2, X3, X4, X5, W1, W2, W3, and W4 are as described. The present invention relates generally to inhibitors of histone deacetylase and to methods...
Histone methyltransferases in cancer
DEFF Research Database (Denmark)
Albert, Mareike; Helin, Kristian
regulators, namely histone methyltransferases (HMTs). Several HMTs have been linked to different types of cancer; however, in most cases we only have limited knowledge regarding the molecular mechanisms by which the HMTs contribute to disease development. We summarize the current knowledge regarding some...
Inhibition of histone deacetylation alters Arabidopsis root growth in response to auxin via PIN1 degradation.
Nguyen, Hoai Nguyen; Kim, Jun Hyeok; Jeong, Chan Young; Hong, Suk-Whan; Lee, Hojoung
Our results showed the histone deacetylase inhibitors (HDIs) control root development in Arabidopsis via regulation of PIN1 degradation. Epigenetic regulation plays a crucial role in the expression of many genes in response to exogenous or endogenous signals in plants as well as other organisms. One of epigenetic mechanisms is modifications of histone, such as acetylation and deacetylation, are catalyzed by histone acetyltransferase (HAT) and histone deacetylase (HDAC), respectively. The Arabidopsis HDACs, HDA6, and HDA19, were reported to function in physiological processes, including embryo development, abiotic stress response, and flowering. In this study, we demonstrated that histone deacetylase inhibitors (HDIs) inhibit primary root elongation and lateral root emergence. In response to HDIs treatment, the PIN1 protein was almost abolished in the root tip. However, the PIN1 gene did not show decreased expression in the presence of HDIs, whereas IAA genes exhibited increases in transcript levels. In contrast, we observed a stable level of gene expression of stress markers (KIN1 and COR15A) and a cell division marker (CYCB1). Taken together, these results suggest that epigenetic regulation may control auxin-mediated root development through the 26S proteasome-mediated degradation of PIN1 protein.
Rapid purification of recombinant histones.
Klinker, Henrike; Haas, Caroline; Harrer, Nadine; Becker, Peter B; Mueller-Planitz, Felix
The development of methods to assemble nucleosomes from recombinant histones decades ago has transformed chromatin research. Nevertheless, nucleosome reconstitution remains time consuming to this day, not least because the four individual histones must be purified first. Here, we present a streamlined purification protocol of recombinant histones from bacteria. We termed this method "rapid histone purification" (RHP) as it circumvents isolation of inclusion bodies and thereby cuts out the most time-consuming step of traditional purification protocols. Instead of inclusion body isolation, whole cell extracts are prepared under strongly denaturing conditions that directly solubilize inclusion bodies. By ion exchange chromatography, the histones are purified from the extracts. The protocol has been successfully applied to all four canonical Drosophila and human histones. RHP histones and histones that were purified from isolated inclusion bodies had similar purities. The different purification strategies also did not impact the quality of octamers reconstituted from these histones. We expect that the RHP protocol can be readily applied to the purification of canonical histones from other species as well as the numerous histone variants.
Hippocampal histone acetylation regulates object recognition and the estradiol-induced enhancement of object recognition.
Zhao, Zaorui; Fan, Lu; Fortress, Ashley M; Boulware, Marissa I; Frick, Karyn M
Histone acetylation has recently been implicated in learning and memory processes, yet necessity of histone acetylation for such processes has not been demonstrated using pharmacological inhibitors of histone acetyltransferases (HATs). As such, the present study tested whether garcinol, a potent HAT inhibitor in vitro, could impair hippocampal memory consolidation and block the memory-enhancing effects of the modulatory hormone 17β-estradiol E2. We first showed that bilateral infusion of garcinol (0.1, 1, or 10 μg/side) into the dorsal hippocampus (DH) immediately after training impaired object recognition memory consolidation in ovariectomized female mice. A behaviorally effective dose of garcinol (10 μg/side) also significantly decreased DH HAT activity. We next examined whether DH infusion of a behaviorally subeffective dose of garcinol (1 ng/side) could block the effects of DH E2 infusion on object recognition and epigenetic processes. Immediately after training, ovariectomized female mice received bilateral DH infusions of vehicle, E2 (5 μg/side), garcinol (1 ng/side), or E2 plus garcinol. Forty-eight hours later, garcinol blocked the memory-enhancing effects of E2. Garcinol also reversed the E2-induced increase in DH histone H3 acetylation, HAT activity, and levels of the de novo methyltransferase DNMT3B, as well as the E2-induced decrease in levels of the memory repressor protein histone deacetylase 2. Collectively, these findings suggest that histone acetylation is critical for object recognition memory consolidation and the beneficial effects of E2 on object recognition. Importantly, this work demonstrates that the role of histone acetylation in memory processes can be studied using a HAT inhibitor.
Ethyl acetate production by the elusive alcohol acetyltransferase from yeast.
Kruis, Aleksander J; Levisson, Mark; Mars, Astrid E; van der Ploeg, Max; Garcés Daza, Fernando; Ellena, Valeria; Kengen, Servé W M; van der Oost, John; Weusthuis, Ruud A
Ethyl acetate is an industrially relevant ester that is currently produced exclusively through unsustainable processes. Many yeasts are able to produce ethyl acetate, but the main responsible enzyme has remained elusive, hampering the engineering of novel production strains. Here we describe the discovery of a new enzyme (Eat1) from the yeast Wickerhamomyces anomalus that resulted in high ethyl acetate production when expressed in Saccharomyces cerevisiae and Escherichia coli. Purified Eat1 showed alcohol acetyltransferase activity with ethanol and acetyl-CoA. Homologs of eat1 are responsible for most ethyl acetate synthesis in known ethyl acetate-producing yeasts, including S. cerevisiae, and are only distantly related to known alcohol acetyltransferases. Eat1 is therefore proposed to compose a novel alcohol acetyltransferase family within the α/β hydrolase superfamily. The discovery of this novel enzyme family is a crucial step towards the development of biobased ethyl acetate production and will also help in selecting improved S. cerevisiae brewing strains. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.
Histone deacetylases and atherosclerosis.
Zheng, Xia-xia; Zhou, Tian; Wang, Xin-An; Tong, Xiao-hong; Ding, Jia-wang
Atherosclerosis is the most common pathological process that leads to cardiovascular diseases, a disease of large- and medium-sized arteries that is characterized by a formation of atherosclerotic plaques consisting of necrotic cores, calcified regions, accumulated modified lipids, smooth muscle cells (SMCs), endothelial cells, leukocytes, and foam cells. Recently, the question about how to suppress the occurrence of atherosclerosis and alleviate the progress of cardiovascular disease becomes the hot topic. Accumulating evidence suggests that histone deacetylases(HDACs) play crucial roles in arteriosclerosis. This review summarizes the effect of HDACs and HDAC inhibitors(HDACi) on the progress of atherosclerosis. Copyright © 2015. Published by Elsevier Ireland Ltd.
Targeting Histone Deacetylases: A Novel Approach in Parkinson's Disease
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Sorabh Sharma
Full Text Available The worldwide prevalence of movement disorders is increasing day by day. Parkinson's disease (PD is the most common movement disorder. In general, the clinical manifestations of PD result from dysfunction of the basal ganglia. Although the exact underlying mechanisms leading to neural cell death in this disease remains unknown, the genetic causes are often established. Indeed, it is becoming increasingly evident that chromatin acetylation status can be impaired during the neurological disease conditions. The acetylation and deacetylation of histone proteins are carried out by opposing actions of histone acetyltransferases (HATs and histone deacetylases (HDACs, respectively. In the recent past, studies with HDAC inhibitors result in beneficial effects in both in vivo and in vitro models of PD. Various clinical trials have also been initiated to investigate the possible therapeutic potential of HDAC inhibitors in patients suffering from PD. The possible mechanisms assigned for these neuroprotective actions of HDAC inhibitors involve transcriptional activation of neuronal survival genes and maintenance of histone acetylation homeostasis, both of which have been shown to be dysregulated in PD. In this review, the authors have discussed the putative role of HDAC inhibitors in PD and associated abnormalities and suggest new directions for future research in PD.
Histone turnover within nonproliferating cells
International Nuclear Information System (INIS)
Commerford, S.L.; Carsten, A.L.; Cronkite, E.P.
The turnover of DNA and histones in the livers and brains of mice has been determined. These mice had been exposed to constant levels of tritiated water from conception until they were 8 months old. At this point, exposure to tritium was discontinued, and the tritium remaining in DNA and histones was measured at various intervals afterward. The half-lives calculated for these components (with 95% confidence limits given in parentheses) were 117 (85 to 188) days for liver histone, 318 (241 to 466) days for liver DNA, 159 (129 to 208) days for brain histone and 593 (376 to 1406) days for brain DNA. The difference between histone and DNA turnover is statistically significant for both tissues and indicates that histone turnover within tissues cannot be solely accounted for by cell turnover within the tissue but also must include histone turnover within living cells. The half-life of histone within cells is estimated to be 117 (88 to 178) days in liver and 223 (187 to 277) days in brain
Histone acetylation mediates epigenetic regulation of transcriptional reprogramming in insects during metamorphosis, wounding and infection
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Mukherjee Krishnendu
Full Text Available Abstract Background Gene expression in eukaryotes is regulated by histone acetylation/deacetylation, an epigenetic process mediated by histone acetyltransferases (HATs and histone deacetylases (HDACs whose opposing activities are tightly regulated. The acetylation of histones by HATs increases DNA accessibility and promotes gene expression, whereas the removal of acetyl groups by HDACs has the opposite effect. Results We explored the role of HDACs and HATs in epigenetic reprogramming during metamorphosis, wounding and infection in the lepidopteran model host Galleria mellonella. We measured the expression of genes encoding components of HATs and HDACs to monitor the transcriptional activity of each enzyme complex and found that both enzymes were upregulated during pupation. Specific HAT inhibitors were able to postpone pupation and to reduce insect survival following wounding, whereas HDAC inhibitors accelerated pupation and increased survival. The administration of HDAC inhibitors modulated the expression of effector genes with key roles in tissue remodeling (matrix metalloproteinase, the regulation of sepsis (inhibitor of metalloproteinases from insects and host defense (antimicrobial peptides, and simultaneously induced HAT activity, suggesting that histone acetylation is regulated by a feedback mechanism. We also discovered that both the entomopathogenic fungus Metarhizium anisopliae and the human bacterial pathogen Listeria monocytogenes can delay metamorphosis in G. mellonella by skewing the HDAC/HAT balance. Conclusions Our study provides for the first evidence that pathogenic bacteria can interfere with the regulation of HDACs and HATs in insects which appear to manipulate host immunity and development. We conclude that histone acetylation/deacetylation in insects mediates transcriptional reprogramming during metamorphosis and in response to wounding and infection.
Comparative effects of histone deacetylases inhibitors and resveratrol on Trypanosoma cruzi replication, differentiation, infectivity and gene expression.
Campo, Vanina A
Histone post-translational modification, mediated by histone acetyltransferases and deacetylases, is one of the most studied factors affecting gene expression. Recent data showing differential histone acetylation states during the Trypanosoma cruzi cell cycle suggest a role for epigenetics in the control of this process. As a starting point to study the role of histone deacetylases in the control of gene expression and the consequences of their inhibition and activation in the biology of T. cruzi, two inhibitors for different histone deacetylases: trichostatin A for class I/II and sirtinol for class III and the activator resveratrol for class III, were tested on proliferative and infective forms of this parasite. The two inhibitors tested caused histone hyperacetylation whereas resveratrol showed the opposite effect on both parasite forms, indicating that a biologically active in vivo level of these compounds was achieved. Histone deacetylase inhibitors caused life stage-specific effects, increasing trypomastigotes infectivity and blocking metacyclogenesis. Moreover, these inhibitors affected specific transcript levels, with sirtinol causing the most pronounced change. On the other hand, resveratrol showed strong anti-parasitic effects. This compound diminished epimastigotes growth, promoted metacyclogenesis, reduced in vitro infection and blocked differentiation and/or replication of intracellular amastigotes. In conclusion, the data presented here supports the notion that these compounds can modulate T. cruzi gene expression, differentiation, infection and histones deacetylase activity. Furthermore, among the compounds tested in this study, the results point to Resveratrol as promising trypanocidal drug candidate. Copyright © 2017 The Author. Published by Elsevier Ltd.. All rights reserved.
Gene expression profiling of epigenetic chromatin modification enzymes and histone marks by cigarette smoke: implications for COPD and lung cancer.
Sundar, Isaac K; Rahman, Irfan
Chromatin-modifying enzymes mediate DNA methylation and histone modifications on recruitment to specific target gene loci in response to various stimuli. The key enzymes that regulate chromatin accessibility for maintenance of modifications in DNA and histones, and for modulation of gene expression patterns in response to cigarette smoke (CS), are not known. We hypothesize that CS exposure alters the gene expression patterns of chromatin-modifying enzymes, which then affects multiple downstream pathways involved in the response to CS. We have, therefore, analyzed chromatin-modifying enzyme profiles and validated by quantitative real-time PCR (qPCR). We also performed immunoblot analysis of targeted histone marks in C57BL/6J mice exposed to acute and subchronic CS, and of lungs from nonsmokers, smokers, and patients with chronic obstructive pulmonary disease (COPD). We found a significant increase in expression of several chromatin modification enzymes, including DNA methyltransferases, histone acetyltransferases, histone methyltransferases, and SET domain proteins, histone kinases, and ubiquitinases. Our qPCR validation data revealed a significant downregulation of Dnmt1, Dnmt3a, Dnmt3b, Hdac2, Hdac4, Hat1, Prmt1, and Aurkb We identified targeted chromatin histone marks (H3K56ac and H4K12ac), which are induced by CS. Thus CS-induced genotoxic stress differentially affects the expression of epigenetic modulators that regulate transcription of target genes via DNA methylation and site-specific histone modifications. This may have implications in devising epigenetic-based therapies for COPD and lung cancer. Copyright © 2016 the American Physiological Society.
Blood pressure regulation by CD4+ lymphocytes expressing choline acetyltransferase
Olofsson, Peder S.; Steinberg, Benjamin E.; Sobbi, Roozbeh; Cox, Maureen A.; Ahmed, Mohamed N.; Oswald, Michaela; Szekeres, Ferenc; Hanes, William M.; Introini, Andrea; Liu, Shu Fang; Holodick, Nichol E.; Rothstein, Thomas L.; L?vdahl, Cecilia; Chavan, Sangeeta S.; Yang, Huan
Blood pressure regulation is known to be maintained by a neuro-endocrine circuit, but whether immune cells contribute to blood pressure homeostasis has not been defined. We previously described that CD4+ T lymphocytes that express choline acetyltransferase (ChAT), which catalyzes the synthesis of the vasorelaxant acetylcholine, relay neural signals 1 . Here we show that these CD4+ CD44high CD62Llow T helper cells by gene expression are a distinct T cell population defined by ChAT (CD4 TChAT)....
Identification of lysine acetyltransferase substrates using bioorthogonal chemical proteomics.
Grammel, Markus; Hang, Howard C
Bioorthogonal chemical proteomics is a valuable method to identify enzyme-specific substrates, a challenging task by traditional biochemical standards. The addition of recombinant enzyme and alkynyl chemical reporter to complex protein mixtures, such as cell lysates, allows the detection and identification of modified substrates. Proteins that have been modified with the chemical reporter can be selectively labeled with fluorescent dyes for detection or affinity tags for biochemical enrichment and subsequent identification by mass spectrometry. Here, we describe the detection and identification of substrates of the lysine acetyltransferase p300 in nuclear extracts using the chemical reporter 4-pentynoyl-CoA.
Histone chaperone networks shaping chromatin function
DEFF Research Database (Denmark)
Hammond, Colin; Strømme, Caroline Bianchi; Huang, Hongda
The association of histones with specific chaperone complexes is important for their folding, oligomerization, post-translational modification, nuclear import, stability, assembly and genomic localization. In this way, the chaperoning of soluble histones is a key determinant of histone availability...... and fate, which affects all chromosomal processes, including gene expression, chromosome segregation and genome replication and repair. Here, we review the distinct structural and functional properties of the expanding network of histone chaperones. We emphasize how chaperones cooperate in the histone...... chaperone network and via co-chaperone complexes to match histone supply with demand, thereby promoting proper nucleosome assembly and maintaining epigenetic information by recycling modified histones evicted from chromatin....
Bisbenzamidine derivative, pentamidine represses DNA damage response through inhibition of histone H2A acetylation
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Ohba Reiko
Full Text Available Abstract Background MRE11 is an important nuclease which functions in the end-resection step of homologous recombination (HR repair of DNA double-strand breaks (DSBs. As MRE11-deficient ATLD cells exhibit hyper radio-sensitivity and impaired DSB repair, MRE11 inhibitors could possibly function as potent radio-sensitizers. Therefore, we investigated whether a bisbenzamidine derivative, pentamidine, which can inhibit endoexonuclease activity, might influence DSB-induced damage responses via inhibition of MRE11. Results We first clarified that pentamidine inhibited MRE11 nuclease activity and also reduced ATM kinase activity in vitro. Pentamidine increased the radio-sensitivity of HeLa cells, suggesting that this compound could possibly influence DNA damage response factors in vivo. Indeed, we found that pentamidine reduced the accumulation of γ-H2AX, NBS1 and phospho-ATM at the sites of DSBs. Furthermore, pentamidine decreased HR activity in vivo. Pentamidine was found to inhibit the acetylation of histone H2A which could contribute both to inhibition of IR-induced focus formation and HR repair. These results suggest that pentamidine might exert its effects by inhibiting histone acetyltransferases. We found that pentamidine repressed the activity of Tip60 acetyltransferase which is known to acetylate histone H2A and that knockdown of Tip60 by siRNA reduced HR activity. Conclusion These results indicate that inhibition of Tip60 as well as hMRE11 nuclease by pentamidine underlies the radiosensitizing effects of this compound making it an excellent sensitizer for radiotherapy or chemotherapy.
Mutations in the Chromatin Regulator Gene BRPF1 Cause Syndromic Intellectual Disability and Deficient Histone Acetylation.
Yan, Kezhi; Rousseau, Justine; Littlejohn, Rebecca Okashah; Kiss, Courtney; Lehman, Anna; Rosenfeld, Jill A; Stumpel, Constance T R; Stegmann, Alexander P A; Robak, Laurie; Scaglia, Fernando; Nguyen, Thi Tuyet Mai; Fu, He; Ajeawung, Norbert F; Camurri, Maria Vittoria; Li, Lin; Gardham, Alice; Panis, Bianca; Almannai, Mohammed; Sacoto, Maria J Guillen; Baskin, Berivan; Ruivenkamp, Claudia; Xia, Fan; Bi, Weimin; Cho, Megan T; Potjer, Thomas P; Santen, Gijs W E; Parker, Michael J; Canham, Natalie; McKinnon, Margaret; Potocki, Lorraine; MacKenzie, Jennifer J; Roeder, Elizabeth R; Campeau, Philippe M; Yang, Xiang-Jiao
Identification of over 500 epigenetic regulators in humans raises an interesting question regarding how chromatin dysregulation contributes to different diseases. Bromodomain and PHD finger-containing protein 1 (BRPF1) is a multivalent chromatin regulator possessing three histone-binding domains, one non-specific DNA-binding module, and several motifs for interacting with and activating three lysine acetyltransferases. Genetic analyses of fish brpf1 and mouse Brpf1 have uncovered an important role in skeletal, hematopoietic, and brain development, but it remains unclear how BRPF1 is linked to human development and disease. Here, we describe an intellectual disability disorder in ten individuals with inherited or de novo monoallelic BRPF1 mutations. Symptoms include infantile hypotonia, global developmental delay, intellectual disability, expressive language impairment, and facial dysmorphisms. Central nervous system and spinal abnormalities are also seen in some individuals. These clinical features overlap with but are not identical to those reported for persons with KAT6A or KAT6B mutations, suggesting that BRPF1 targets these two acetyltransferases and additional partners in humans. Functional assays showed that the resulting BRPF1 variants are pathogenic and impair acetylation of histone H3 at lysine 23, an abundant but poorly characterized epigenetic mark. We also found a similar deficiency in different lines of Brpf1-knockout mice. These data indicate that aberrations in the chromatin regulator gene BRPF1 cause histone H3 acetylation deficiency and a previously unrecognized intellectual disability syndrome. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.
Chromatin replication and histone dynamics
DEFF Research Database (Denmark)
Alabert, Constance; Jasencakova, Zuzana; Groth, Anja
organization into chromatin. We reveal how specialized replication-coupled mechanisms rapidly assemble newly synthesized DNA into nucleosomes, while the complete restoration of chromatin organization including histone marks is a continuous process taking place throughout the cell cycle. Because failure...
The acetyltransferase HAT1 moderates the NF-κB response by regulating the transcription factor PLZF.
Sadler, Anthony J; Suliman, Bandar A; Yu, Liang; Yuan, Xiangliang; Wang, Die; Irving, Aaron T; Sarvestani, Soroush T; Banerjee, Ashish; Mansell, Ashley S; Liu, Jun-Ping; Gerondakis, Steve; Williams, Bryan R G; Xu, Dakang
To date, the activities of protein kinases have formed the core of our understanding of cell signal transduction. Comprehension of the extent of protein acetylation has raised expectations that this alternate post-transcriptional modification will be shown to rival phosphorylation in its importance in mediating cellular responses. However, limited instances have been identified. Here we show that signalling from Toll-like or TNF-α receptors triggers the calcium/calmodulin-dependent protein kinase (CaMK2) to activate histone acetyltransferase-1 (HAT1), which then acetylates the transcriptional regulator PLZF. Acetylation of PLZF promotes the assembly of a repressor complex incorporating HDAC3 and the NF-κB p50 subunit that limits the NF-κB response. Accordingly, diminishing the activity of CaMK2, the expression levels of PLZF or HAT1, or mutating key residues that are covalently modified in PLZF and HAT1, curtails control of the production of inflammatory cytokines. These results identify a central role for acetylation in controlling the inflammatory NF-κB transcriptional programme.
Differential effects of garcinol and curcumin on histone and p53 modifications in tumour cells
International Nuclear Information System (INIS)
Collins, Hilary M; Kundu, Tapas K; Heery, David M; Abdelghany, Magdy K; Messmer, Marie; Yue, Baigong; Deeves, Sian E; Kindle, Karin B; Mantelingu, Kempegowda; Aslam, Akhmed; Winkler, G Sebastiaan
summary, although garcinol and curcumin can both inhibit histone acetyltransferase activities, our results show that these compounds have differential effects on cancer cells in culture. Garcinol treatment alters expression of chromatin modifying enzymes in MCF7 cells, resulting in reprogramming of key histone and p53 PTMs and growth arrest, underscoring its potential as a cancer chemopreventive agent
Crystal structure of homoserine O-acetyltransferase from Leptospira interrogans
International Nuclear Information System (INIS)
Wang Mingzhu; Liu Lin; Wang Yanli; Wei Zhiyi; Zhang Ping; Li Yikun; Jiang Xiaohua; Xu Hang; Gong Weimin
Homoserine O-acetyltransferase (HTA, EC 188.8.131.52) initiates methionine biosynthesis pathway by catalyzing the transfer of acetyl group from acetyl-CoA to homoserine. This study reports the crystal structure of HTA from Leptospira interrogans determined at 2.2 A resolution using selenomethionyl single-wavelength anomalous diffraction method. HTA is modular and consists of two structurally distinct domains-a core α/β domain containing the catalytic site and a helical bundle called the lid domain. Overall, the structure fold belongs to α/β hydrolase superfamily with the characteristic 'catalytic triad' residues in the active site. Detailed structure analysis showed that the catalytic histidine and serine are both present in two conformations, which may be involved in the catalytic mechanism for acetyl transfer
Quantitating the specificity and selectivity of Gcn5-mediated acetylation of histone H3.
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Yin-Ming Kuo
Full Text Available Lysine acetyltransferases (KATs play a unique role in regulating gene transcription as well as maintaining the epigenetic state of the cell. KATs such as Gcn5 and p300/CBP can modify multiple residues on a single histone; however, order and specificity of acetylation can be altered by factors such as histone chaperones, subunit proteins or external stimulus. While the importance of acetylation is well documented, it has been difficult to quantitatively measure the specificity and selectivity of acetylation at different residues within a histone. In this paper, we demonstrate a label-free quantitative high throughput mass spectrometry-based assay capable of quantitatively monitoring all known acetylation sites of H3 simultaneously. Using this assay, we are able to analyze the steady-state enzyme kinetics of Gcn5, an evolutionarily conserved KAT. In doing so, we measured Gcn5-mediated acetylation at six residues (K14>K9 ≈ K23> K18> K27 ≈ K36 and the catalytic efficiency (k(cat/K(m for K9, K14, K18, and K23 as well as the nonenzymatic acetylation rate. We observed selectivity differences of up to -4 kcal/mol between K14 and K18, the highest and lowest measurable k(cat/K(m. These data provide a first look at quantitating the specificity and selectivity of multiple lysines on a single substrate (H3 by Gcn5.
Beyond Histones: New Substrate Proteins of Lysine Deacetylases in Arabidopsis Nuclei
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Magdalena Füßl
Full Text Available The reversible acetylation of lysine residues is catalyzed by the antagonistic action of lysine acetyltransferases and deacetylases, which can be considered as master regulators of their substrate proteins. Lysine deacetylases, historically referred to as histone deacetylases, have profound functions in regulating stress defenses and development in plants. Lysine acetylation of the N-terminal histone tails promotes gene transcription and decondensation of chromatin, rendering the DNA more accessible to the transcription machinery. In plants, the classical lysine deacetylases from the RPD3/HDA1-family have thus far mainly been studied in the context of their deacetylating activities on histones, and their versatility in molecular activities is still largely unexplored. Here we discuss the potential impact of lysine acetylation on the recently identified nuclear substrate proteins of lysine deacetylases from the Arabidopsis RPD3/HDA1-family. Among the deacetylase substrate proteins, many interesting candidates involved in nuclear protein import, transcriptional regulation, and chromatin remodeling have been identified. These candidate proteins represent key starting points for unraveling new molecular functions of the Arabidopsis lysine deacetylases. Site-directed engineering of lysine acetylation sites on these target proteins might even represent a new approach for optimizing plant growth under climate change conditions.
Curcumin loaded NLC induces histone hypoacetylation in the CNS after intraperitoneal administration in mice.
Puglia, Carmelo; Frasca, Giuseppina; Musumeci, Teresa; Rizza, Luisa; Puglisi, Giovanni; Bonina, Francesco; Chiechio, Santina
The natural p300-specific histone acetyltransferase (HAT) inhibitor, curcumin (CUR), has been widely investigated for its potential therapeutic effect as an anticancer and anti-inflammatory agent. Notwithstanding this interesting pharmacological profile, CUR shows some drawbacks, such as poor absorption and a very fast metabolism and elimination, that limit its clinical use. Aim of the present study was to formulate CUR loaded nanostructured lipid carriers (NLC-CUR) in order to improve the bioavailability and stability of this compound after systemic administration with increased effects in the central nervous system (CNS). NLC-CUR were prepared and characterized on their physicochemical properties by PCS and DSC analyses. Thus, NLC-CUR were systemically injected and the effects in the CNS were compared with a CUR control formulation containing 0.05% DMSO (DMSO-CUR). Our results demonstrate that CUR is able to decrease histone acetylation in the CNS when included in NLCs. Western blot analysis shows that intraperitoneal injection of NLC-CUR (100mg/kg) in mice induces a marked hypoacetylation of histone 4 (H4) at lysine 12 (K12) in the spinal cord compared with control group. Notably, DMSO-CUR (100mg/kg) did not change the H4K12 acetylation level in the CNS. Our study suggests a novel approach to ameliorate the pharmacokinetics of CUR that allows a better permeation in the CNS. Copyright © 2012 Elsevier B.V. All rights reserved.
p300/CBP Histone Acetyltransferase Activity Is Required for Newly Acquired and Reactivated Fear Memories in the Lateral Amygdala
Maddox, Stephanie A.; Watts, Casey S.; Schafe, Glenn E.
Modifications in chromatin structure have been widely implicated in memory and cognition, most notably using hippocampal-dependent memory paradigms including object recognition, spatial memory, and contextual fear memory. Relatively little is known, however, about the role of chromatin-modifying enzymes in amygdala-dependent memory formation.…
Histone modifications: Cycling with chromosomal replication
DEFF Research Database (Denmark)
Thon, Genevieve
Histone modifications tend to be lost during chromosome duplication. Several recent studies suggest that the RNA interference pathway becomes active during the weakened transcriptional repression occurring at centromeres in S phase, resulting in the re-establishment of histone modifications...
Histone Lysine Methylation and Neurodevelopmental Disorders
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Jeong-Hoon Kim
Full Text Available Methylation of several lysine residues of histones is a crucial mechanism for relatively long-term regulation of genomic activity. Recent molecular biological studies have demonstrated that the function of histone methylation is more diverse and complex than previously thought. Moreover, studies using newly available genomics techniques, such as exome sequencing, have identified an increasing number of histone lysine methylation-related genes as intellectual disability-associated genes, which highlights the importance of accurate control of histone methylation during neurogenesis. However, given the functional diversity and complexity of histone methylation within the cell, the study of the molecular basis of histone methylation-related neurodevelopmental disorders is currently still in its infancy. Here, we review the latest studies that revealed the pathological implications of alterations in histone methylation status in the context of various neurodevelopmental disorders and propose possible therapeutic application of epigenetic compounds regulating histone methylation status for the treatment of these diseases.
Loss of Nat4 and its associated histone H4 N-terminal acetylation mediates calorie restriction-induced longevity.
Molina-Serrano, Diego; Schiza, Vassia; Demosthenous, Christis; Stavrou, Emmanouil; Oppelt, Jan; Kyriakou, Dimitris; Liu, Wei; Zisser, Gertrude; Bergler, Helmut; Dang, Weiwei; Kirmizis, Antonis
Changes in histone modifications are an attractive model through which environmental signals, such as diet, could be integrated in the cell for regulating its lifespan. However, evidence linking dietary interventions with specific alterations in histone modifications that subsequently affect lifespan remains elusive. We show here that deletion of histone N-alpha-terminal acetyltransferase Nat4 and loss of its associated H4 N-terminal acetylation (N-acH4) extend yeast replicative lifespan. Notably, nat4Δ-induced longevity is epistatic to the effects of calorie restriction (CR). Consistent with this, (i) Nat4 expression is downregulated and the levels of N-acH4 within chromatin are reduced upon CR, (ii) constitutive expression of Nat4 and maintenance of N-acH4 levels reduces the extension of lifespan mediated by CR, and (iii) transcriptome analysis indicates that nat4Δ largely mimics the effects of CR, especially in the induction of stress-response genes. We further show that nicotinamidase Pnc1, which is typically upregulated under CR, is required for nat4Δ-mediated longevity. Collectively, these findings establish histone N-acH4 as a regulator of cellular lifespan that links CR to increased stress resistance and longevity. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.
Loss of p300 and CBP disrupts histone acetylation at the mouse Sry promoter and causes XY gonadal sex reversal.
Carré, Gwenn-Aël; Siggers, Pam; Xipolita, Marilena; Brindle, Paul; Lutz, Beat; Wells, Sara; Greenfield, Andy
CREB-binding protein (CBP, CREBBP, KAT3A) and its closely related paralogue p300 (EP300, KAT3B), together termed p300/CBP, are histone/lysine acetyl-transferases that control gene expression by modifying chromatin-associated proteins. Here, we report roles for both of these chromatin-modifying enzymes in mouse sex determination, the process by which the embryonic gonad develops into a testis or an ovary. By targeting gene ablation to embryonic gonadal somatic cells using an inducible Cre line, we show that gonads lacking either gene exhibit major abnormalities of XY gonad development at 14.5 dpc, including partial sex reversal. Embryos lacking three out of four functional copies of p300/Cbp exhibit complete XY gonadal sex reversal and have greatly reduced expression of the key testis-determining genes Sry and Sox9. An analysis of histone acetylation at the Sry promoter in mutant gonads at 11.5 dpc shows a reduction in levels of the positive histone mark H3K27Ac. Our data suggest a role for CBP/p300 in testis determination mediated by control of histone acetylation at the Sry locus and reveal a novel element in the epigenetic control of Sry and mammalian sex determination. They also suggest possible novel causes of human disorders of sex development (DSD). © The Author 2017. Published by Oxford University Press.
Histone displacement during nucleotide excision repair
DEFF Research Database (Denmark)
Dinant, C.; Bartek, J.; Bekker-Jensen, S.
, thus allowing repair proteins to efficiently access DNA. On the other hand, after completion of the repair, the chromatin must be returned to its previous undamaged state. Chromatin remodeling can refer to three separate but interconnected processes, histone post-translational modifications, insertion...... of histone variants and histone displacement (including nucleosome sliding). Here we review current knowledge, and speculate about current unknowns, regarding those chromatin remodeling activities that physically displace histones before, during and after NER....
Specific phosphorylation of histone demethylase KDM3A determines target gene expression in response to heat shock.
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Mo-bin Cheng
Full Text Available Histone lysine (K residues, which are modified by methyl- and acetyl-transferases, diversely regulate RNA synthesis. Unlike the ubiquitously activating effect of histone K acetylation, the effects of histone K methylation vary with the number of methyl groups added and with the position of these groups in the histone tails. Histone K demethylases (KDMs counteract the activity of methyl-transferases and remove methyl group(s from specific K residues in histones. KDM3A (also known as JHDM2A or JMJD1A is an H3K9me2/1 demethylase. KDM3A performs diverse functions via the regulation of its associated genes, which are involved in spermatogenesis, metabolism, and cell differentiation. However, the mechanism by which the activity of KDM3A is regulated is largely unknown. Here, we demonstrated that mitogen- and stress-activated protein kinase 1 (MSK1 specifically phosphorylates KDM3A at Ser264 (p-KDM3A, which is enriched in the regulatory regions of gene loci in the human genome. p-KDM3A directly interacts with and is recruited by the transcription factor Stat1 to activate p-KDM3A target genes under heat shock conditions. The demethylation of H3K9me2 at the Stat1 binding site specifically depends on the co-expression of p-KDM3A in the heat-shocked cells. In contrast to heat shock, IFN-γ treatment does not phosphorylate KDM3A via MSK1, thereby abrogating its downstream effects. To our knowledge, this is the first evidence that a KDM can be modified via phosphorylation to determine its specific binding to target genes in response to thermal stress.
The Effects of Pharmacological Inhibition of Histone Deacetylase 3 (HDAC3 in Huntington's Disease Mice.
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Haiqun Jia
Full Text Available An important epigenetic modification in Huntington's disease (HD research is histone acetylation, which is regulated by histone acetyltransferase and histone deacetylase (HDAC enzymes. HDAC inhibitors have proven effective in HD model systems, and recent work is now focused on functional dissection of the individual HDAC enzymes in these effects. Histone deacetylase 3 (HDAC3, a member of the class I subfamily of HDACs, has previously been implicated in neuronal toxicity and huntingtin-induced cell death. Hence, we tested the effects of RGFP966 ((E-N-(2-amino-4-fluorophenyl-3-(1-cinnamyl-1H-pyrazol-4-ylacrylamide, a benzamide-type HDAC inhibitor that selectively targets HDAC3, in the N171-82Q transgenic mouse model of HD. We found that RGFP966 at doses of 10 and 25 mg/kg improves motor deficits on rotarod and in open field exploration, accompanied by neuroprotective effects on striatal volume. In light of previous studies implicating HDAC3 in immune function, we measured gene expression changes for 84 immune-related genes elicited by RGFP966 using quantitative PCR arrays. RGFP966 treatment did not cause widespread changes in cytokine/chemokine gene expression patterns, but did significantly alter the striatal expression of macrophage migration inhibitory factor (Mif, a hormone immune modulator associated with glial cell activation, in N171-82Q transgenic mice, but not WT mice. Accordingly, RGFP966-treated mice showed decreased glial fibrillary acidic protein (GFAP immunoreactivity, a marker of astrocyte activation, in the striatum of N171-82Q transgenic mice compared to vehicle-treated mice. These findings suggest that the beneficial actions of HDAC3 inhibition could be related, in part, with lowered Mif levels and its associated downstream effects.
Identification and Interrogation of Combinatorial Histone Modifications
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Kelly R Karch
Full Text Available Histone proteins are dynamically modified to mediate a variety of cellular processes including gene transcription, DNA damage repair, and apoptosis. Regulation of these processes occurs through the recruitment of non-histone proteins to chromatin by specific combinations of histone post-translational modifications (PTMs. Mass spectrometry has emerged as an essential tool to discover and quantify histone PTMs both within and between samples in an unbiased manner. Developments in mass spectrometry that allow for characterization of large histone peptides or intact protein has made it possible to determine which modifications occur simultaneously on a single histone polypeptide. A variety of techniques from biochemistry, biophysics, and chemical biology have been employed to determine the biological relevance of discovered combinatorial codes. This review first describes advancements in the field of mass spectrometry that have facilitated histone PTM analysis and then covers notable approaches to probe the biological relevance of these modifications in their nucleosomal context.
Structures and functions of insect arylalkylamine N-acetyltransferase (iaaNAT; a key enzyme for physiological and behavioral switch in arthropods
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Susumu eHiragaki
Full Text Available The evolution of N-acetyltransfeases (NATs seems complex. Vertebrate arylalkylamine N-acetyltransferase (aaNAT has been extensively studied since it Leads to the synthesis of melatonin, a multifunctional neurohormone prevalent in photoreceptor cells, and is known as as a chemical token of the night. Melatonin also serves as a scavenger for reactive oxygen species. This is also true with invertebrates. NAT therefore has distinct functional implications in circadian function, as timezymes (aaNAT, and also xenobiotic reactions (arylamine NAT or simply NAT. NATs belong to a broader enzyme group, the GCN5-related N-acetyltransferase superfamily. Due to low sequence homology and a seemingly fast rate of structural differentiation, the nomenclature for NATs can be confusing. The advent of bioinformatics, however, has helped to classify this group of enzymes; vertebrates have two distinct subgroups, the timezyme type and the xenobiotic type, which has a wider substrate range including imidazolamine, pharmacological drugs, environmental toxicants and even histone. Insect aaNAT (iaaNAT form their own clade in the phylogeny, distinct from vertebrate aaNATs. Arthropods are unique, since the phylum has exoskeleton in which quinones derived from N-acetylated monoamines function in coupling chitin and arthropodins. Monoamine oxidase (MAO activity is limited in insects, but NAT-mediated degradation prevails. However, unexpectedly iaaNAT occurs not only among arthropods but also among basal deuterostomia, and is therefore more apomorphic. Our analyses illustrate that iaaNATs has unique physiological roles but at the same time it plays a role in a timezyme function, at least in photoperiodism. Photoperiodism has been considered as a function of circadian system but the detailed molecular mechanism is not well understood. We propose a molecular hypothesis for photoperiodism in Antheraea pernyi based on the transcription regulation of NAT interlocked by the
Kinetic characterisation of arylamine N-acetyltransferase from Pseudomonas aeruginosa
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Sim Edith
Full Text Available Abstract Background Arylamine N-acetyltransferases (NATs are important drug- and carcinogen-metabolising enzymes that catalyse the transfer of an acetyl group from a donor, such as acetyl coenzyme A, to an aromatic or heterocyclic amine, hydrazine, hydrazide or N-hydroxylamine acceptor substrate. NATs are found in eukaryotes and prokaryotes, and they may also have an endogenous function in addition to drug metabolism. For example, NAT from Mycobacterium tuberculosis has been proposed to have a role in cell wall lipid biosynthesis, and is therefore of interest as a potential drug target. To date there have been no studies investigating the kinetic mechanism of a bacterial NAT enzyme. Results We have determined that NAT from Pseudomonas aeruginosa, which has been described as a model for NAT from M. tuberculosis, follows a Ping Pong Bi Bi kinetic mechanism. We also describe substrate inhibition by 5-aminosalicylic acid, in which the substrate binds both to the free form of the enzyme and the acetyl coenzyme A-enzyme complex in non-productive reaction pathways. The true kinetic parameters for the NAT-catalysed acetylation of 5-aminosalicylic acid with acetyl coenzyme A as the co-factor have been established, validating earlier approximations. Conclusion This is the first reported study investigating the kinetic mechanism of a bacterial NAT enzyme. Additionally, the methods used herein can be applied to investigations of the interactions of NAT enzymes with new chemical entities which are NAT ligands. This is likely to be useful in the design of novel potential anti-tubercular agents.
Carnitine acetyltransferase: A new player in skeletal muscle insulin resistance?
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Sofia Mikkelsen Berg
Full Text Available Carnitine acetyltransferase (CRAT deficiency has previously been shown to result in muscle insulin resistance due to accumulation of long-chain acylcarnitines. However, differences in the acylcarnitine profile and/or changes in gene expression and protein abundance of CRAT in myotubes obtained from obese patients with type 2 diabetes mellitus (T2DM and glucose-tolerant obese and lean controls remain unclear. The objective of the study was to examine whether myotubes from obese patients with T2DM express differences in gene expression and protein abundance of CRAT and in acylcarnitine species pre-cultured under glucose and insulin concentrations similar to those observed in healthy individuals in the over-night fasted, resting state. Primary myotubes obtained from obese persons with or without T2DM and lean controls (n=9 in each group were cultivated and harvested for LC-MS-based profiling of acylcarnitines. The mRNA expression and protein abundance of CRAT were determined by qPCR and Western Blotting, respectively. Our results suggest that the mRNA levels and protein abundance of CRAT were similar between groups. Of the 14 different acylcarnitine species measured by LC-MS, the levels of palmitoylcarnitine (C16 and octadecanoylcarnitine (C18 were slightly reduced in myotubes derived from T2DM patients (p<0.05 compared to glucose-tolerant obese and lean controls. This suggests that the CRAT function is not the major contributor to primary insulin resistance in cultured myotubes obtained from obese T2DM patients.
Chandrika, Nulu Naga Prafulla; Sundaravelpandian, Kalaipandian; Schmidt, Wolfgang
Post-translational modifications of core histones are important for various DNA-templated processes such as transcription and repair. We recently reported that the ALFIN LIKE 6 (AL6) gene, identified in a forward genetic screen, is critical for phosphate deficiency-induced root hair formation and several other processes associated with the regulation of cellular phosphate homeostasis. AL6 contains a Plant Homeo Domain (PHD) finger that can bind to trimethylated lysine 4 of histone H3 (H3K4me3). Homozygous mutants defective in AL6 expression form very short root hairs under phosphate-deficient conditions, presumably caused by altered expression of putative primary and secondary down-stream targets of AL6. In this Addendum, we speculate about possible roles of AL6, H3K4 trimethylation and other chromatin modifications in the adaptation of plants to low phosphate availability. PMID:23531693
Identification of lysine acetyltransferase p300 substrates using 4-pentynoyl-coenzyme A and bioorthogonal proteomics.
Yang, Yu-Ying; Yu-Ying, Yang; Grammel, Markus; Markus, Grammel; Hang, Howard C; Howard, Hang C
Proteomic studies have identified a plethora of lysine acetylated proteins in eukaryotes and bacteria. Determining the individual lysine acetyltransferases responsible for each protein acetylation mark is crucial for elucidating the underlying regulatory mechanisms, but has been challenging due to limited biochemical methods. Here, we describe the application of a bioorthogonal chemical proteomics method to profile and identify substrates of individual lysine acetyltransferases. Addition of 4-pentynoyl-coenzyme A, an alkynyl chemical reporter for protein acetylation, to cell extracts, together with purified lysine acetyltransferase p300, enabled the fluorescent profiling and identification of protein substrates via Cu(I)-catalyzed alkyne-azide cycloaddition. We identified several known protein substrates of the acetyltransferase p300 as well as the lysine residues that were modified. Interestingly, several new candidate p300 substrates and their sites of acetylation were also discovered using this approach. Our results demonstrate that bioorthogonal chemical proteomics allows the rapid substrate identification of individual protein acetyltransferases in vitro. Copyright © 2011 Elsevier Ltd. All rights reserved.
Allosteric regulation of a protein acetyltransferase in Micromonospora aurantiaca by the amino acids cysteine and arginine.
Xu, Jun-Yu; You, Di; Leng, Pei-Qiang; Ye, Bang-Ce
ACT domains (amino acid-binding domains) are linked to a wide range of metabolic enzymes that are regulated by amino acid concentration. Seventy proteins with ACT-GCN5-related N-acetyltransferase (GNAT) domain organization were found in actinomycetales. In this study, we investigate the ACT-containing GNAT acetyltransferase, Micau_1670 (MaKat), from Micromonospora aurantiaca ATCC 27029. Arginine and cysteine were identified as ligands by monitoring the conformational changes that occur upon amino acids binding to the ACT domain in the MaKat protein using FRET assay. It was found that MaKat is an amino acid-regulated protein acetyltransferase, whereas arginine and cysteine stimulated the activity of MaKat with regard to acetylation of acetyl-CoA synthetase (Micau_0428). Our research reveals the biochemical characterization of a protein acetyltransferase that contains a fusion of a GNAT domain with an ACT domain and provides a novel signaling pathway for regulating cellular protein acetylation. These findings indicate that acetylation of proteins and acetyltransferase activity may be tightly linked to cellular concentrations of some amino acids in actinomycetales. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
The Histone Demethylase UTX Promotes Brown Adipocyte Thermogenic Program Via Coordinated Regulation of H3K27 Demethylation and Acetylation.
Zha, Lin; Li, Fenfen; Wu, Rui; Artinian, Liana; Rehder, Vincent; Yu, Liqing; Liang, Houjie; Xue, Bingzhong; Shi, Hang
Brown adipocytes function to dissipate energy as heat through adaptive thermogenesis. Understanding the molecular mechanisms underlying the brown fat thermogenic program may provide insights for the development of therapeutic approaches in the treatment of obesity. Most studies investigating the mechanisms underlying brown fat development focus on genetic mechanisms; little is known about the epigenetic mechanisms in this process. We have discovered that ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX), a histone demethylase for di- or tri-methylated histone 3 lysine 27 (H3K27me2/3), plays a potential role in regulating brown adipocyte thermogenic program. We found that UTX is up-regulated during brown adipocyte differentiation and by cold exposure in both brown adipose tissue (BAT) and white adipose tissue (WAT) of mice, suggesting a potential role in thermogenesis. Inactivation of UTX down-regulates brown fat specific gene expression, while overexpression of UTX does the opposite. Notably, activation of β adrenergic signaling recruits UTX to the UCP1 and PGC1α promoters, leading to decreased H3K27me3, a histone transcriptional repressive mark. UTX demethylates H3K27me3 and subsequently interacts with the histone acetyltransferase (HAT) protein CBP, resulting in increased H3K27 acetylation (H3K27ac), a histone transcriptional active mark. UTX positively regulate brown adipocyte thermogenic program through coordinated control of demethylating H3K27me3 and acetylating H3K27, switching the transcriptional repressive state to the transcriptional active state at the promoters of UCP1 and PGC1α. We conclude that UTX may play a potential role in regulation of brown adipocyte gene expression and may mediate β adrenergic activation of brown fat function. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Total Chemical Synthesis of Modified Histones.
Qi, Yun-Kun; Ai, Hua-Song; Li, Yi-Ming; Yan, Baihui
In the post-genome era, epigenetics has received increasing attentions in recent years. The post-translational modifications (PTMs) of four core histones play central roles in epigenetic regulation of eukaryotic genome by either directly altering the biophysical properties of nucleosomes or by recruiting other effector proteins. In order to study the biological functions and structural mechanisms of these histone PTMs, an obligatory step is to prepare a sufficient amount of homogeneously modified histones. This task cannot be fully accomplished either by recombinant technology or enzymatic modification. In this context, synthetic chemists have developed novel protein synthetic tools and state-of-the-art chemical ligation strategies for the preparation of homologous modified histones. In this review, we summarize the recent advances in the preparation of modified histones, focusing on the total chemical synthesis strategies. The importance and potential of synthetic chemistry for the study of histone code will be also discussed.
N-Acetylaspartate Metabolism Outside the Brain: Lipogenesis, Histone Acetylation, and Cancer
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Juliane G. Bogner-Strauss
Full Text Available N-acetylaspartate (NAA is a highly abundant brain metabolite. Aberrant NAA concentrations have been detected in many pathological conditions and although the function of NAA has been extensively investigated in the brain it is still controversial. Only recently, a role of NAA has been reported outside the brain. In brown adipocytes, which show high expression of the NAA-producing and the NAA-cleaving enzyme, the metabolism of NAA has been implicated in lipid synthesis and histone acetylation. Increased expression of N-acetyltransferase 8-like (Nat8l, the gene encoding the NAA synthesizing enzyme induces de novo lipogenesis and the brown adipocyte phenotype. Accordingly silencing of aspartoacylase, the NAA-cleaving enzyme, reduced brown adipocyte differentiation mechanistically by decreasing histone acetylation and gene transcription. Notably, the expression of Nat8l and the amount of NAA were also shown to be increased in several tumors and inversely correlate with patients' survival. Additionally, Nat8l silencing reduced cell proliferation in tumor and non-tumor cells, while NAA supplementation could rescue it. However, the mechanism behind has not yet been clarified. It remains to be addressed whether NAA per se and/or its catabolism to acetate and aspartate, metabolites that have both been implicated in tumor growth, are valuable targets for future therapies.
Histone acetylation: molecular mnemonics on the chromatin.
Gräff, Johannes; Tsai, Li-Huei
Long-lasting memories require specific gene expression programmes that are, in part, orchestrated by epigenetic mechanisms. Of the epigenetic modifications identified in cognitive processes, histone acetylation has spurred considerable interest. Whereas increments in histone acetylation have consistently been shown to favour learning and memory, a lack thereof has been causally implicated in cognitive impairments in neurodevelopmental disorders, neurodegeneration and ageing. As histone acetylation and cognitive functions can be pharmacologically restored by histone deacetylase inhibitors, this epigenetic modification might constitute a molecular memory aid on the chromatin and, by extension, a new template for therapeutic interventions against cognitive frailty.
Acetylation of Mammalian ADA3 Is Required for Its Functional Roles in Histone Acetylation and Cell Proliferation.
Mohibi, Shakur; Srivastava, Shashank; Bele, Aditya; Mirza, Sameer; Band, Hamid; Band, Vimla
Alteration/deficiency in activation 3 (ADA3) is an essential component of specific histone acetyltransferase (HAT) complexes. We have previously shown that ADA3 is required for establishing global histone acetylation patterns and for normal cell cycle progression (S. Mohibi et al., J Biol Chem 287:29442-29456, 2012, http://dx.doi.org/10.1074/jbc.M112.378901). Here, we report that these functional roles of ADA3 require its acetylation. We show that ADA3 acetylation, which is dynamically regulated in a cell cycle-dependent manner, reflects a balance of coordinated actions of its associated HATs, GCN5, PCAF, and p300, and a new partner that we define, the deacetylase SIRT1. We use mass spectrometry and site-directed mutagenesis to identify major sites of ADA3 acetylated by GCN5 and p300. Acetylation-defective mutants are capable of interacting with HATs and other components of HAT complexes but are deficient in their ability to restore ADA3-dependent global or locus-specific histone acetylation marks and cell proliferation in Ada3-deleted murine embryonic fibroblasts (MEFs). Given the key importance of ADA3-containing HAT complexes in the regulation of various biological processes, including the cell cycle, our study presents a novel mechanism to regulate the function of these complexes through dynamic ADA3 acetylation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
Integrated omics approaches to characterize a nuclear receptor corepressor-associated histone deacetylase in mouse skeletal muscle.
Gong, Yingyun; Cao, Rui; Ding, Guolian; Hong, Sungguan; Zhou, Wenjun; Lu, Wenyun; Damle, Manashree; Fang, Bin; Wang, Chuhan C; Qian, Justin; Lie, Natasha; Lanzillotta, Cristina; Rabinowitz, Joshua D; Sun, Zheng
Nuclear receptors regulate gene expression by differentially binding to coactivators or corepressors in a ligand-dependent manner, which further recruits a set of epigenome-modifying enzymes that remodel chromatin conformation. Histone acetylation is a major epigenomic change controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDAC3 is the only HDAC that confers the enzymatic activity to the complexes nucleated by nuclear receptor corepressors NCoR and SMRT. To address the metabolic function of HDAC3, we have deleted it specifically in mouse skeletal muscles. We have performed the following omics profiling in skeletal muscles of these mice: (1) RNA-seq profiling of total RNA; (2) Global nuclear run-on (GRO-seq) analysis of nascent RNAs; (3) Chromatin immuno-precipitation (ChIP-seq) of HDAC3 at both early evening and early morning; (4) proteomics profiling with mass spectrometry; (5) snap-shot metabolomics profiling of water-soluble metabolites at the basal condition; (6) snap-shot metabolomics profiling of lipid species at the basal condition; (7) kinetic fluxomics analysis of glucose utilization using 13 C 6 -glucose In vivo during treadmill running exercise. These approaches have provided several novel insights into how nuclear receptors regulate circadian rhythm of skeletal muscle fuel metabolism, which has been published elsewhere. Here we present the original datasets and technical details during the execution, analysis, and interpretation of these omics studies. Copyright © 2017 Elsevier B.V. All rights reserved.
Rewiring AMPK and Mitochondrial Retrograde Signaling for Metabolic Control of Aging and Histone Acetylation in Respiratory-Defective Cells
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R. Magnus N. Friis
Full Text Available Abnormal respiratory metabolism plays a role in numerous human disorders. We find that regulation of overall histone acetylation is perturbed in respiratory-incompetent (ρ0 yeast. Because histone acetylation is highly sensitive to acetyl-coenzyme A (acetyl-CoA availability, we sought interventions that suppress this ρ0 phenotype through reprogramming metabolism. Nutritional intervention studies led to the discovery that genetic coactivation of the mitochondrion-to-nucleus retrograde (RTG response and the AMPK (Snf1 pathway prevents abnormal histone deacetylation in ρ0 cells. Metabolic profiling of signaling mutants uncovered links between chromatin-dependent phenotypes of ρ0 cells and metabolism of ATP, acetyl-CoA, glutathione, branched-chain amino acids, and the storage carbohydrate trehalose. Importantly, RTG/AMPK activation reprograms energy metabolism to increase the supply of acetyl-CoA to lysine acetyltransferases and extend the chronological lifespan of ρ0 cells. Our results strengthen the framework for rational design of nutrient supplementation schemes and drug-discovery initiatives aimed at mimicking the therapeutic benefits of dietary interventions.
N-acetyltransferase 2 gene polymorphism and presbycusis.
Unal, Murat; Tamer, Lülüfer; Doğruer, Zeynep Nil; Yildirim, Hatice; Vayisoğlu, Yusuf; Camdeviren, Handan
The enzyme of N-acetyltransferase (NAT) is involved in the metabolism and detoxification of cytotoxic and carcinogenic compounds as well as reactive oxygen species (ROS). The excessive amount of ROS generation occurs in the ageing inner ear. The exact etiopathogenesis of presbycusis is not known, but it is generally accepted that it is the result of series of insults, such as physiologic age-related degeneration, noise exposure, medical disorders and their treatment, as well as hereditary susceptibility. The effect of aging shows a wide interindividual range; we aimed to investigate whether profiles of NAT2 genotypes may be associated with the risk of presbycusis. Hospital-based, case-control study. We examined 68 adults with presbycusis and 98 healthy controls. DNA was extracted from whole blood, and the polymorphisms of NAT2*5A, NAT2*6A, NAT2*7A/B, and NAT2*14A were determined using a real-time polymerase chain reaction and fluorescence resonance energy transfer with a Light-Cycler Instrument. Associations between specific genotypes and the development of presbycusis were examined by use of logistic regression analyses to calculate odds ratios and 95% confidence intervals. Gene polymorphisms at NAT2*5A, NAT2*7A/B, and NAT2*14A in subjects with presbycusis were not significantly different from in the controls (P > .05). However, in NAT2*6A, the risk of presbycusis was 15.2-fold more in individuals with mutant allele than subjects with wild genotype (P = .013). Individuals with NAT2*6A heterozygote allele had a 0.34-fold less risk in the development of presbycusis than subjects with mutant allele (P = .032) We demonstrated a significant association between the NAT2*6A polymorphism and age-related hearing loss in this population. However, the sample size was relatively small, and further studies need to investigate the exact role of NAT2 gene polymorphism in the etiopathogenesis of the presbycusis.
PSG gene expression is up-regulated by lysine acetylation involving histone and nonhistone proteins.
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Soledad A Camolotto
Full Text Available BACKGROUND: Lysine acetylation is an important post-translational modification that plays a central role in eukaryotic transcriptional activation by modifying chromatin and transcription-related factors. Human pregnancy-specific glycoproteins (PSG are the major secreted placental proteins expressed by the syncytiotrophoblast at the end of pregnancy and represent early markers of cytotrophoblast differentiation. Low PSG levels are associated with complicated pregnancies, thus highlighting the importance of studying the mechanisms that control their expression. Despite several transcription factors having been implicated as key regulators of PSG gene family expression; the role of protein acetylation has not been explored. METHODOLOGY/PRINCIPAL FINDINGS: Here, we explored the role of acetylation on PSG gene expression in the human placental-derived JEG-3 cell line. Pharmacological inhibition of histone deacetylases (HDACs up-regulated PSG protein and mRNA expression levels, and augmented the amount of acetylated histone H3 associated with PSG 5'regulatory regions. Moreover, PSG5 promoter activation mediated by Sp1 and KLF6, via the core promoter element motif (CPE, -147/-140, was markedly enhanced in the presence of the HDAC inhibitor trichostatin A (TSA. This effect correlated with an increase in Sp1 acetylation and KLF6 nuclear localization as revealed by immunoprecipitation and subcellular fractionation assays. The co-activators PCAF, p300, and CBP enhanced Sp1-dependent PSG5 promoter activation through their histone acetylase (HAT function. Instead, p300 and CBP acetyltransferase domain was dispensable for sustaining co-activation of PSG5 promoter by KLF6. CONCLUSIONS/SIGNIFICANCE: Results are consistent with a regulatory role of lysine acetylation on PSG expression through a relaxed chromatin state and an increase in the transcriptional activity of Sp1 and KLF6 following an augmented Sp1 acetylation and KLF6 nuclear localization.
Histone H2A mobility is regulated by its tails and acetylation of core histone tails
International Nuclear Information System (INIS)
Higashi, Tsunehito; Matsunaga, Sachihiro; Isobe, Keisuke; Morimoto, Akihiro; Shimada, Tomoko; Kataoka, Shogo; Watanabe, Wataru; Uchiyama, Susumu; Itoh, Kazuyoshi; Fukui, Kiichi
Histone tail domains play important roles in cellular processes, such as replication, transcription, and chromosome condensation. Histone H2A has one central and two tail domains, and their functions have mainly been studied from a biochemical perspective. In addition, analyses based on visualization have been employed for functional analysis of some chromatin proteins. In this study, we analyzed histone H2A mobility in vivo by two-photon FRAP, and elucidated that the histone H2A N- and C-terminal tails regulate its mobility. We found that histone H2A mobility was increased following treatment of host cells with a histone deacetylase inhibitor. Our results support a model in which core histone tails directly regulate transcription by interacting with nucleosome DNA via electrostatic interactions
Extracellular histones induce erythrocyte fragility and anemia.
Kordbacheh, Farzaneh; O'Meara, Connor H; Coupland, Lucy A; Lelliott, Patrick M; Parish, Christopher R
Extracellular histones have been shown to play an important pathogenic role in many diseases, primarily through their cytotoxicity toward nucleated cells and their ability to promote platelet activation with resultant thrombosis and thrombocytopenia. In contrast, little is known about the effect of extracellular histones on erythrocyte function. We demonstrate in this study that histones promote erythrocyte aggregation, sedimentation, and using a novel in vitro shear stress model, we show that histones induce erythrocyte fragility and lysis in a concentration-dependent manner. Furthermore, histones impair erythrocyte deformability based on reduced passage of erythrocytes through an artificial spleen. These in vitro results were mirrored in vivo with the injection of histones inducing anemia within minutes of administration, with a concomitant increase in splenic hemoglobin content. Thrombocytopenia and leukopenia were also observed. These findings suggest that histones binding to erythrocytes may contribute to the elevated erythrocyte sedimentation rates observed in inflammatory conditions. Furthermore, histone-induced increases in red blood cell lysis and splenic clearance may be a significant factor in the unexplained anemias seen in critically ill patients. © 2017 by The American Society of Hematology.
Histone modifications and nuclear architecture: A review
Czech Academy of Sciences Publication Activity Database
Bártová, Eva; Kroupová, Jana; Harničarová, Andrea; Galiová-Šustáčková, Gabriela; Kozubek, Stanislav
Roč. 56, č. 8 (2008), s. 711-721 ISSN 0722-186X R&D Projects: GA ČR(CZ) GA204/06/0978; GA MŠk(CZ) LC535 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : histones * histone modifications * nuclear architecture Subject RIV: BO - Biophysics
Structure and Functions of Linker Histones.
Lyubitelev, A V; Nikitin, D V; Shaytan, A K; Studitsky, V M; Kirpichnikov, M P
Linker histones such as variants H1, H5, and other similar proteins play an important role in regulation of chromatin structure and dynamics. However, interactions of linker histones with DNA and proteins, as well as specific functions of their different variants, are poorly studied. This is because they acquire tertiary structure only when interacting with a nucleosome, and because of limitations of currently available methods. However, deeper investigation of linker histones and their interactions with other proteins will address a number of important questions - from structure of compacted chromatin to regulation of early embryogenesis. In this review, structures of histone H1 variants and its interaction with chromatin DNA are considered. A possible functional significance of different H1 variants, a role of these proteins in maintaining interphase chromatin structure, and interactions of linker histones with other cellular proteins are also discussed.
Aminoglycoside resistance profile and structural architecture of the aminoglycoside acetyltransferase AAC(6')-Im.
Smith, Clyde A; Bhattacharya, Monolekha; Toth, Marta; Stewart, Nichole K; Vakulenko, Sergei B
Aminoglycoside 6'-acetyltransferase-Im (AAC(6')-Im) is the closest monofunctional homolog of the AAC(6')-Ie acetyltransferase of the bifunctional enzyme AAC(6')-Ie/APH(2")-Ia. The AAC(6')-Im acetyltransferase confers 4- to 64-fold higher MICs to 4,6-disubstituted aminoglycosides and the 4,5-disubstituted aminoglycoside neomycin than AAC(6')-Ie, yet unlike AAC(6')-Ie, the AAC(6')-Im enzyme does not confer resistance to the atypical aminoglycoside fortimicin. The structure of the kanamycin A complex of AAC(6')-Im shows that the substrate binds in a shallow positively-charged pocket, with the N6' amino group positioned appropriately for an efficient nucleophilic attack on an acetyl-CoA cofactor. The AAC(6')-Ie enzyme binds kanamycin A in a sufficiently different manner to position the N6' group less efficiently, thereby reducing the activity of this enzyme towards the 4,6-disubstituted aminoglycosides. Conversely, docking studies with fortimicin in both acetyltransferases suggest that the atypical aminoglycoside might bind less productively in AAC(6')-Im, thus explaining the lack of resistance to this molecule.
Modulation of Escherichia coli serine acetyltransferase catalytic activity in the cysteine synthase complex
Czech Academy of Sciences Publication Activity Database
Benoni, Roberto; De Bei, O.; Paredi, G.; Hayes, C. S.; Franko, N.; Mozzarelli, A.; Bettati, S.; Campanini, B.
Roč. 591, č. 9 (2017), s. 1212-1224 ISSN 0014-5793 Institutional support: RVO:61388963 Keywords : cysteine synthase * protein-protein interaction * serine acetyltransferase Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 3.623, year: 2016
Orofacial clefts and spina bifida: N-acetyltransferase phenotype, maternal smoking, and medication use.
Rooij, I.A.L.M. van; Groenen, P.; Drongelen, M. van; Morsche, R.H.M. te; Peters, W.H.M.; Steegers-Theunissen, R.P.M.
BACKGROUND: Orofacial clefts and spina bifida are midline defects with a multifactorial etiology. Maternal smoking and medication use periconceptionally have been studied as risk factors for these malformations. The biotransformation enzyme N-acetyltransferase 2 (NAT2), plays a part in the
Sequence analysis of the N-acetyltransferase 2 gene (NAT2) among ...
African Journals Online (AJOL)
Yazun Bashir Jarrar
Nov 26, 2017 ... Sequence analysis of the N-acetyltransferase 2 gene (NAT2) among Jordanian volunteers. Yazun Bashir Jarrar, Ayat Ahmed Balasmeh and Wassan Jarrar. Department of Pharmacy, College of Pharmacy, AlZaytoonah University of Jordan, Amman, Jordan. ABSTRACT. The present study aimed to identify ...
MS_HistoneDB, a manually curated resource for proteomic analysis of human and mouse histones.
El Kennani, Sara; Adrait, Annie; Shaytan, Alexey K; Khochbin, Saadi; Bruley, Christophe; Panchenko, Anna R; Landsman, David; Pflieger, Delphine; Govin, Jérôme
Histones and histone variants are essential components of the nuclear chromatin. While mass spectrometry has opened a large window to their characterization and functional studies, their identification from proteomic data remains challenging. Indeed, the current interpretation of mass spectrometry data relies on public databases which are either not exhaustive (Swiss-Prot) or contain many redundant entries (UniProtKB or NCBI). Currently, no protein database is ideally suited for the analysis of histones and the complex array of mammalian histone variants. We propose two proteomics-oriented manually curated databases for mouse and human histone variants. We manually curated >1700 gene, transcript and protein entries to produce a non-redundant list of 83 mouse and 85 human histones. These entries were annotated in accordance with the current nomenclature and unified with the "HistoneDB2.0 with Variants" database. This resource is provided in a format that can be directly read by programs used for mass spectrometry data interpretation. In addition, it was used to interpret mass spectrometry data acquired on histones extracted from mouse testis. Several histone variants, which had so far only been inferred by homology or detected at the RNA level, were detected by mass spectrometry, confirming the existence of their protein form. Mouse and human histone entries were collected from different databases and subsequently curated to produce a non-redundant protein-centric resource, MS_HistoneDB. It is dedicated to the proteomic study of histones in mouse and human and will hopefully facilitate the identification and functional study of histone variants.
Histone deacetylases suppress CGG repeat-induced neurodegeneration via transcriptional silencing in models of fragile X tremor ataxia syndrome.
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Peter K Todd
Full Text Available Fragile X Tremor Ataxia Syndrome (FXTAS is a common inherited neurodegenerative disorder caused by expansion of a CGG trinucleotide repeat in the 5'UTR of the fragile X syndrome (FXS gene, FMR1. The expanded CGG repeat is thought to induce toxicity as RNA, and in FXTAS patients mRNA levels for FMR1 are markedly increased. Despite the critical role of FMR1 mRNA in disease pathogenesis, the basis for the increase in FMR1 mRNA expression is unknown. Here we show that overexpressing any of three histone deacetylases (HDACs 3, 6, or 11 suppresses CGG repeat-induced neurodegeneration in a Drosophila model of FXTAS. This suppression results from selective transcriptional repression of the CGG repeat-containing transgene. These findings led us to evaluate the acetylation state of histones at the human FMR1 locus. In patient-derived lymphoblasts and fibroblasts, we determined by chromatin immunoprecipitation that there is increased acetylation of histones at the FMR1 locus in pre-mutation carriers compared to control or FXS derived cell lines. These epigenetic changes correlate with elevated FMR1 mRNA expression in pre-mutation cell lines. Consistent with this finding, histone acetyltransferase (HAT inhibitors repress FMR1 mRNA expression to control levels in pre-mutation carrier cell lines and extend lifespan in CGG repeat-expressing Drosophila. These findings support a disease model whereby the CGG repeat expansion in FXTAS promotes chromatin remodeling in cis, which in turn increases expression of the toxic FMR1 mRNA. Moreover, these results provide proof of principle that HAT inhibitors or HDAC activators might be used to selectively repress transcription at the FMR1 locus.
Molecular mechanisms and potential functions of histone demethylases
DEFF Research Database (Denmark)
Kooistra, Susanne Marije; Helin, Kristian
Histone modifications are thought to regulate chromatin structure, transcription and other nuclear processes. Histone methylation was originally believed to be an irreversible modification that could only be removed by histone eviction or by dilution during DNA replication. However, the isolation...... of two families of enzymes that can demethylate histones has changed this notion. The biochemical activities of these histone demethylases towards specific Lys residues on histones, and in some cases non-histone substrates, have highlighted their importance in developmental control, cell-fate decisions...
Histone modifications in response to DNA damage
International Nuclear Information System (INIS)
Altaf, Mohammed; Saksouk, Nehme; Cote, Jacques
The packaging of the eukaryotic genome into highly condensed chromatin makes it inaccessible to the factors required for gene transcription, DNA replication, recombination and repair. Eukaryotes have developed intricate mechanisms to overcome this repressive barrier imposed by chromatin. Histone modifying enzymes and ATP-dependent chromatin remodeling complexes play key roles here as they regulate many nuclear processes by altering the chromatin structure. Significantly, these activities are integral to the process of DNA repair where histone modifications act as signals and landing platforms for various repair proteins. This review summarizes the recent developments in our understanding of histone modifications and their role in the maintenance of genome integrity
Dynamics of Histone Tails within Chromatin
Bernier, Morgan; North, Justin; Page, Michael; Jaroniec, Christopher; Hammel, Christopher; Poirier, Michael
Genetic information in humans is encoded within DNA molecules that is wrapped around histone octamer proteins and compacted into a highly conserved structural polymer, chromatin. The physical and material properties of chromatin appear to influence gene expression by altering the accessibility of proteins to the DNA. The tails of the histones are flexible domains that are thought to play a role in regulating DNA accessibility and compaction; however the molecular mechanisms for these phenomena are not understood. I will present CW-EPR studies on site directed spin labeled nucleosomes that probe the structure and dynamics of these histone tails within nucleosomes.
Histone demethylases in development and disease
DEFF Research Database (Denmark)
Pedersen, Marianne Terndrup; Helin, Kristian
Histone modifications serve as regulatory marks that are instrumental for the control of transcription and chromatin architecture. Strict regulation of gene expression patterns is crucial during development and differentiation, where diverse cell types evolve from common predecessors. Since...... the first histone lysine demethylase was discovered in 2004, a number of demethylases have been identified and implicated in the control of gene expression programmes and cell fate decisions. Histone demethylases are now emerging as important players in developmental processes and have been linked to human...
Interactions of acetylated histones with DNA as revealed by UV laser induced histone-DNA crosslinking
International Nuclear Information System (INIS)
Stefanovsky, V.Yu.; Dimitrov, S.I.; Angelov, D.; Pashev, I.G.
The interaction of acetylated histones with DNA in chromatin has been studied by UV laser-induced crosslinking histones to DNA. After irradiation of the nuclei, the covalently linked protein-DNA complexes were isolated and the presence of histones in them demonstrated immunochemically. When chromatin from irradiated nuclei was treated with clostripain, which selectively cleaved the N-terminal tails of core histones, no one of them was found covalently linked to DNA, thus showing that crosslinking proceeded solely via the N-terminal regions. However, the crosslinking ability of the laser was preserved both upon physiological acetylation of histones, known to be restricted to the N-terminal tails, and with chemically acetylated chromatin. This finding is direct evidence that the postsynthetic histone acetylation does not release the N-terminal tails from interaction with DNA
A plant dialect of the histone language.
Loidl, Peter
The genome contains all the information needed to build an organism. However, during differentiation and development, additional epigenetic information determines the functional state of cells and tissues. This epigenetic information can be introduced by cytosine methylation and by marking nucleosomal histones. The code written on histones consists of post-translational modifications, including acetylation and methylation. In contrast to the universal nature of the DNA code, the histone language and its decoding machinery differ among animals, plants and fungi. Plant cells have retained totipotency to generate the entire plant and maintained the ability to dedifferentiate, which suggests that the establishment and maintenance of epigenetic information differs from animals. Here, I aim to summarize the histone code and plant-specific aspects of setting and translating the code.
An Organellar Nα-Acetyltransferase, Naa60, Acetylates Cytosolic N Termini of Transmembrane Proteins and Maintains Golgi Integrity
Henriette Aksnes; Petra Van Damme; Marianne Goris; Kristian K. Starheim; Michaël Marie; Svein Isungset Støve; Camilla Hoel; Thomas Vikestad Kalvik; Kristine Hole; Nina Glomnes; Clemens Furnes; Sonja Ljostveit; Mathias Ziegler; Marc Niere; Kris Gevaert
N-terminal acetylation is a major and vital protein modification catalyzed by N-terminal acetyltransferases (NATs). NatF, or N alpha-acetyltransferase 60 (Naa60), was recently identified as a NAT in multicellular eukaryotes. Here, we find that Naa60 differs from all other known NATs by its Golgi localization. A new membrane topology assay named PROMPT and a selective membrane permeabilization assay established that Naa60 faces the cytosolic side of intracellular membranes. An Nt-acetylome ana...
System-wide Studies of N-Lysine Acetylation in Rhodopseudomonas palustris Reveals Substrate Specificity of Protein Acetyltransferases
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Crosby, Heidi A [University of Wisconsin, Madison; Pelletier, Dale A [ORNL; Hurst, Gregory {Greg} B [ORNL; Escalante-Semerena, Jorge C [University of Wisconsin, Madison
Background: Protein acetylation is widespread in prokaryotes. Results: Six new acyl-CoA synthetases whose activities are controlled by acetylation were identified, and their substrate preference established. A new protein acetyltransferase was also identified and its substrate specificity determined. Conclusion: Protein acetyltransferases acetylate a conserved lysine residue in protein substrates. Significance: The R. palustris Pat enzyme specifically acetylates AMP-forming acyl-CoA synthetases and regulates fatty acid metabolism.
Genistein cooperates with the histone deacetylase inhibitor vorinostat to induce cell death in prostate cancer cells
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Phillip Cornel J
Full Text Available Abstract Background Among American men, prostate cancer is the most common, non-cutaneous malignancy that accounted for an estimated 241,000 new cases and 34,000 deaths in 2011. Previous studies have suggested that Wnt pathway inhibitory genes are silenced by CpG hypermethylation, and other studies have suggested that genistein can demethylate hypermethylated DNA. Genistein is a soy isoflavone with diverse effects on cellular proliferation, survival, and gene expression that suggest it could be a potential therapeutic agent for prostate cancer. We undertook the present study to investigate the effects of genistein on the epigenome of prostate cancer cells and to discover novel combination approaches of other compounds with genistein that might be of translational utility. Here, we have investigated the effects of genistein on several prostate cancer cell lines, including the ARCaP-E/ARCaP-M model of the epithelial to mesenchymal transition (EMT, to analyze effects on their epigenetic state. In addition, we investigated the effects of combined treatment of genistein with the histone deacetylase inhibitor vorinostat on survival in prostate cancer cells. Methods Using whole genome expression profiling and whole genome methylation profiling, we have determined the genome-wide differences in genetic and epigenetic responses to genistein in prostate cancer cells before and after undergoing the EMT. Also, cells were treated with genistein, vorinostat, and combination treatment, where cell death and cell proliferation was determined. Results Contrary to earlier reports, genistein did not have an effect on CpG methylation at 20 μM, but it did affect histone H3K9 acetylation and induced increased expression of histone acetyltransferase 1 (HAT1. In addition, genistein also had differential effects on survival and cooperated with the histone deacteylase inhibitor vorinostat to induce cell death and inhibit proliferation. Conclusion Our results suggest that
Replicating chromatin: a tale of histones
DEFF Research Database (Denmark)
Groth, Anja
framework of chromatin and carry information to specify higher-order organization and gene expression. When replication forks traverse the chromosomes, nucleosomes are transiently disrupted, allowing the replication machinery to gain access to DNA. Histone recycling, together with new deposition, ensures...... reassembly on nascent DNA strands. The aim of this review is to discuss how histones - new and old - are handled at the replication fork, highlighting new mechanistic insights and revisiting old paradigms....
Histone Lysine Methylation in Diabetic Nephropathy
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Guang-dong Sun
Full Text Available Diabetic nephropathy (DN belongs to debilitating microvascular complications of diabetes and is the leading cause of end-stage renal diseases worldwide. Furthermore, outcomes from the DCCT/EDIC study showed that DN often persists and progresses despite intensive glucose control in many diabetes patients, possibly as a result of prior episode of hyperglycemia, which is called "metabolic memory." The underlying mechanisms responsible for the development and progression of DN remain poorly understood. Activation of multiple signaling pathways and key transcription factors can lead to aberrant expression of DN-related pathologic genes in target renal cells. Increasing evidence suggests that epigenetic mechanisms in chromatin such as DNA methylation, histone acetylation, and methylation can influence the pathophysiology of DN and metabolic memory. Exciting researches from cell culture and experimental animals have shown that key histone methylation patterns and the related histone methyltransferases and histone demethylases can play important roles in the regulation of inflammatory and profibrotic genes in renal cells under diabetic conditions. Because histone methylation is dynamic and potentially reversible, it can provide a window of opportunity for the development of much-needed novel therapeutic potential for DN in the future. In this minireview, we discuss recent advances in the field of histone methylation and its roles in the pathogenesis and progression of DN.
Extracellular histones in tissue injury and inflammation.
Allam, Ramanjaneyulu; Kumar, Santhosh V R; Darisipudi, Murthy N; Anders, Hans-Joachim
Neutrophil NETosis is an important element of host defense as it catapults chromatin out of the cell to trap bacteria, which then are killed, e.g., by the chromatin's histone component. Also, during sterile inflammation TNF-alpha and other mediators trigger NETosis, which elicits cytotoxic effects on host cells. The same mechanism should apply to other forms of regulated necrosis including pyroptosis, necroptosis, ferroptosis, and cyclophilin D-mediated regulated necrosis. Beyond these toxic effects, extracellular histones also trigger thrombus formation and innate immunity by activating Toll-like receptors and the NLRP3 inflammasome. Thereby, extracellular histones contribute to the microvascular complications of sepsis, major trauma, small vessel vasculitis as well as acute liver, kidney, brain, and lung injury. Finally, histones prevent the degradation of extracellular DNA, which promotes autoimmunization, anti-nuclear antibody formation, and autoimmunity in susceptible individuals. Here, we review the current evidence on the pathogenic role of extracellular histones in disease and discuss how to target extracellular histones to improve disease outcomes.
Crystallization of ornithine acetyltransferase from yeast by counter-diffusion and preliminary X-ray study
International Nuclear Information System (INIS)
Maes, Dominique; Crabeel, Marjolaine; Van de Weerdt, Cécile; Martial, Joseph; Peeters, Eveline; Charlier, Daniël; Decanniere, Klaas; Vanhee, Celine; Wyns, Lode; Zegers, Ingrid
A study on the crystallization of ornithine acetyltransferase from yeast, catalysing the fifth step in microbial arginine synthesis, is presented. The use of the counter-diffusion technique removes the disorder present in one dimension in crystals grown by either batch or hanging-drop techniques. A study is presented on the crystallization of ornithine acetyltransferase from yeast, which catalyzes the fifth step in microbial arginine synthesis. The use of the counter-diffusion technique removes the disorder present in one dimension in crystals grown by either the batch or hanging-drop techniques. This makes the difference between useless crystals and crystals that allow successful determination of the structure of the protein. The crystals belong to space group P4, with unit-cell parameters a = b = 66.98, c = 427.09 Å, and a data set was collected to 2.76 Å
Crystallization of ornithine acetyltransferase from yeast by counter-diffusion and preliminary X-ray study
Energy Technology Data Exchange (ETDEWEB)
Maes, Dominique, E-mail: firstname.lastname@example.com; Crabeel, Marjolaine [Laboratorium voor Ultrastructuur, Vrije Universiteit Brussel (VUB) and Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Pleinlaan 2, B-1050 Brussels (Belgium); Van de Weerdt, Cécile; Martial, Joseph [Laboratoire de Biologie Moléculaire et de Génie Génétique, Université de Liège, Allée de la Chimie 3, B-4000 Liège (Belgium); Peeters, Eveline; Charlier, Daniël [Erfelijkheidsleer en Microbiologie, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussels (Belgium); Decanniere, Klaas; Vanhee, Celine; Wyns, Lode; Zegers, Ingrid [Laboratorium voor Ultrastructuur, Vrije Universiteit Brussel (VUB) and Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Pleinlaan 2, B-1050 Brussels (Belgium)
A study on the crystallization of ornithine acetyltransferase from yeast, catalysing the fifth step in microbial arginine synthesis, is presented. The use of the counter-diffusion technique removes the disorder present in one dimension in crystals grown by either batch or hanging-drop techniques. A study is presented on the crystallization of ornithine acetyltransferase from yeast, which catalyzes the fifth step in microbial arginine synthesis. The use of the counter-diffusion technique removes the disorder present in one dimension in crystals grown by either the batch or hanging-drop techniques. This makes the difference between useless crystals and crystals that allow successful determination of the structure of the protein. The crystals belong to space group P4, with unit-cell parameters a = b = 66.98, c = 427.09 Å, and a data set was collected to 2.76 Å.
Novel 3-N-Aminoglycoside Acetyltransferase Gene, aac(3)-Ic, from a Pseudomonas aeruginosa Integron
Riccio, Maria Letizia; Docquier, Jean-Denis; Dell'Amico, Emanuela; Luzzaro, Francesco; Amicosante, Gianfranco; Rossolini, Gian Maria
A novel gene, aac(3)-Ic, encoding an AAC(3)-I aminoglycoside 3-N-acetyltransferase, was identified on a gene cassette inserted into a Pseudomonas aeruginosa integron that also carries a blaVIM-2 and a cmlA7 gene cassette. The aac(3)-Ic gene product is 59 and 57% identical to AAC(3)-Ia and AAC(3)-Ib, respectively, and confers resistance to gentamicin and sisomicin.
New Integron-Associated Gene Cassette Encoding a 3-N-Aminoglycoside Acetyltransferase
Levings, Renee S.; Partridge, Sally R.; Lightfoot, Diane; Hall, Ruth M.; Djordjevic, Steven P.
A fifth gene cassette containing an aacC gene, aacCA5, was found in an aacCA5-aadA7 cassette array in a class 1 integron isolated from a multiply drug resistant Salmonella enterica serovar Kentucky strain. The AacC-A5 or AAC(3)-Ie acetyltransferase encoded by aacCA5 is related to other AAC(3)-I enzymes and confers resistance to gentamicin.
Induction of spermidine/spermine N1-acetyltransferase by methylglyoxal bis(guanylhydrazone).
Pegg, A E; Erwin, B G; Persson, L
The anti-tumor agent methylglyoxal bis(guanylhydrazone) was found to be a competitive inhibitor of spermidine/spermine N1-acetyltransferase with a Ki of about 8 microM. Treatment of rats with this drug lead to a very large increase in the total amount of spermidine/spermine N1-acetyltransferase in liver, kidney and spleen. The total increase as measured using a specific antiserum amounted to 700-fold in liver and 100-fold in kidney within 18 h of treatment with 80 mg/kg doses. At least part of this induction was due to a pronounced increase in the half-life of the acetyltransferase which increased from 15 min to more than 12 h. The very large increase in the amount of the enzyme is likely to overwhelm the direct inhibition, and a net increase in the acetylation of polyamines by this enzyme would be expected to occur after treatment with methylglyoxal bis(guanylhydrazone). The acetylated polyamines are known to be rapidly degraded by polyamine oxidase producing putrescine. Direct evidence that a substantial part of the increase in the content of putrescine in the liver of rats treated with methylglyoxal bis(guanylhydrazone) occurs via the induction of this acetylase/oxidase pathway was obtained. These results indicate that methylglyoxal bis(guanylhydrazone) affects cellular polyamine levels not only by means of its inhibitory effect on S-adenosylmethionine decarboxylase and diamine oxidase but also by the induction of spermidine/spermine N1-acetyltransferase. They also raise the possibility that the enormous increase in this enzyme which occurs with higher doses may contribute to the very severe toxicity of methylglyoxal bis(guanylhydrazone).
Developmental exposure to 50 parts-per-billion arsenic influences histone modifications and associated epigenetic machinery in a region- and sex-specific manner in the adult mouse brain
International Nuclear Information System (INIS)
Tyler, Christina R.; Hafez, Alexander K.; Solomon, Elizabeth R.; Allan, Andrea M.
Epidemiological studies report that arsenic exposure via drinking water adversely impacts cognitive development in children and, in adults, can lead to greater psychiatric disease susceptibility, among other conditions. While it is known that arsenic toxicity has a profound effect on the epigenetic landscape, very few studies have investigated its effects on chromatin architecture in the brain. We have previously demonstrated that exposure to a low level of arsenic (50 ppb) during all three trimesters of fetal/neonatal development induces deficits in adult hippocampal neurogenesis in the dentate gyrus (DG), depressive-like symptoms, and alterations in gene expression in the adult mouse brain. As epigenetic processes control these outcomes, here we assess the impact of our developmental arsenic exposure (DAE) paradigm on global histone posttranslational modifications and associated chromatin-modifying proteins in the dentate gyrus and frontal cortex (FC) of adult male and female mice. DAE influenced histone 3 K4 trimethylation with increased levels in the male DG and FC and decreased levels in the female DG (no change in female FC). The histone methyltransferase MLL exhibited a similar sex- and region-specific expression profile as H3K4me3 levels, while histone demethylase KDM5B expression trended in the opposite direction. DAE increased histone 3 K9 acetylation levels in the male DG along with histone acetyltransferase (HAT) expression of GCN5 and decreased H3K9ac levels in the male FC along with decreased HAT expression of GCN5 and PCAF. DAE decreased expression of histone deacetylase enzymes HDAC1 and HDAC2, which were concurrent with increased H3K9ac levels but only in the female DG. Levels of H3 and H3K9me3 were not influenced by DAE in either brain region of either sex. These findings suggest that exposure to a low, environmentally relevant level of arsenic during development leads to long-lasting changes in histone methylation and acetylation in the adult
Simplified Method for Rapid Purification of Soluble Histones
Directory of Open Access Journals (Sweden)
Nives Ivić
Full Text Available Functional and structural studies of histone-chaperone complexes, nucleosome modifications, their interactions with remodelers and regulatory proteins rely on obtaining recombinant histones from bacteria. In the present study, we show that co-expression of Xenopus laevis histone pairs leads to production of soluble H2AH2B heterodimer and (H3H42 heterotetramer. The soluble histone complexes are purified by simple chromatographic techniques. Obtained H2AH2B dimer and H3H4 tetramer are proficient in histone chaperone binding and histone octamer and nucleosome formation. Our optimized protocol enables rapid purification of multiple soluble histone variants with a remarkable high yield and simplifies histone octamer preparation. We expect that this simple approach will contribute to the histone chaperone and chromatin research. This work is licensed under a Creative Commons Attribution 4.0 International License.
Dynamic intramolecular regulation of the histone chaperone nucleoplasmin controls histone binding and release.
Warren, Christopher; Matsui, Tsutomu; Karp, Jerome M; Onikubo, Takashi; Cahill, Sean; Brenowitz, Michael; Cowburn, David; Girvin, Mark; Shechter, David
Nucleoplasmin (Npm) is a highly conserved histone chaperone responsible for the maternal storage and zygotic release of histones H2A/H2B. Npm contains a pentameric N-terminal core domain and an intrinsically disordered C-terminal tail domain. Though intrinsically disordered regions are common among histone chaperones, their roles in histone binding and chaperoning remain unclear. Using an NMR-based approach, here we demonstrate that the Xenopus laevis Npm tail domain controls the binding of histones at its largest acidic stretch (A2) via direct competition with both the C-terminal basic stretch and basic nuclear localization signal. NMR and small-angle X-ray scattering (SAXS) structural analyses allowed us to construct models of both the tail domain and the pentameric complex. Functional analyses demonstrate that these competitive intramolecular interactions negatively regulate Npm histone chaperone activity in vitro. Together these data establish a potentially generalizable mechanism of histone chaperone regulation via dynamic and specific intramolecular shielding of histone interaction sites.
Organ distribution of histones after intravenous infusion of FITC histones or after sepsis.
Fattahi, Fatemeh; Grailer, Jamison J; Jajou, Lawrence; Zetoune, Firas S; Andjelkovic, Anuska V; Ward, Peter A
Histones appear in plasma during infectious or non-infectious sepsis and are associated with multiorgan injury. In the current studies, intravenous infusion of histones resulted in their localization in major organs. In vitro exposure of mouse macrophages to histones caused a buildup of histones on cell membranes followed by localization into cytosol and into the nucleus. After polymicrobial sepsis (cecal ligation and puncture), histones appeared in plasma as well as in a multiorgan pattern, peaking at 8 h followed by decline. In lungs, histones and neutrophils appeared together, with evidence for formation of neutrophil extracellular traps (NETs), which represent an innate immune response to trap and kill bacteria and other infectious agents. In liver, there was intense NET formation, featuring linear patterns containing histones and strands of DNA. When neutrophils were activated in vitro with C5a or phorbol myristate acetate, NET formation ensued. While formation of NETs represents entrapment and killing of infectious agents, the simultaneous release from neutrophils of histones often results in tissue/organ damage.
Organ Distribution of Histones after Intravenous Infusion of FITC-Histones or after Sepsis
Fattahi, Fatemeh; Grailer, Jamison J.; Jajou, Lawrence; Zetoune, Firas S.; Andjelkovic, Anuska V.; Ward, Peter A.
Histones appear in plasma during infectious or non-infectious sepsis and are associated with multiorgan injury. In the current studies, intravenous infusion of histones resulted in their localization in major organs. In vitro exposure of mouse macrophages to histones caused a buildup of histones on cell membranes followed by localization into cytosol and into the nucleus. After polymicrobial sepsis (cecal ligation and puncture, CLP), histones appeared in plasma as well as in a multiorgan pattern, peaking at 8 hr followed by decline. In lungs, histones and neutrophils appeared together, with evidence for formation of neutrophil extracellular traps (NETs), which represent an innate immune response to trap and kill bacteria and other infectious agents. In liver, there was intense NET formation, featuring linear patterns containing histones and strands of DNA. When neutrophils were activated in vitro with C5a or phorbol myristate acetate, NET formation ensued. While formation of NETs represents entrapment and killing of infectious agents, the simultaneous release from neutrophils of histones often results in tissue/organ damage. PMID:25680340
Pattern of change in histone 3 lysine 9 acetylation and histone ...
Indian Academy of Sciences (India)
Aug 11, 2014 ... [Li Y., Wang J., Xie Y., Liu S. and Tian Y. 2014 Pattern of change in histone 3 lysine 9 acetylation and histone deacetylases in development ..... 2004 Expression and functional characterization of recombinant human HDAC1 and HDAC3. Life Sci. 74, 2693–. 2705. Li Y., Qi J., Liu K., Li B., Wang H. and Jia J.
Replication stress interferes with histone recycling and predeposition marking of new histones
DEFF Research Database (Denmark)
Jasencakova, Zuzana; Scharf, Annette N D; Ask, Katrine
To restore chromatin on new DNA during replication, recycling of histones evicted ahead of the fork is combined with new histone deposition. The Asf1 histone chaperone, which buffers excess histones under stress, is a key player in this process. Yet how histones handled by human Asf1 are modified...... remains unclear. Here we identify marks on histones H3-H4 bound to Asf1 and changes induced upon replication stress. In S phase, distinct cytosolic and nuclear Asf1b complexes show ubiquitous H4K5K12diAc and heterogeneous H3 marks, including K9me1, K14ac, K18ac, and K56ac. Upon acute replication arrest......, the predeposition mark H3K9me1 and modifications typical of chromatin accumulate in Asf1 complexes. In parallel, ssDNA is generated at replication sites, consistent with evicted histones being trapped with Asf1. During recovery, histones stored with Asf1 are rapidly used as replication resumes. This shows...
Effect of Increased Yeast Alcohol Acetyltransferase Activity on Flavor Profiles of Wine and Distillates
Lilly, M.; Lambrechts, M. G.; Pretorius, I. S.
The distinctive flavor of wine, brandy, and other grape-derived alcoholic beverages is affected by many compounds, including esters produced during alcoholic fermentation. The characteristic fruity odors of the fermentation bouquet are primarily due to a mixture of hexyl acetate, ethyl caproate (apple-like aroma), iso-amyl acetate (banana-like aroma), ethyl caprylate (apple-like aroma), and 2-phenylethyl acetate (fruity, flowery flavor with a honey note). The objective of this study was to investigate the feasibility of improving the aroma of wine and distillates by overexpressing one of the endogenous yeast genes that controls acetate ester production during fermentation. The synthesis of acetate esters by the wine yeast Saccharomyces cerevisiae during fermentation is ascribed to at least three acetyltransferase activities, namely, alcohol acetyltransferase (AAT), ethanol acetyltransferase, and iso-amyl AAT. To investigate the effect of increased AAT activity on the sensory quality of Chenin blanc wines and distillates from Colombar base wines, we have overexpressed the alcohol acetyltransferase gene (ATF1) of S. cerevisiae. The ATF1 gene, located on chromosome XV, was cloned from a widely used commercial wine yeast strain of S. cerevisiae, VIN13, and placed under the control of the constitutive yeast phosphoglycerate kinase gene (PGK1) promoter and terminator. Chromoblot analysis confirmed the integration of the modified copy of ATF1 into the genome of three commercial wine yeast strains (VIN7, VIN13, and WE228). Northern blot analysis indicated constitutive expression of ATF1 at high levels in these yeast transformants. The levels of ethyl acetate, iso-amyl acetate, and 2-phenylethyl acetate increased 3- to 10-fold, 3.8- to 12-fold, and 2- to 10-fold, respectively, depending on the fermentation temperature, cultivar, and yeast strain used. The concentrations of ethyl caprate, ethyl caprylate, and hexyl acetate only showed minor changes, whereas the acetic acid
Effect of increased yeast alcohol acetyltransferase activity on flavor profiles of wine and distillates.
Lilly, M; Lambrechts, M G; Pretorius, I S
The distinctive flavor of wine, brandy, and other grape-derived alcoholic beverages is affected by many compounds, including esters produced during alcoholic fermentation. The characteristic fruity odors of the fermentation bouquet are primarily due to a mixture of hexyl acetate, ethyl caproate (apple-like aroma), iso-amyl acetate (banana-like aroma), ethyl caprylate (apple-like aroma), and 2-phenylethyl acetate (fruity, flowery flavor with a honey note). The objective of this study was to investigate the feasibility of improving the aroma of wine and distillates by overexpressing one of the endogenous yeast genes that controls acetate ester production during fermentation. The synthesis of acetate esters by the wine yeast Saccharomyces cerevisiae during fermentation is ascribed to at least three acetyltransferase activities, namely, alcohol acetyltransferase (AAT), ethanol acetyltransferase, and iso-amyl AAT. To investigate the effect of increased AAT activity on the sensory quality of Chenin blanc wines and distillates from Colombar base wines, we have overexpressed the alcohol acetyltransferase gene (ATF1) of S. cerevisiae. The ATF1 gene, located on chromosome XV, was cloned from a widely used commercial wine yeast strain of S. cerevisiae, VIN13, and placed under the control of the constitutive yeast phosphoglycerate kinase gene (PGK1) promoter and terminator. Chromoblot analysis confirmed the integration of the modified copy of ATF1 into the genome of three commercial wine yeast strains (VIN7, VIN13, and WE228). Northern blot analysis indicated constitutive expression of ATF1 at high levels in these yeast transformants. The levels of ethyl acetate, iso-amyl acetate, and 2-phenylethyl acetate increased 3- to 10-fold, 3.8- to 12-fold, and 2- to 10-fold, respectively, depending on the fermentation temperature, cultivar, and yeast strain used. The concentrations of ethyl caprate, ethyl caprylate, and hexyl acetate only showed minor changes, whereas the acetic acid
Regulation of replication fork progression through histone supply and demand
DEFF Research Database (Denmark)
Groth, Anja; Corpet, Armelle; Cook, Adam J L
DNA replication in eukaryotes requires nucleosome disruption ahead of the replication fork and reassembly behind. An unresolved issue concerns how histone dynamics are coordinated with fork progression to maintain chromosomal stability. Here, we characterize a complex in which the human histone c...... progression and histone supply and demand.......1 chaperone function, histone supply, and replicative unwinding of DNA in chromatin. We propose that Asf1, as a histone acceptor and donor, handles parental and new histones at the replication fork via an Asf1-(H3-H4)-MCM2-7 intermediate and thus provides a means to fine-tune replication fork...
The histones of the endosymbiont alga of Peridinium balticum (Dinophyceae).
Rizzo, P J; Morris, R L; Zweidler, A
The histones of the endosymbiont nucleus of the binucleate dinoflagellate Peridinium balticum were characterized by amino acid analysis and peptide mapping, and compared to calf thymus histones. Using these and various other criteria we have identified two H1-like histones as well as the highly conserved histones H3 and H4. A 13,000 dalton component in sodium dodecyl sulphate (SDS) gels can be separated into two components in Triton-containing gels. We suggest that these histones (HPb1 and HPb2) correspond to the vertebrate histones H2A and H2B, respectively.
Histones bundle F-actin filaments and affect actin structure.
Directory of Open Access Journals (Sweden)
Edna Blotnick
Full Text Available Histones are small polycationic proteins complexed with DNA located in the cell nucleus. Upon apoptosis they are secreted from the cells and react with extracellular polyanionic compounds. Actin which is a polyanionic protein, is also secreted from necrotic cells and interacts with histones. We showed that both histone mixture (histone type III and the recombinant H2A histone bundles F-actin, increases the viscosity of the F-actin containing solution and polymerizes G-actin. The histone-actin bundles are relatively insensitive to increase of ionic strength, unlike other polycation, histatin, lysozyme, spermine and LL-37 induced F-actin bundles. The histone-actin bundles dissociate completely only in the presence of 300-400 mM NaCl. DNA, which competes with F-actin for histones, disassembles histone induced actin bundles. DNase1, which depolymerizes F- to G-actin, actively unbundles the H2A histone induced but slightly affects the histone mixture induced actin bundles. Cofilin decreases the amount of F-actin sedimented by low speed centrifugation, increases light scattering and viscosity of F-actin-histone mixture containing solutions and forms star like superstructures by copolymerizing G-actin with H2A histone. The results indicate that histones are tightly attached to F-actin by strong electrostatic and hydrophobic forces. Since both histones and F-actin are present in the sputum of patients with cystic fibrosis, therefore, the formation of the stable histone-actin bundles can contribute to the pathology of this disease by increasing the viscosity of the sputum. The actin-histone interaction in the nucleus might affect gene expression.
Histones bundle F-actin filaments and affect actin structure.
Blotnick, Edna; Sol, Asaf; Muhlrad, Andras
Histones are small polycationic proteins complexed with DNA located in the cell nucleus. Upon apoptosis they are secreted from the cells and react with extracellular polyanionic compounds. Actin which is a polyanionic protein, is also secreted from necrotic cells and interacts with histones. We showed that both histone mixture (histone type III) and the recombinant H2A histone bundles F-actin, increases the viscosity of the F-actin containing solution and polymerizes G-actin. The histone-actin bundles are relatively insensitive to increase of ionic strength, unlike other polycation, histatin, lysozyme, spermine and LL-37 induced F-actin bundles. The histone-actin bundles dissociate completely only in the presence of 300-400 mM NaCl. DNA, which competes with F-actin for histones, disassembles histone induced actin bundles. DNase1, which depolymerizes F- to G-actin, actively unbundles the H2A histone induced but slightly affects the histone mixture induced actin bundles. Cofilin decreases the amount of F-actin sedimented by low speed centrifugation, increases light scattering and viscosity of F-actin-histone mixture containing solutions and forms star like superstructures by copolymerizing G-actin with H2A histone. The results indicate that histones are tightly attached to F-actin by strong electrostatic and hydrophobic forces. Since both histones and F-actin are present in the sputum of patients with cystic fibrosis, therefore, the formation of the stable histone-actin bundles can contribute to the pathology of this disease by increasing the viscosity of the sputum. The actin-histone interaction in the nucleus might affect gene expression.
Histones induce rapid and profound thrombocytopenia in mice
Bhandari, Ashish A.
Histones are released from dying cells and contribute to antimicrobial defense during infection. However, extracellular histones are a double-edged sword because they also damage host tissue and may cause death. We studied the interactions of histones with platelets. Histones bound to platelets, induced calcium influx, and recruited plasma adhesion proteins such as fibrinogen to induce platelet aggregation. Hereby fibrinogen cross-linked histone-bearing platelets and triggered microaggregation. Fibrinogen interactions with αIIbβ3 integrins were not required for this process but were necessary for the formation of large platelet aggregates. Infused histones associated with platelets in vivo and caused a profound thrombocytopenia within minutes after administration. Mice lacking platelets or αIIbβ3 integrins were protected from histone-induced death but not from histone-induced tissue damage. Heparin, at high concentrations, prevented histone interactions with platelets and protected mice from histone-induced thrombocytopenia, tissue damage, and death. Heparin and histones are evolutionary maintained. Histones may combine microbicidal with prothrombotic properties to fight invading microbes and maintain hemostasis after injury. Heparin may provide an innate counter mechanism to neutralize histones and diminish collateral tissue damage. PMID:21700775
Histone Methylation and Epigenetic Silencing in Breast Cancer
National Research Council Canada - National Science Library
Simon, Jeffrey A; Lange, Carol A
.... EZH2 is a histone methyltransferase which modifies lysine-27 of histone H3 an epigenetic mark which is generally linked to gene silencing and is implicated in tumor suppressor silencing during breast cancer progression...
Histone H4 Lysine 20 methylation
DEFF Research Database (Denmark)
Jørgensen, Stine; Schotta, Gunnar; Sørensen, Claus Storgaard
of histones have emerged as key regulators of genomic integrity. Intense research during the past few years has revealed histone H4 lysine 20 methylation (H4K20me) as critically important for the biological processes that ensure genome integrity, such as DNA damage repair, DNA replication and chromatin...... compaction. The distinct H4K20 methylation states are mediated by SET8/PR-Set7 that catalyses monomethylation of H4K20, whereas SUV4-20H1 and SUV4-20H2 enzymes mediate further H4K20 methylation to H4K20me2 and H4K20me3. Disruption of these H4K20-specific histone methyltransferases leads to genomic...
The emerging functions of histone demethylases
DEFF Research Database (Denmark)
Agger, Karl; Christensen, Jesper; Cloos, Paul Ac
Epigenetic information refers to heritable changes in gene function that are stable between cell divisions but which is not a result of changes in the DNA sequence. Part of the epigenetic mechanism has been ascribed to modifications of histones or DNA that affects the transcription of specific...... genes. In this context, post-translational modifications of histone tails, in particular methylation of lysines, are regarded as important for the storage of epigenetic information. Regulation of this information plays an important role during cellular differentiation where cells with different...... characteristic features evolve from the same ancestor, despite identical genomic material. The characterization of several enzymes catalyzing histone lysine methylation have supported this concept by showing the requirement of these enzymes for normal development and their involvement in diseases such as cancer...
Chemical and semisynthesis of modified histones.
Maity, Suman Kumar; Jbara, Muhammad; Brik, Ashraf
Post-translational modifications (PTMs) of histones play critical roles in the epigenetic regulation of eukaryotic genome by directly altering the biophysical properties of chromatin or by recruiting effector proteins. The large number of PTMs and the inherent complexity in their population and signaling processes make it highly challenging to understand epigenetics-related processes. To address these challenges, accesses to homogeneously modified histones are obligatory. Over the last decade, synthetic protein chemists have been devising novel synthetic tools and applying state-of-the-art chemoselective ligation strategies to prepare precious materials useful in answering fundamental questions in this area. In this short review, we cover some of the recent breakthroughs in these directions in particular the synthesis and semi-synthesis of modified histones and their use to unravel the mysteries of epigenetics. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
Nickel compounds induce histone ubiquitination by inhibiting histone deubiquitinating enzyme activity
International Nuclear Information System (INIS)
Ke Qingdong; Ellen, Thomas P.; Costa, Max
Nickel (Ni) compounds are known carcinogens but underlying mechanisms are not clear. Epigenetic changes are likely to play an important role in nickel ion carcinogenesis. Previous studies have shown epigenetic effects of nickel ions, including the loss of histone acetylation and a pronounced increase in dimethylated H3K9 in nickel-exposed cells. In this study, we demonstrated that both water-soluble and insoluble nickel compounds induce histone ubiquitination (uH2A and uH2B) in a variety of cell lines. Investigations of the mechanism by which nickel increases histone ubiquitination in cells reveal that nickel does not affect cellular levels of the substrates of this modification, i.e., ubiquitin, histones, and other non-histone ubiquitinated proteins. In vitro ubiquitination and deubiquitination assays have been developed to further investigate possible effects of nickel on enzymes responsible for histone ubiquitination. Results from the in vitro assays demonstrate that the presence of nickel did not affect the levels of ubiquitinated histones in the ubiquitinating assay. Instead, the addition of nickel significantly prevents loss of uH2A and uH2B in the deubiquitinating assay, suggesting that nickel-induced histone ubiquitination is the result of inhibition of (a) putative deubiquitinating enzyme(s). Additional supporting evidence comes from the comparison of the response to nickel ions with a known deubiquitinating enzyme inhibitor, iodoacetamide (IAA). This study is the first to demonstrate such effects of nickel ions on histone ubiquitination. It also sheds light on the possible mechanisms involved in altering the steady state of this modification. The study provides further evidence that supports the notion that nickel ions alter epigenetic homeostasis in cells, which may lead to altered programs of gene expression and carcinogenesis
New N-Acetyltransferase Fold in the Structure and Mechanism of the Phosphonate Biosynthetic Enzyme FrbF
Energy Technology Data Exchange (ETDEWEB)
Bae, Brian; Cobb, Ryan E.; DeSieno, Matthew A.; Zhao, Huimin; Nair, Satish K. (UIUC)
The enzyme FrbF from Streptomyces rubellomurinus has attracted significant attention due to its role in the biosynthesis of the antimalarial phosphonate FR-900098. The enzyme catalyzes acetyl transfer onto the hydroxamate of the FR-900098 precursors cytidine 5'-monophosphate-3-aminopropylphosphonate and cytidine 5'-monophosphate-N-hydroxy-3-aminopropylphosphonate. Despite the established function as a bona fide N-acetyltransferase, FrbF shows no sequence similarity to any member of the GCN5-like N-acetyltransferase (GNAT) superfamily. Here, we present the 2.0 {angstrom} resolution crystal structure of FrbF in complex with acetyl-CoA, which demonstrates a unique architecture that is distinct from those of canonical GNAT-like acetyltransferases. We also utilized the co-crystal structure to guide structure-function studies that identified the roles of putative active site residues in the acetyltransferase mechanism. The combined biochemical and structural analyses of FrbF provide insights into this previously uncharacterized family of N-acetyltransferases and also provide a molecular framework toward the production of novel N-acyl derivatives of FR-900098.
Histones as mediators of host defense, inflammation and thrombosis
Hoeksema, Marloes; van Eijk, Martin; Haagsman, Henk P; Hartshorn, Kevan L
Histones are known for their ability to bind to and regulate expression of DNA. However, histones are also present in cytoplasm and extracellular fluids where they serve host defense functions and promote inflammatory responses. Histones are a major component of neutrophil extracellular traps that contribute to bacterial killing but also to inflammatory injury. Histones can act as antimicrobial peptides and directly kill bacteria, fungi, parasites and viruses, in vitro and in a variety of ani...
Biological significance of lysine mono-, di- and trimethylation on histone and non-histone proteins
International Nuclear Information System (INIS)
Perez-Burgos, L.
Histones are the proteins that compact DNA into the repeating unit of chromatin known as the nucleosome. The N-termini of histones are subject to a series of post-translational modifications, one of which is methylation. This modification is termed 'epigenetic' because it extends the information encoded in the genome. Lysines can be mono-, di- or tri-methylated at different positions on histones H1, H3 and H4. In order to study the biological role of histone lysine methylation, antibodies were generated against mono-, di- and trimethylated H3-K9 and H3-27. Indeed, different chromatin domains in the mouse nucleus are enriched in distinct forms of histone lysine methylation, such as pericentric heterochromatin and the inactive X chromosome. Interestingly, heterochromatin in Arabidopsis thaliana is enriched in the mono- and di-, but not the trimethylated form of H3-K9. Furthermore, there exists a hierarchy of epigenetic modifications in which H3-K9 trimethylation is found to be upstream of DNA methylation on mouse major satellites. Histone lysine methylation is also involved in gene regulation upon development. One example is the chicken 61538;-globin locus, a region of facultative chromatin that undergoes a loss of di- and trimethylated H3-K27 in mature red blood cells, concomitant with expression of the 61538;-globin genes. SET-domain proteins are enzymes that methylate histones, but some of them are also able to methylate non-histone substrates. In particular, p53 is methylated by Set9 on lysine 372, G9a and Glp-1 on lysine 373 and by Smyd2 on lysine 370. Smyd2 transcript levels are greatly increased upon irradiation and dimethylated p53-370 specifically binds to 53BP1, a protein involved in recognizing DNA double-stranded breaks upon ionizing radiation. These results argue for a novel role of p53-K370 methylation in the biology of DNA damage. In summary, lysine methylation is a post-translational modification that can occur both on histone and non-histone proteins
Comprehensive Catalog of Currently Documented Histone Modifications.
Zhao, Yingming; Garcia, Benjamin A
Modern techniques in molecular biology, genomics, and mass spectrometry-based proteomics have identified a large number of novel histone posttranslational modifications (PTMs), many of whose functions are still under intense investigation. Here, we catalog histone PTMs under two classes: first, those whose functions have been fairly well studied and, second, those PTMs that have been more recently identified but whose functions remain unclear. We hope that this will be a useful resource for researchers from all biological or technical backgrounds, aiding in their chromatin and epigenetic pursuits. Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.
Histone redistribution and conformational effect on chromatin induced by formaldehyde.
Polacow, I; Cabasso, L; Li, H J
Histone redistributions between endogenous DNA in calf thymus chromatin and exogenous DNA from Clostridium perfringens (69% A + T) or from Micrococcus luteus (30% A + T) induced by 0.6 M NaCl or by 2% formaldehyde were studied by thermal denaturation. The observed redistribution occurred on histone Hl when the exogenous DNA was (A + T)-richer than the DNA in chromatin, and when the mixture was exposed to 0.6 M NaCl or formaldehyde. When a (G + C)-richer DNA was added as the acceptor for histones, no substantial transfer of histones from chromatin DNA to exogenous DNA was found. Thus the activation energy of histone dissociation from chromatin DNA seems to be substantially lowered by 0.6 M NaCl or formaldehyde such that histones (mostly histone Hl) can be dissociated and bind the (A + T)-richer DNA and form a more stable complex. It is suggested that the formaldehyde effect on histones may be due to the loss of positive charges on lysine and arginin residues (probably more on lysine than on arginine) in histones after their rapid reaction with formaldehyde. Formaldehyde treatment of chromatin also distorts the DNA conformation, as revealed by circular dichroism (CD) studies. This structural effect occurs mainly on those base pairs bound by histones other than Hl, or within the chromatin subunit. Histone redistribution is treated as a thermodynamic phenomenon of histone binding to DNA. The validity of using formaldehyde to study chromatin structure is discussed.
Biochemical studies on histones of the central nervous system. 2
International Nuclear Information System (INIS)
Schmitt, M.; Matthies, H.
There are no qualitative differences in the electrophoretic patterns of histones from neurones and glia. A 25% increased acetylation rate is found in neutronal histones as compared to glial histones after incubation of chopped brain in a [ 14 C]-acetate containing medium. This result probably reflects different condensation states of the chromatins of both cell types, as demonstrated by electron microscopy. (author)
Distribution pattern of histone H3 phosphorylation at serine 10
Indian Academy of Sciences (India)
Histones are the major eukaryotic DNA-binding proteins. Posttranslational modifications on N-terminal tails of histones that form nucleosomes are often associated with distinct biological functions. Some theories suggest that one of these modifications, the phosphorylation of histone H3 at serine 10 (H3S10ph) plays a role ...
Histones as mediators of host defense, inflammation and thrombosis
Hoeksema, Marloes; Eijk, Martin van; Haagsman, Henk P; Hartshorn, Kevan L
Histones are known for their ability to bind to and regulate expression of DNA. However, histones are also present in cytoplasm and extracellular fluids where they serve host defense functions and promote inflammatory responses. Histones are a major component of neutrophil extracellular traps that
Phosphorylation of rat thymus histones, its control and the effects thereon of γ-irradiation
International Nuclear Information System (INIS)
Fonagy, A.; Ord, M.G.; Stocken, L.A.
The phosphate content of rat thymus histones was determined. As expected for a replicating tissue, histones 1 and 2B were more phosphorylated and had higher 32 P uptakes than did histones from resting liver nuclei; the other histones all showed 32 P uptake, but the phosphate content and uptake of histone 2A was about half that for liver histone 2A. When thymus nuclei were incubated in a slightly hypo-osmotic medium, non-histone proteins and phosphorylated histones were released into solution; this was enhanced if ATP was present in the medium. [γ- 32 P]ATP was incorporated into non-histone proteins, including Pl, and into the ADP-ribosylated form of histone 1; negligible 32 P was incorporated into the other, bound, histones. Histones 1 and 2B added to the incubation medium were extensively, and histones 2A and 4 slightly, phosphorylated. Histones released by increasing the ionic strength of the medium were phosphorylated. Added lysozyme and cytochrome c were neither bound nor phosphorylated, but added non-histone protein Pl was phosphorylated, causing other histones to be released from the nuclei, especially histones 2A and 3. The released histones were phosphorylated. γ-irradiation decreased 32 P uptake into the non-ADP-ribosylated histones 1 and 4; phosphorylation of histone 1 in vitro was unaffected. The importance of non-histone proteins, ATP availability and nuclear protein kinases to the control of histone phosphorylation in vivo is discussed. (author)
Role of extracellular histones in the cardiomyopathy of sepsis.
Kalbitz, Miriam; Grailer, Jamison J; Fattahi, Fatemeh; Jajou, Lawrence; Herron, Todd J; Campbell, Katherine F; Zetoune, Firas S; Bosmann, Markus; Sarma, J Vidya; Huber-Lang, Markus; Gebhard, Florian; Loaiza, Randall; Valdivia, Hector H; Jalife, José; Russell, Mark W; Ward, Peter A
The purpose of this study was to define the relationship in polymicrobial sepsis (in adult male C57BL/6 mice) between heart dysfunction and the appearance in plasma of extracellular histones. Procedures included induction of sepsis by cecal ligation and puncture and measurement of heart function using echocardiogram/Doppler parameters. We assessed the ability of histones to cause disequilibrium in the redox status and intracellular [Ca(2+)]i levels in cardiomyocytes (CMs) (from mice and rats). We also studied the ability of histones to disturb both functional and electrical responses of hearts perfused with histones. Main findings revealed that extracellular histones appearing in septic plasma required C5a receptors, polymorphonuclear leukocytes (PMNs), and the Nacht-, LRR-, and PYD-domains-containing protein 3 (NLRP3) inflammasome. In vitro exposure of CMs to histones caused loss of homeostasis of the redox system and in [Ca(2+)]i, as well as defects in mitochondrial function. Perfusion of hearts with histones caused electrical and functional dysfunction. Finally, in vivo neutralization of histones in septic mice markedly reduced the parameters of heart dysfunction. Histones caused dysfunction in hearts during polymicrobial sepsis. These events could be attenuated by histone neutralization, suggesting that histones may be targets in the setting of sepsis to reduce cardiac dysfunction. © FASEB.
Histones as mediators of host defense, inflammation and thrombosis.
Hoeksema, Marloes; van Eijk, Martin; Haagsman, Henk P; Hartshorn, Kevan L
Histones are known for their ability to bind to and regulate expression of DNA. However, histones are also present in cytoplasm and extracellular fluids where they serve host defense functions and promote inflammatory responses. Histones are a major component of neutrophil extracellular traps that contribute to bacterial killing but also to inflammatory injury. Histones can act as antimicrobial peptides and directly kill bacteria, fungi, parasites and viruses, in vitro and in a variety of animal hosts. In addition, histones can trigger inflammatory responses in some cases acting through Toll-like receptors or inflammasome pathways. Extracellular histones mediate organ injury (lung, liver), sepsis physiology, thrombocytopenia and thrombin generation and some proteins can bind histones and reduce these potentially harmful effects.
Partial microduplication in the histone acetyltransferase complex member KANSL1 is associated with congenital heart defects in 22q11.2 microdeletion syndrome patients.
León, Luis E; Benavides, Felipe; Espinoza, Karena; Vial, Cecilia; Alvarez, Patricia; Palomares, Mirta; Lay-Son, Guillermo; Miranda, Macarena; Repetto, Gabriela M
22q11.2 microdeletion syndrome (22q11.2DS) is the most common microdeletion disorder in humans, with an incidence of 1/4000 live births. It is caused by a heterozygous deletion of 1.5-3 Mb on chromosome region 22q11.2. Patients with the deletion present features that include neuropsychiatric problems, craniofacial abnormalities and cardiovascular malformations. However, the phenotype is highly variable and the factors related to the clinical heterogeneity are not fully understood. About 65% of patients with 22q11.2DS have congenital heart defects (CHD). The main goal of this study was to identify common CNVs in 22q11.2DS patients that could be associated with the incomplete penetrance of CHD. Analysis of genomic DNA from 253 patients with 22q11.2DS using array technology showed an association between a microduplication located in region 17q21.31 and CHD (p-value = 0.023, OR = 2.75, 95% CI = 1.17-7.03). This region includes the first three exons of KANSL1 gene. Bioinformatic analysis showed that KANSL1 and CRKL, a gene in the commonly deleted region of 22q11.2DS, are part of the same regulatory module in a miRNA-mRNA network. These results show that a KANSL1 microduplication, in combination with the 22q11.2 deletion, is associated with increased risk of CHD in these patients, suggesting that KANSL1 plays a role as a modifier gene in 22q11.2DS patients.
Immunolocalization of choline acetyltransferase of common type in the central brain mass of Octopus vulgaris.
Casini, A; Vaccaro, R; D'Este, L; Sakaue, Y; Bellier, J P; Kimura, H; Renda, T G
Acetylcholine, the first neurotransmitter to be identified in the vertebrate frog, is widely distributed among the animal kingdom. The presence of a large amount of acetylcholine in the nervous system of cephalopods is well known from several biochemical and physiological studies. However, little is known about the precise distribution of cholinergic structures due to a lack of a suitable histochemical technique for detecting acetylcholine. The most reliable method to visualize the cholinergic neurons is the immunohistochemical localization of the enzyme choline acetyltransferase, the synthetic enzyme of acetylcholine. Following our previous study on the distribution patterns of cholinergic neurons in the Octopus vulgaris visual system, using a novel antibody that recognizes choline acetyltransferase of the common type (cChAT), now we extend our investigation on the octopus central brain mass. When applied on sections of octopus central ganglia, immunoreactivity for cChAT was detected in cell bodies of all central brain mass lobes with the notable exception of the subfrontal and subvertical lobes. Positive varicosed nerves fibers where observed in the neuropil of all central brain mass lobes.
Immunolocalization of choline acetyltransferase of common type in the central brain mass of Octopus vulgaris
Directory of Open Access Journals (Sweden)
A. Casini
Full Text Available Acetylcholine, the first neurotransmitter to be identified in the vertebrate frog, is widely distributed among the animal kingdom. The presence of a large amount of acetylcholine in the nervous system of cephalopods is well known from several biochemical and physiological studies. However, little is known about the precise distribution of cholinergic structures due to a lack of a suitable histochemical technique for detecting acetylcholine. The most reliable method to visualize the cholinergic neurons is the immunohistochemical localization of the enzyme choline acetyltransferase, the synthetic enzyme of acetylcholine. Following our previous study on the distribution patterns of cholinergic neurons in the Octopus vulgaris visual system, using a novel antibody that recognizes choline acetyltransferase of the common type (cChAT, now we extend our investigation on the octopus central brain mass. When applied on sections of octopus central ganglia, immunoreactivity for cChAT was detected in cell bodies of all central brain mass lobes with the notable exception of the subfrontal and subvertical lobes. Positive varicosed nerves fibers where observed in the neuropil of all central brain mass lobes.
Cadmium Induces Histone H3 Lysine Methylation by Inhibiting Histone Demethylase Activity
Xiao, Chunlian; Liu, Yin; Xie, Chengfeng; Tu, Wei; Xia, Yujie; Costa, Max; Zhou, Xue
Cadmium is an established human lung carcinogen with weak mutagenicity. However, the mechanisms underlying cadmium-induced carcinogenesis remain obscure. It has been suggested that epigenetic mechanisms may play a role in cadmium-induced carcinogenesis. In this study, we investigated the effects of cadmium on histone methylation and histone demethylases, and the role of histone methylation in transformation of immortalized normal human bronchial epithelial (BEAS-2B) cells. Exposure to 0.625, 1.25, 2.5, and 5.0 μM of cadmium for 6, 24, and 48 h increased global trimethylated histone H3 on lysine 4 (H3K4me3) and dimethylated histone H3 on lysine 9 (H3K9me2) in BEAS-2B cells compared with untreated cells, and most of these changes remained after the removal of cadmium (P cadmium inhibited the activities of histone H3 on lysine 4 (H3K4) and histone H3 on lysine 9 (H3K9) demethylases which were detected by histone demethylation assay. However, there was no significant change in the protein levels of the H3K4 demethylase lysine-specific demethylase 5A (KDM5A) and the H3K9 demethylase lysine-specific demethylase 3A (KDM3A). Interestingly, during transformation of BEAS-2B cells by 20 weeks of exposure to 2.0 μM cadmium as assessed by anchorage-independent growth in soft agar, global H3K4me3, and H3K9me2 were significantly increased at 4 weeks (P cadmium increases global H3K4me3 and H3K9me2 by inhibiting the activities of histone demethylases, and aberrant histone methylation that occurs early (48 h) and at 4 weeks is associated with cadmium-induced transformation of BEAS-2B cells at the early stage. PMID:25673502
Replication-uncoupled histone deposition during adenovirus DNA replication.
Komatsu, Tetsuro; Nagata, Kyosuke
In infected cells, the chromatin structure of the adenovirus genome DNA plays critical roles in its genome functions. Previously, we reported that in early phases of infection, incoming viral DNA is associated with both viral core protein VII and cellular histones. Here we show that in late phases of infection, newly synthesized viral DNA is also associated with histones. We also found that the knockdown of CAF-1, a histone chaperone that functions in the replication-coupled deposition of histones, does not affect the level of histone H3 bound on viral chromatin, although CAF-1 is accumulated at viral DNA replication foci together with PCNA. Chromatin immunoprecipitation assays using epitope-tagged histone H3 demonstrated that histone variant H3.3, which is deposited onto the cellular genome in a replication-independent manner, is selectively associated with both incoming and newly synthesized viral DNAs. Microscopic analyses indicated that histones but not USF1, a transcription factor that regulates viral late gene expression, are excluded from viral DNA replication foci and that this is achieved by the oligomerization of the DNA binding protein (DBP). Taken together, these results suggest that histone deposition onto newly synthesized viral DNA is most likely uncoupled with viral DNA replication, and a possible role of DBP oligomerization in this replication-uncoupled histone deposition is discussed.
The Role of Extracellular Histones in Influenza Virus Pathogenesis.
Ashar, Harshini K; Mueller, Nathan C; Rudd, Jennifer M; Snider, Timothy A; Achanta, Mallika; Prasanthi, Maram; Pulavendran, Sivasami; Thomas, Paul G; Ramachandran, Akhilesh; Malayer, Jerry R; Ritchey, Jerry W; Rajasekhar, Rachakatla; Chow, Vincent T K; Esmon, Charles T; Teluguakula, Narasaraju
Although exaggerated host immune responses have been implicated in influenza-induced lung pathogenesis, the etiologic factors that contribute to these events are not completely understood. We previously demonstrated that neutrophil extracellular traps exacerbate pulmonary injury during influenza pneumonia. Histones are the major protein components of neutrophil extracellular traps and are known to have cytotoxic effects. Here, we examined the role of extracellular histones in lung pathogenesis during influenza. Mice infected with influenza virus displayed high accumulation of extracellular histones, with widespread pulmonary microvascular thrombosis. Occluded pulmonary blood vessels with vascular thrombi often exhibited endothelial necrosis surrounded by hemorrhagic effusions and pulmonary edema. Histones released during influenza induced cytotoxicity and showed strong binding to platelets within thrombi in infected mouse lungs. Nasal wash samples from influenza-infected patients also showed increased accumulation of extracellular histones, suggesting a possible clinical relevance of elevated histones in pulmonary injury. Although histones inhibited influenza growth in vitro, in vivo treatment with histones did not yield antiviral effects and instead exacerbated lung pathology. Blocking with antihistone antibodies caused a marked decrease in lung pathology in lethal influenza-challenged mice and improved protection when administered in combination with the antiviral agent oseltamivir. These findings support the pathogenic effects of extracellular histones in that pulmonary injury during influenza was exacerbated. Targeting histones provides a novel therapeutic approach to influenza pneumonia. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
Regulation of spermidine/spermine N1-acetyltransferase in L6 cells by polyamines and related compounds.
Erwin, B G; Pegg, A E
Exposure of rat L6 cells in culture to exogenous polyamines led to a very large increase in the activity of spermidine/spermine N1-acetyltransferase. Spermine was more potent than spermidine in bringing about this increase, but in both cases the elevated acetyltransferase activity increased the cellular conversion of spermidine into putrescine. The N1-acetyltransferase turned over very rapidly in the L6 cells, with a half-life of 9 min after spermidine and 18 min after spermine. A wide variety of synthetic polyamine analogues also brought about a substantial induction of spermidine/spermine N1-acetyltransferase activity. These included sym-norspermidine, sym-norspermine, sym-homospermidine, N4-substituted spermidine derivatives, 1,3,6-triaminohexane, 1,4,7-triaminoheptane and deoxyspergualin, which were comparable with spermidine in their potency, and N1N8-bis(ethyl)spermidine, N1N9-bis(ethyl)homospermidine, methylglyoxal bis(guanylhydrazone), ethylglyoxal bis(guanylhydrazone) and 1,1'-[(methylethanediylidene)dinitrilo]bis(3-amino-guanidine ), which were even more active than spermidine. It is suggested that these polyamine analogues may bring about a decrease in cellular polyamines not only by inhibiting biosynthesis but by stimulating the degradation of spermidine into putrescine. PMID:3800951
Histone deacetylase inhibitors in multiple myeloma
Directory of Open Access Journals (Sweden)
Sarah Deleu
Full Text Available Novel drugs such as bortezomib and high dose chemotherapy combined with stem cell transplantation improved the outcome of multiple myeloma patients in the past decade. However, multiple myeloma often remains incurable due to the development of drug resistance governed by the bone marrow micro-environment. Therefore targeting new pathways to overcome this resistance is needed. Histone deacetylase (HDAC inhibitors represent a new class of anti-myeloma agents. Inhibiting HDACs results in histone hyperacetylation and alterations in chromatine structure, which, in turn, cause growth arrest differentiation and/or apoptosis in several tumor cells. Here we summarize the molecular actions of HDACi as a single agent or in combination with other drugs in different in vitro and in vivo myeloma models and in (preclinical trials.
Histone H1--DNA interaction. On the mechanism of DNA strands crosslinking by histone H1.
Glotov, B O; Nikolaev, L G; Severin, E S
Crosslinking of DNA fibers by histone H1 or phosphorylated on Ser-37 histone H1, and by the individual fragments of the H1 polypeptide chain was studied by the method of turbidimetry. The dependence of the turbidity of DNA-protein complexes on the ionic strength in solution suggests that the condensation of H1.DNA complexes in vitro is apparently due to both specific histone-DNA interactions with the contribution of hydrogen and/or hydrophobic bonds and the formation of polycationic "bridges" fastening the DNA fibers. The effectiveness of the condensation is postulated to be a function of a proportion between the two mechanisms which in turn can be controlled by slight changes in ionic surroundings. The sharp dependence of shrinkage of H1.DNA complexes on ionic strength at "physiological" salt concentrations could provide a mechanism to regulate density and consequently the total activity of chromatin in the cell nuclei. The phosphorylation of histone H1 on Ser-37 by a specific histone kinase does not noticeably affect the pattern of DNA crosslinking by the H1. Images PMID:27766
Biochemical Analysis Reveals the Multifactorial Mechanism of Histone H3 Clipping by Chicken Liver Histone H3 Protease
Chauhan, Sakshi
Proteolytic clipping of histone H3 has been identified in many organisms. Despite several studies, the mechanism of clipping, the substrate specificity, and the significance of this poorly understood epigenetic mechanism are not clear. We have previously reported histone H3 specific proteolytic clipping and a protein inhibitor in chicken liver. However, the sites of clipping are still not known very well. In this study, we attempt to identify clipping sites in histone H3 and to determine the mechanism of inhibition by stefin B protein, a cysteine protease inhibitor. By employing site-directed mutagenesis and in vitro biochemical assays, we have identified three distinct clipping sites in recombinant human histone H3 and its variants (H3.1, H3.3, and H3t). However, post-translationally modified histones isolated from chicken liver and Saccharomyces cerevisiae wild-type cells showed different clipping patterns. Clipping of histone H3 N-terminal tail at three sites occurs in a sequential manner. We have further observed that clipping sites are regulated by the structure of the N-terminal tail as well as the globular domain of histone H3. We also have identified the QVVAG region of stefin B protein to be very crucial for inhibition of the protease activity. Altogether, our comprehensive biochemical studies have revealed three distinct clipping sites in histone H3 and their regulation by the structure of histone H3, histone modifications marks, and stefin B.
Identification and characterization of the genes encoding the core histones and histone variants of Neurospora crassa.
Hays, Shan M; Swanson, Johanna; Selker, Eric U
We have identified and characterized the complete complement of genes encoding the core histones of Neurospora crassa. In addition to the previously identified pair of genes that encode histones H3 and H4 (hH3 and hH4-1), we identified a second histone H4 gene (hH4-2), a divergently transcribed pair of genes that encode H2A and H2B (hH2A and hH2B), a homolog of the F/Z family of H2A variants (hH2Az), a homolog of the H3 variant CSE4 from Saccharomyces cerevisiae (hH3v), and a highly diverged ...
novel 6'-n-aminoglycoside acetyltransferase AAC(6')-Iaj from a clinical isolate of Pseudomonas aeruginosa.
Tada, Tatsuya; Miyoshi-Akiyama, Tohru; Shimada, Kayo; Shimojima, Masahiro; Kirikae, Teruo
Pseudomonas aeruginosa NCGM1588 has a novel chromosomal class 1 integron, In151, which includes the aac(6')-Iaj gene. The encoded protein, AAC(6')-Iaj, was found to consist of 184 amino acids, with 70% identity to AAC(6')-Ia. Escherichia coli transformed with a plasmid containing the aac(6')-Iaj gene acquired resistance to all aminoglycosides tested except gentamicin. Of note, aac(6')-Iaj contributed to the resistance to arbekacin. Thin-layer chromatography revealed that AAC(6')-Iaj acetylated all aminoglycosides tested except gentamicin. These findings indicated that AAC(6')-Iaj is a functional acetyltransferase that modifies the amino groups at the 6' positions of aminoglycosides and contributes to aminoglycoside resistance of P. aeruginosa NCGM1588, including arbekacin.
Cigarette Smoking, N-Acetyltransferase 2 Acetylation Status, and Bladder Cancer Risk
DEFF Research Database (Denmark)
Marcus, P.M.; Hayes, R.B.; Vineis, P.
Tobacco use is an established cause of bladder cancer. The ability to detoxify aromatic amines, which are present in tobacco and are potent bladder carcinogens, is compromised in persons with the N-acetyltransferase 2 slow acetylation polymorphism. The relationship of cigarette smoking with bladder...... cancer risk therefore has been hypothesized to be stronger among slow acetylators. The few studies to formally explore such a possibility have produced inconsistent results, however. To assess this potential gene-environment interaction in as many bladder cancer studies as possible and to summarize...... results, we conducted a meta-analysis using data from 16 bladder cancer studies conducted in the general population (n = 1999 cases), Most had been conducted in European countries. Because control subjects were unavailable for a number of these studies, we used a case-series design, which can be used...
The adipogenic acetyltransferase Tip60 targets activation function 1 of peroxisome proliferator-activated receptor gamma
DEFF Research Database (Denmark)
van Beekum, Olivier; Brenkman, Arjan B; Grøntved, Lars
The transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) plays a key role in the regulation of lipid and glucose metabolism in adipocytes, by regulating their differentiation, maintenance, and function. The transcriptional activity of PPARgamma is dictated by the set...... in cells, and through use of chimeric proteins, we established that coactivation by Tip60 critically depends on the N-terminal activation function 1 of PPARgamma, a domain involved in isotype-specific gene expression and adipogenesis. Chromatin immunoprecipitation experiments showed that the endogenous Tip...... of proteins with which this nuclear receptor interacts under specific conditions. Here we identify the HIV-1 Tat-interacting protein 60 (Tip60) as a novel positive regulator of PPARgamma transcriptional activity. Using tandem mass spectrometry, we found that PPARgamma and the acetyltransferase Tip60 interact...
Mechanism by which arylamine N-acetyltransferase 1 ablation causes insulin resistance in mice
DEFF Research Database (Denmark)
Camporez, João Paulo; Wang, Yongliang; Faarkrog, Kasper
A single-nucleotide polymorphism in the human arylamine N-acetyltransferase 2 (Nat2) gene has recently been identified as associated with insulin resistance in humans. To understand the cellular and molecular mechanisms by which alterations in Nat2 activity might cause insulin resistance, we...... examined murine ortholog Nat1 knockout (KO) mice. Nat1 KO mice manifested whole-body insulin resistance, which could be attributed to reduced muscle, liver, and adipose tissue insulin sensitivity. Hepatic and muscle insulin resistance were associated with marked increases in both liver and muscle...... adipose tissue, and hepatocytes. Taken together, these studies demonstrate that Nat1 deletion promotes reduced mitochondrial activity and is associated with ectopic lipid-induced insulin resistance. These results provide a potential genetic link among mitochondrial dysfunction with increased ectopic lipid...
Ligand promiscuity through the eyes of the aminoglycoside N3 acetyltransferase IIa.
Norris, Adrianne L; Serpersu, Engin H
Aminoglycoside-modifying enzymes (AGMEs) are expressed in many pathogenic bacteria and cause resistance to aminoglycoside (AG) antibiotics. Remarkably, the substrate promiscuity of AGMEs is quite variable. The molecular basis for such ligand promiscuity is largely unknown as there is not an obvious link between amino acid sequence or structure and the antibiotic profiles of AGMEs. To address this issue, this article presents the first kinetic and thermodynamic characterization of one of the least promiscuous AGMEs, the AG N3 acetyltransferase-IIa (AAC-IIa) and its comparison to two highly promiscuous AGMEs, the AG N3-acetyltransferase-IIIb (AAC-IIIb) and the AG phosphotransferase(3')-IIIa (APH). Despite having similar antibiotic selectivities, AAC-IIIb and APH catalyze different reactions and share no homology to one another. AAC-IIa and AAC-IIIb catalyze the same reaction and are very similar in both amino acid sequence and structure. However, they demonstrate strong differences in their substrate profiles and kinetic and thermodynamic properties. AAC-IIa and APH are also polar opposites in terms of ligand promiscuity but share no sequence or apparent structural homology. However, they both are highly dynamic and may even contain disordered segments and both adopt well-defined conformations when AGs are bound. Contrary to this AAC-IIIb maintains a well-defined structure even in apo form. Data presented herein suggest that the antibiotic promiscuity of AGMEs may be determined neither by the flexibility of the protein nor the size of the active site cavity alone but strongly modulated or controlled by the effects of the cosubstrate on the dynamic and thermodynamic properties of the enzyme. Copyright Copyright © 2013 The Protein Society.
Structural and functional characterization of TRI3 trichothecene 15-O-acetyltransferase from Fusarium sporotrichioides
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Garvey, Graeme S.; McCormick, Susan P.; Alexander, Nancy J.; Rayment, Ivan; (US-Agriculture); (UW)
Fusarium head blight is a devastating disease of cereal crops whose worldwide incidence is increasing and at present there is no satisfactory way of combating this pathogen or its associated toxins. There is a wide variety of trichothecene mycotoxins and they all contain a 12,13-epoxytrichothecene skeleton but differ in their substitutions. Indeed, there is considerable variation in the toxin profile across the numerous Fusarium species that has been ascribed to differences in the presence or absence of biosynthetic enzymes and their relative activity. This article addresses the source of differences in acetylation at the C15 position of the trichothecene molecule. Here, we present the in vitro structural and biochemical characterization of TRI3, a 15-O-trichothecene acetyltransferase isolated from F. sporotrichioides and the 'in vivo' characterization of Deltatri3 mutants of deoxynivalenol (DON) producing F. graminearum strains. A kinetic analysis shows that TRI3 is an efficient enzyme with the native substrate, 15-decalonectrin, but is inactive with either DON or nivalenol. The structure of TRI3 complexed with 15-decalonectrin provides an explanation for this specificity and shows that Tri3 and Tri101 (3-O-trichothecene acetyltransferase) are evolutionarily related. The active site residues are conserved across all sequences for TRI3 orthologs, suggesting that differences in acetylation at C15 are not due to differences in Tri3. The tri3 deletion mutant shows that acetylation at C15 is required for DON biosynthesis even though DON lacks a C15 acetyl group. The enzyme(s) responsible for deacetylation at the 15 position of the trichothecene mycotoxins have not been identified.
Mechanisms of transcriptional repression by histone lysine methylation
DEFF Research Database (Denmark)
Hublitz, Philip; Albert, Mareike; Peters, Antoine H F M
During development, covalent modification of both, histones and DNA contribute to the specification and maintenance of cell identity. Repressive modifications are thought to stabilize cell type specific gene expression patterns, reducing the likelihood of reactivation of lineage-unrelated genes......, transcription factor binding and the antagonizing activities of distinct epigenetic regulators such as histone methyltransferases (HMTs) and histone demethylases (HDMs). Subsequently, we compare chromatin signatures associated with different types of transcriptional outcomes from stable repression to highly...
Mutations in Histone Acetylase Modifier BRPF1 Cause an Autosomal-Dominant Form of Intellectual Disability with Associated Ptosis.
Mattioli, Francesca; Schaefer, Elise; Magee, Alex; Mark, Paul; Mancini, Grazia M; Dieterich, Klaus; Von Allmen, Gretchen; Alders, Marielle; Coutton, Charles; van Slegtenhorst, Marjon; Vieville, Gaëlle; Engelen, Mark; Cobben, Jan Maarten; Juusola, Jane; Pujol, Aurora; Mandel, Jean-Louis; Piton, Amélie
Intellectual disability (ID) is a common neurodevelopmental disorder exhibiting extreme genetic heterogeneity, and more than 500 genes have been implicated in Mendelian forms of ID. We performed exome sequencing in a large family affected by an autosomal-dominant form of mild syndromic ID with ptosis, growth retardation, and hypotonia, and we identified an inherited 2 bp deletion causing a frameshift in BRPF1 (c.1052_1053del) in five affected family members. BRPF1 encodes a protein modifier of two histone acetyltransferases associated with ID: KAT6A (also known as MOZ or MYST3) and KAT6B (MORF or MYST4). The mRNA transcript was not significantly reduced in affected fibroblasts and most likely produces a truncated protein (p.Val351Glyfs ∗ 8). The protein variant shows an aberrant cellular location, loss of certain protein interactions, and decreased histone H3K23 acetylation. We identified BRPF1 deletions or point mutations in six additional individuals with a similar phenotype. Deletions of the 3p25 region, containing BRPF1 and SETD5, cause a defined ID syndrome where most of the clinical features are attributed to SETD5 deficiency. We compared the clinical symptoms of individuals carrying mutations or small deletions of BRPF1 alone or SETD5 alone with those of individuals with deletions encompassing both BRPF1 and SETD5. We conclude that both genes contribute to the phenotypic severity of 3p25 deletion syndrome but that some specific features, such as ptosis and blepharophimosis, are mostly driven by BRPF1 haploinsufficiency. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.
Histones of Chlamydomonas reinhardtii. Synthesis, acetylation, and methylation
International Nuclear Information System (INIS)
Waterborg, J.H.; Robertson, A.J.; Tatar, D.L.; Borza, C.M.; Davie, J.R.
Histones of the green alga Chlamydomonas reinhardtii were prepared by a new method and fractionated by reversed-phase high-performance liquid chromatography. Acid-urea-Triton gel analysis and tritiated acetate labeling demonstrated high levels of steady-state acetylation for the single histone H3 protein, in contrast to low levels on histones H4 and H2B. Twenty percent of histone H3 is subject to dynamic acetylation with, on average, three acetylated lysine residues per protein molecule. Histone synthesis in light-dark-synchronized cultures was biphasic with pattern differences between two histone H1 variants, between two H2A variants, and between H2B and ubiquitinated H2B. Automated protein sequence analysis of histone H3 demonstrated a site-specific pattern of steady-state acetylation between 7 and 17% at five of the six amino-terminal lysines and of monomethylation between 5 and 81% at five of the eight amino-terminal lysines in a pattern that may limit dynamic acetylation. An algal histone H3 sequence was confirmed by protein sequencing with a since threonine as residue 28 instead of the serine(28)-alanine(29) sequence, present in all other known plant and animal H3 histones
Targeting post-translational modifications of histones for cancer therapy.
Hsu, Y-C; Hsieh, Y-H; Liao, C-C; Chong, L-W; Lee, C-Y; Yu, Y-L; Chou, R-H
Post-translational modifications (PTMs) on histones including acetylation, methylation, phosphorylation, citrullination, ubiquitination, ADP ribosylation, and sumoylation, play important roles in different biological events including chromatin dynamics, DNA replication, and transcriptional regulation. Aberrant histones PTMs leads to abnormal gene expression and uncontrolled cell proliferation, followed by development of cancers. Therefore, targeting the enzymes required for specific histone PTMs holds a lot of potential for cancer treatment. In this review article, we retrospect the latest studies in the regulations of acetylation, methylation, and phosphorylation of histones. We also summarize inhibitors/drugs that target these modifications for cancer treatment.
Rapid divergence of histones in Hydrozoa (Cnidaria) and evolution of a novel histone involved in DNA damage response in hydra.
Reddy, Puli Chandramouli; Ubhe, Suyog; Sirwani, Neha; Lohokare, Rasika; Galande, Sanjeev
Histones are fundamental components of chromatin in all eukaryotes. Hydra, an emerging model system belonging to the basal metazoan phylum Cnidaria, provides an ideal platform to understand the evolution of core histone components at the base of eumetazoan phyla. Hydra exhibits peculiar properties such as tremendous regenerative capacity, lack of organismal senescence and rarity of malignancy. In light of the role of histone modifications and histone variants in these processes it is important to understand the nature of histones themselves and their variants in hydra. Here, we report identification of the complete repertoire of histone-coding genes in the Hydra magnipapillata genome. Hydra histones were classified based on their copy numbers, gene structure and other characteristic features. Genomic organization of canonical histone genes revealed the presence of H2A-H2B and H3-H4 paired clusters in high frequency and also a cluster with all core histones along with H1. Phylogenetic analysis of identified members of H2A and H2B histones suggested rapid expansion of these groups in Hydrozoa resulting in the appearance of unique subtypes. Amino acid sequence level comparisons of H2A and H2B forms with bilaterian counterparts suggest the possibility of a highly mobile nature of nucleosomes in hydra. Absolute quantitation of transcripts confirmed the high copy number of histones and supported the canonical nature of H2A. Furthermore, functional characterization of H2A.X.1 and a unique variant H2A.X.2 in the gastric region suggest their role in the maintenance of genome integrity and differentiation processes. These findings provide insights into the evolution of histones and their variants in hydra. Copyright © 2017 Elsevier GmbH. All rights reserved.
DNA and factor VII-activating protease protect against the cytotoxicity of histones
Marsman, Gerben; von Richthofen, Helen; Bulder, Ingrid; Lupu, Florea; Hazelzet, Jan; Luken, Brenda M.; Zeerleder, Sacha
Circulating histones have been implicated as major mediators of inflammatory disease because of their strong cytotoxic effects. Histones form the protein core of nucleosomes; however, it is unclear whether histones and nucleosomes are equally cytotoxic. Several plasma proteins that neutralize
Open and Closed: The Roles of Linker Histones in Plants and Animals
Over, Ryan S.; Michaels, Scott D.
Linker histones play key roles alongside core histones in the regulation and maintenance of chromatin. Here, we illustrate our current understanding of the contributions of linker histones to the cell cycle, development, and chromatin structure in plants and animals.
Pattern of change in histone 3 lysine 9 acetylation and histone ...
Indian Academy of Sciences (India)
Aug 11, 2014 ... Kim Y. S., Kim M. J., Koo T. H., Kim J. D., Koun S., Ham H. J. et al. 2012 Histone deacetylase is required for the activation of Wnt/β- catenin signaling crucial for heart valve formation in zebrafish embryos. Biochem. Biophys. Res. Commun. 423, 140–146. Kratz A., Arner E., Saito R., Kubosaki A., Kawai J., ...
Antifungal properties of wheat histones (H1-H4) and purified wheat histone H1
Wheat (Triticum sp.) histones H1, H2, H3, and H4 were extracted. H1 was further purified. Their activities against fungi with varying degrees of wheat pathogenicity were determined. They included Aspergillus flavus, A. fumigatus, A. niger, F. oxysporum, F. verticillioides, F. solani, F. graminearu...
Structural Basis of Histone Demethylase KDM6B Histone 3 Lysine 27 Specificity
DEFF Research Database (Denmark)
Jones, Sarah E; Olsen, Lars; Gajhede, Michael
KDM subfamily 6 enzymes KDM6A and KDM6B specifically catalyze demethylation of di- and trimethylated lysine on histone 3 lysine 27 (H3K27me3/2) and play an important role in repression of developmental genes. Despite identical amino acid sequence in the immediate surroundings of H3K9me3/2 (ARKS...
histoneHMM : Differential analysis of histone modifications with broad genomic footprints
Heinig, Matthias; Colomé-Tatché, Maria; Taudt, Aaron; Rintisch, Carola; Schafer, Sebastian; Pravenec, Michal; Hubner, Norbert; Vingron, Martin; Johannes, Frank
Background: ChIP-seq has become a routine method for interrogating the genome-wide distribution of various histone modifications. An important experimental goal is to compare the ChIP-seq profiles between an experimental sample and a reference sample, and to identify regions that show differential
Erasing the methyl mark: histone demethylases at the center of cellular differentiation and disease
DEFF Research Database (Denmark)
Cloos, Paul A C; Christensen, Jesper; Agger, Karl
The enzymes catalyzing lysine and arginine methylation of histones are essential for maintaining transcriptional programs and determining cell fate and identity. Until recently, histone methylation was regarded irreversible. However, within the last few years, several families of histone...... demethylases erasing methyl marks associated with gene repression or activation have been identified, underscoring the plasticity and dynamic nature of histone methylation. Recent discoveries have revealed that histone demethylases take part in large multiprotein complexes synergizing with histone deacetylases......, histone methyltransferases, and nuclear receptors to control developmental and transcriptional programs. Here we review the emerging biochemical and biological functions of the histone demethylases and discuss their potential involvement in human diseases, including cancer....
Chemical Probes of Histone Lysine Methyltransferases
Growing evidence suggests that histone methyltransferases (HMTs, also known as protein methyltransferases (PMTs)) play an important role in diverse biological processes and human diseases by regulating gene expression and the chromatin state. Therefore, HMTs have been increasingly recognized by the biomedical community as a class of potential therapeutic targets. High quality chemical probes of HMTs, as tools for deciphering their physiological functions and roles in human diseases and testing therapeutic hypotheses, are critical for advancing this promising field. In this review, we focus on the discovery, characterization, and biological applications of chemical probes for HMTs. PMID:25423077
Histone methylation and aging: Lessons learned from model systems
McCauley, Brenna S.; Dang, Weiwei
Aging induces myriad cellular and, ultimately, physiological changes that cause a decline in an organism's functional capabilities. Although the aging process and pathways that regulate it have been extensively studied, only in the last decade have we begun to appreciate that dynamic histone methylation may contribute to this process. In this review, we discuss recent work implicating histone methylation in aging. Loss of certain histone methyltransferases and demethylases changes lifespan in invertebrates, and alterations in histone methylation in aged organisms regulate lifespan and aging phenotypes, including oxidative stress-induced hormesis in yeast, insulin signaling in Caenorhabiditis elegans and mammals, and the senescence-associated secretory phenotype in mammals. In all cases where histone methylation has been shown to impact aging and aging phenotypes, it does so by regulating transcription, suggesting that this is a major mechanism of its action in this context. Histone methylation additionally regulates or is regulated by other cellular pathways that contribute to or combat aging. Given the numerous processes that regulate aging and histone methylation, and are in turn regulated by them, the role of histone methylation in aging is almost certainly underappreciated. PMID:24859460
The histone H5 variant in Xenopus laevis
Moorman, A. F.; de Boer, P. A.; Linders, M. T.; Charles, R.
The presumptive histone H5 of Xenopus laevis has been characterized by SDS and acid-urea-Triton polyacrylamide gel electrophoresis and compared with chicken histone H5. Chicken H5 has a lower electrophoretic mobility compared to that of Xenopus H5 in both gel systems. It is shown, using a polyclonal
Histone H1 regulates chromatin condensation in Leishmania parasites.
Masina, Slavica; Zangger, Haroun; Rivier, Denis; Fasel, Nicolas
We investigated the functional role of the Leishmania histone H1 and demonstrate for the first time that addition of histone H1 has a strong effect on microccocal digestion, chromatin condensation of parasite nuclei and that its overexpression can modulate parasite infectivity in vivo.
Histone lysine demethylases as targets for anticancer therapy
DEFF Research Database (Denmark)
Højfeldt, Jonas W; Agger, Karl; Helin, Kristian
interesting drug targets. The successful introduction of DNA methylation and histone deacetylase (HDAC) inhibitors for the treatment of specific subtypes of cancer has paved the way for the use of epigenetic therapy. Here, we highlight key biological findings demonstrating the roles of members of the histone...
Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones.
Liokatis, Stamatios
Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications (PTMs). They are newly identified species with unknown means of establishment and functional implications. Current analytical methods are inadequate to detect the copy-specific occurrence of PTMs on the nucleosomal sister histones. This protocol presents a biochemical method for the in vitro reconstitution of nucleosomes containing differentially isotope-labeled sister histones. The generated complex can be also asymmetrically modified, after including a premodified histone pool during refolding of histone subcomplexes. These asymmetric nucleosome preparations can be readily reacted with histone-modifying enzymes to study modification cross-talk mechanisms imposed by the asymmetrically pre-incorporated PTM using nuclear magnetic resonance (NMR) spectroscopy. Particularly, the modification reactions in real-time can be mapped independently on the two sister histones by performing different types of NMR correlation experiments, tailored for the respective isotope type. This methodology provides the means to study crosstalk mechanisms that contribute to the formation and propagation of asymmetric PTM patterns on nucleosomal complexes.
Biotinylation is a natural, albeit rare, modification of human histones
Kuroishi, Toshinobu; Rios-Avila, Luisa; Pestinger, Valerie; Wijeratne, Subhashinee S. K.; Zempleni, Janos
Previous studies suggest that histones H3 and H4 are posttranslationally modified by binding of the vitamin biotin, catalyzed by holocarboxylase synthetase (HCS). Albeit a rare epigenetic mark, biotinylated histones were repeatedly shown to be enriched in repeat regions and repressed loci, participating in the maintenance of genome stability and gene regulation. Recently, a team of investigators failed to detect biotinylated histones and proposed that biotinylation is not a natural modification of histones, but rather an assay artifact. Here, we describe the results of experiments, including the comparison of various analytical protocols, antibodies, cell lines, classes of histones, and radiotracers. These studies provide unambiguous evidence that biotinylation is a natural, albeit rare, histone modification. Less than 0.001% of human histones H3 and H4 are biotinylated, raising concerns that the abundance might too low to elicit biological effects in vivo. We integrated information from this study, previous studies, and ongoing research efforts to present a new working model in which biological effects are caused by a role of HCS in multiprotein complexes in chromatin. In this model, docking of HCS in chromatin causes the occasional binding of biotin to histones as a tracer for HCS binding sites. PMID:21930408
Heparin defends against the toxicity of circulating histones in sepsis.
Wang, Feifei; Zhang, Naipu; Li, Biru; Liu, Lanbo; Ding, Lei; Wang, Ying; Zhu, Yimin; Mo, Xi; Cao, Qing
Although circulating histones were demonstrated as major mediators of death in septic mice models, their roles in septic patients are not clarified. The present study sought to evaluate the clinical relevance of the circulating histone levels in septic children, and the antagonizing effects of heparin on circulating histones. Histone levels in the plasma of septic children were significantly higher than healthy controls, and positively correlated with disease severity. Histone treatment could activate NF-κB pathway of the endothelial cells and induce the secretion of large amount of cytokines that further amplify inflammation, subsequently leading to organ damage. Co-injection of low dose heparin with lethal dose histones could protect mouse from organ damage and death by antagonizing circulating histones, and similar effects were also observed in other septic models. Collectively, these findings indicated that circulating histones might serve as key factors in the pathogenesis of sepsis and their levels in plasma might be a marker for disease progression and prognosis. Furthermore, low dose heparin might be an effective therapy to hamper sepsis progression and reduce the mortality.
Escherichia coli N-Acetylglucosamine-1-Phosphate-Uridyltransferase/Glucosamine-1-Phosphate-Acetyltransferase (GlmU) Inhibitory Activity of Terreic Acid Isolated from Aspergillus terreus.
Sharma, Rashmi; Lambu, Mallikharjuna Rao; Jamwal, Urmila; Rani, Chitra; Chib, Reena; Wazir, Priya; Mukherjee, Debaraj; Chaubey, Asha; Khan, Inshad Ali
Secondary metabolite of Aspergillus terreus, terreic acid, is a reported potent antibacterial that was identified more than 60 years ago, but its cellular target(s) are still unknown. Here we screen its activity against the acetyltransferase domain of a bifunctional enzyme, Escherichia coli N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU). An absorbance-based assay was used to screen terreic acid against the acetyltransferase activity of E. coli GlmU. Terreic acid was found to inhibit the acetyltransferase domain of E. coli GlmU with an IC50 of 44.24 ± 1.85 µM. Mode of inhibition studies revealed that terreic acid was competitive with AcCoA and uncompetitive with GlcN-1-P. It also exhibited concentration-dependent killing of E. coli ATCC 25922 up to 4× minimum inhibitory concentration and inhibited the growth of biofilms generated by E. coli. Characterization of resistant mutants established mutation in the acetyltransferase domain of GlmU. Terreic acid was also found to be metabolically stable in the in vitro incubations with rat liver microsome in the presence of a NADPH regenerating system. The studies reported here suggest that terreic acid is a potent antimicrobial agent and support that E. coli GlmU acetyltransferase is a molecular target of terreic acid, resulting in its antibacterial activity. © 2016 Society for Laboratory Automation and Screening.
A Novel 6'-N-Aminoglycoside Acetyltransferase, AAC(6')-Ial, from a Clinical Isolate of Serratia marcescens.
Tada, Tatsuya; Miyoshi-Akiyama, Tohru; Shimada, Kayo; Dahal, Rajan K; Mishra, Shyam K; Ohara, Hiroshi; Kirikae, Teruo; Pokhrel, Bharat M
Serratia marcescens IOMTU115 has a novel 6'-N-aminoglycoside acetyltransferase-encoding gene, aac(6')-Ial. The encoded protein AAC(6')-Ial has 146 amino acids, with 91.8% identity to the amino acid sequence of AAC(6')-Ic in S. marcescens SM16 and 97.3% identity to the amino acid sequence of AAC(6')-Iap in S. marcescens WW4. The minimum inhibitory concentrations of aminoglycosides for Escherichia coli expressing AAC(6')-Ial were similar to those for E. coli expressing AAC(6')-Ic or AAC(6')-Iap. Thin-layer chromatography showed that AAC(6')-Ial, AAC(6')-Ic, or AAC(6')-Iap acetylated all the aminoglycosides tested, except for apramycin, gentamicin, and lividomycin. Kinetics assays revealed that AAC(6')-Ial is a functional acetyltransferase against aminoglycosides. The aac(6')-Ial gene was located on chromosomal DNA.
Immune activation by histones: plusses and minuses in inflammation.
Pisetsky, David S
Histones are highly cationic proteins that are essential components of the cell nucleus, interacting with DNA to form the nucleosome and regulating transcription. Histones, however, can transit from the cell nucleus during cell death and, once in an extracellular location, can serve as danger signals and activate immune cells. An article in this issue of the European Journal of Immunology [Eur. J. Immunol. 2013. 43: 3336-3342] reports that histones can activate monocyte-derived DCs via the NRLP3 inflammasome to induce the production of IL-1β. As such, histones, which can also stimulate TLRs, may drive events in the immunopathogenesis of a wide range of acute and chronic diseases marked by sterile inflammation. While the mechanism of this stimulation is not known, the positive charge of histones may provide a structural element to promote interaction with cells and activation of downstream signaling systems. © 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.
Acid-Urea Gel Electrophoresis and Western Blotting of Histones.
Hazzalin, Catherine A; Mahadevan, Louis C
Acid-urea gel electrophoresis offers significant advantages over SDS-PAGE for analysis of post-translational protein modifications, being capable of resolving proteins of similar size but varying in charge. Hence, it can be used to separate protein variants with small charge-altering differences in primary sequence, and is particularly useful in the analysis of histones whose charge variation arises from post-translational modification, such as phosphorylation or acetylation. On acid-urea gels, histones that carry multiple modifications, each with a characteristic charge, are resolved into distinct bands, the so-called "histone ladder." Thus, the extent and distribution of different modification states of histones can be visualized. Here, we describe the analysis of histone H3 by acid-urea gel electrophoresis and western blotting.
The role of extracellular histones in haematological disorders.
Alhamdi, Yasir; Toh, Cheng-Hock
Over the past decades, chromosomal alterations have been extensively investigated for their pathophysiological relevance in haematological malignancies. In particular, epigenetic modifications of intra-nuclear histones are now known as key regulators of healthy cell cycles that have also evolved into novel therapeutic targets for certain blood cancers. Thus, for most haematologists, histones are DNA-chained proteins that are buried deep within chromatin. However, the plot has deepened with recent revelations on the function of histones when unchained and released extracellularly upon cell death or from activated neutrophils as part of neutrophil extracellular traps (NETs). Extracellular histones and NETs are increasingly recognized for profound cytotoxicity and pro-coagulant effects. This article highlights the importance of recognizing this new paradigm of extracellular histones as a key player in host defence through its damage-associated molecular patterns, which could translate into novel diagnostic and therapeutic biomarkers in various haematological and critical disorders. © 2016 John Wiley & Sons Ltd.
New histone supply regulates replication fork speed and PCNA unloading
DEFF Research Database (Denmark)
Mejlvang, Jakob; Feng, Yunpeng; Alabert, Constance
Correct duplication of DNA sequence and its organization into chromatin is central to genome function and stability. However, it remains unclear how cells coordinate DNA synthesis with provision of new histones for chromatin assembly to ensure chromosomal stability. In this paper, we show...... that replication fork speed is dependent on new histone supply and efficient nucleosome assembly. Inhibition of canonical histone biosynthesis impaired replication fork progression and reduced nucleosome occupancy on newly synthesized DNA. Replication forks initially remained stable without activation...... of conventional checkpoints, although prolonged histone deficiency generated DNA damage. PCNA accumulated on newly synthesized DNA in cells lacking new histones, possibly to maintain opportunity for CAF-1 recruitment and nucleosome assembly. Consistent with this, in vitro and in vivo analysis showed that PCNA...
Co-expression of G2-EPSPS and glyphosate acetyltransferase GAT genes conferring high tolerance to glyphosate in soybean
Guo, Bingfu; Guo, Yong; Hong, Huilong; Jin, Longguo; Zhang, Lijuan; Chang, Ru-Zhen; Lu, Wei; Lin, Min; Qiu, Li-Juan
Glyphosate is a widely used non-selective herbicide with broad spectrum of weed control around the world. At present, most of the commercial glyphosate tolerant soybeans utilize glyphosate tolerant gene CP4-EPSPS or glyphosate acetyltransferase gene GAT separately. In this study, both glyphosate tolerant gene G2-EPSPS and glyphosate degraded gene GAT were co-transferred into soybean and transgenic plants showed high tolerance to glyphosate. Molecular analysis including PCR, Sothern blot, qRT-...
High-throughput screen identifies small molecule inhibitors targeting acetyltransferase activity of Mycobacterium tuberculosis GlmU.
Rani, Chitra; Mehra, Rukmankesh; Sharma, Rashmi; Chib, Reena; Wazir, Priya; Nargotra, Amit; Khan, Inshad Ali
N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) is a pivotal bifunctional enzyme, its N and C terminal domains catalyzes uridyltransferase and acetyltransferase activities, respectively. Final product of GlmU catalyzed reaction, uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc), acts as sugar donor providing GlcNAc residues in the synthesis of peptidoglycan and a disaccharide linker (D-N-GlcNAc-1-rhamnose), the key structural components of Mycobacterium tuberculosis (M. tuberculosis) cell wall. In the present study, we have searched new inhibitors against acetyltransferase activity of M. tuberculosis GlmU. A subset of 1607 synthetic compounds, selected through dual approach i.e., in-silico and whole cell screen against 20,000 compounds from ChemBridge library, was further screened using an in-vitro high throughput bioassay to identify inhibitors of acetyltransferase domain of M. tuberculosis GlmU. Four compounds were found to inhibit GlmU enzyme specific to acetyltransferase activity, with IC50 values ranging from 9 to 70 μM. Two compounds (6624116, 5655606) also exhibited whole cell activity against drug susceptible as well as drug resistant M. tuberculosis. These two compounds also exhibited increased anti-TB activity when tested in combination with rifampicin, isoniazid and ethambutol, however 5655606 was cytotoxic to eukaryotic cell line. These results demonstrate that identified chemical scaffolds can be used as inhibitors of M. tuberculosis cell wall enzyme after optimizations for future anti-TB drug development program. Copyright © 2015 Elsevier Ltd. All rights reserved.
Identification of critical residues of the serotype modifying O-acetyltransferase of Shigella flexneri
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Thanweer Farzaana
Full Text Available Abstract Background Thirteen serotypes of Shigella flexneri (S. flexneri have been recognised, all of which are capable of causing bacillary dysentery or shigellosis. With the emergence of the newer S. flexneri serotypes, the development of an effective vaccine has only become more challenging. One of the factors responsible for the generation of serotype diversity is an LPS O-antigen modifying, integral membrane protein known as O-acetyltransferase or Oac. Oac functions by adding an acetyl group to a specific O-antigen sugar, thus changing the antigenic signature of the parent S. flexneri strain. Oac is a membrane protein, consisting of hydrophobic and hydrophilic components. Oac bears homology to several known and predicted acetyltransferases with most homology existing in the N-terminal transmembrane (TM regions. Results In this study, the conserved motifs in the TM regions and in hydrophilic loops of S. flexneri Oac were targeted for mutagenesis with the aim of identifying the amino acid residues essential for the function of Oac. We previously identified three critical arginines–R73, R75 and R76 in the cytoplasmic loop 3 of Oac. Re-establishing that these arginines are critical, in this study we suggest a catalytic role for R73 and a structural role for R75 and R76 in O-acetylation. Serine-glycine motifs (SG 52–53, GS 138–139 and SYG 274–276, phenylalanine-proline motifs (FP 78–79 and FPV 282–84 and a tryptophan-threonine motif (WT141-142 found in TM segments and residues RK 110–111, GR 269–270 and D333 found in hydrophilic loops were also found to be critical to Oac function. Conclusions By studying the effect of the mutations on Oac's function and assembly, an insight into the possible roles played by the chosen amino acids in Oac was gained. The transmembrane serine-glycine motifs and hydrophilic residues (RK 110–111, GR 269–270 and D333 were shown to have an affect on Oac assembly which suggests a structural role
Posttranslational Modifications of the Histone 3 Tail and Their Impact on the Activity of Histone Lysine Demethylases In Vitro
DEFF Research Database (Denmark)
Lohse, Brian; Helgstrand, Charlotte; Andersson, Jan Legaard
Posttranslational modifications (PTMs) of the histone H3 tail such as methylation, acetylation and phosphorylation play important roles in epigenetic signaling. Here we study the effect of some of these PTMs on the demethylation rates of methylated lysine 9 in vitro using peptide substrates...... mimicking histone H3. Various combinations with other PTMs were employed to study possible cross-talk effects by comparing enzyme kinetic characteristics. We compared the kinetics of histone tail substrates for truncated histone lysine demethylases KDM4A and KDM4C containing only the catalytic core (cc...... toward bis-trimethylated substrates could be observed. Furthermore, a significant difference in the catalytic activity between dimethylated and trimethylated substrates was found for full length demethylases in line with what has been reported previously for truncated demethylases. Histone peptide...
Recombinant thrombomodulin protects mice against histone-induced lethal thromboembolism.
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Mayumi Nakahara
Full Text Available INTRODUCTION: Recent studies have shown that histones, the chief protein component of chromatin, are released into the extracellular space during sepsis, trauma, and ischemia-reperfusion injury, and act as major mediators of the death of an organism. This study was designed to elucidate the cellular and molecular basis of histone-induced lethality and to assess the protective effects of recombinant thrombomodulin (rTM. rTM has been approved for the treatment of disseminated intravascular coagulation (DIC in Japan, and is currently undergoing a phase III clinical trial in the United States. METHODS: Histone H3 levels in plasma of healthy volunteers and patients with sepsis and DIC were measured using enzyme-linked immunosorbent assay. Male C57BL/6 mice were injected intravenously with purified histones, and pathological examinations were performed. The protective effects of rTM against histone toxicity were analyzed both in vitro and in mice. RESULTS: Histone H3 was not detectable in plasma of healthy volunteers, but significant levels were observed in patients with sepsis and DIC. These levels were higher in non-survivors than in survivors. Extracellular histones triggered platelet aggregation, leading to thrombotic occlusion of pulmonary capillaries and subsequent right-sided heart failure in mice. These mice displayed symptoms of DIC, including thrombocytopenia, prolonged prothrombin time, decreased fibrinogen, fibrin deposition in capillaries, and bleeding. Platelet depletion protected mice from histone-induced death in the first 30 minutes, suggesting that vessel occlusion by platelet-rich thrombi might be responsible for death during the early phase. Furthermore, rTM bound to extracellular histones, suppressed histone-induced platelet aggregation, thrombotic occlusion of pulmonary capillaries, and dilatation of the right ventricle, and rescued mice from lethal thromboembolism. CONCLUSIONS: Extracellular histones cause massive
Mechanism by which arylamineN-acetyltransferase 1 ablation causes insulin resistance in mice.
Camporez, João Paulo; Wang, Yongliang; Faarkrog, Kasper; Chukijrungroat, Natsasi; Petersen, Kitt Falk; Shulman, Gerald I
A single-nucleotide polymorphism in the human arylamine N -acetyltransferase 2 (Nat2) gene has recently been identified as associated with insulin resistance in humans. To understand the cellular and molecular mechanisms by which alterations in Nat2 activity might cause insulin resistance, we examined murine ortholog Nat1 knockout (KO) mice. Nat1 KO mice manifested whole-body insulin resistance, which could be attributed to reduced muscle, liver, and adipose tissue insulin sensitivity. Hepatic and muscle insulin resistance were associated with marked increases in both liver and muscle triglyceride (TAG) and diacylglycerol (DAG) content, which was associated with increased PKCε activation in liver and increased PKCθ activation in skeletal muscle. Nat1 KO mice also displayed reduced whole-body energy expenditure and reduced mitochondrial oxygen consumption in white adipose tissue, brown adipose tissue, and hepatocytes. Taken together, these studies demonstrate that Nat1 deletion promotes reduced mitochondrial activity and is associated with ectopic lipid-induced insulin resistance. These results provide a potential genetic link among mitochondrial dysfunction with increased ectopic lipid deposition, insulin resistance, and type 2 diabetes. Copyright © 2017 the Author(s). Published by PNAS.
The aspirin metabolite salicylate inhibits lysine acetyltransferases and MUC1 induced epithelial to mesenchymal transition.
Fernandez, Harvey R; Lindén, Sara K
MUC1 is a transmembrane mucin that can promote cancer progression, and its upregulation correlates with a worse prognosis in colon cancer. We examined the effects of overexpression of MUC1 in colon cancer cells, finding that it induced epithelial to mesenchymal transition (EMT), including enhanced migration and invasion, and increased Akt phosphorylation. When the clones were treated with the aspirin metabolite salicylate, Akt phosphorylation was decreased and EMT inhibited. As the salicylate motif is necessary for the activity of the lysine acetyltransferase (KAT) inhibitor anacardic acid, we hypothesized these effects were associated with the inhibition of KAT activity. This was supported by anacardic acid treatment producing the same effect on EMT. In vitro KAT assays confirmed that salicylate directly inhibited PCAF/Kat2b, Tip60/Kat5 and hMOF/Kat8, and this inhibition was likely involved in the reversal of EMT in the metastatic prostate cancer cell line PC-3. Salicylate treatment also inhibited EMT induced by cytokines, illustrating the general effect it had on this process. The inhibition of both EMT and KATs by salicylate presents a little explored activity that could explain some of the anti-cancer effects of aspirin.
Molecular Evolution of Aralkylamine N-Acetyltransferase in Fish: A Genomic Survey
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Jia Li
Full Text Available All living organisms synchronize biological functions with environmental changes; melatonin plays a vital role in regulating daily and seasonal variations. Due to rhythmic activity of the timezyme aralkylamine N-acetyltransferase (AANAT, the blood level of melatonin increases at night and decreases during daytime. Whereas other vertebrates have a single form of AANAT, bony fishes possess various isoforms of aanat genes, though the reasons are still unclear. Here, we have taken advantage of multiple unpublished teleost aanat sequences to explore and expand our understanding of the molecular evolution of aanat in fish. Our results confirm that two rounds of whole-genome duplication (WGD led to the existence of three fish isoforms of aanat, i.e., aanat1a, aanat1b, and aanat2; in addition, gene loss led to the absence of some forms from certain special fish species. Furthermore, we suggest the different roles of two aanat1s in amphibious mudskippers, and speculate that the loss of aanat1a, may be related to terrestrial vision change. Several important sites of AANAT proteins and regulatory elements of aanat genes were analyzed for structural comparison and functional forecasting, respectively, which provides insights into the molecular evolution of the differences between AANAT1 and AANAT2.
In Silico Identification and Characterization of N-Terminal Acetyltransferase Genes of Poplar (Populus trichocarpa
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Hang-Yong Zhu
Full Text Available N-terminal acetyltransferase (Nats complex is responsible for protein N-terminal acetylation (Nα-acetylation, which is one of the most common covalent modifications of eukaryotic proteins. Although genome-wide investigation and characterization of Nat catalytic subunits (CS and auxiliary subunits (AS have been conducted in yeast and humans they remain unexplored in plants. Here we report on the identification of eleven genes encoding eleven putative Nat CS polypeptides, and five genes encoding five putative Nat AS polypeptides in Populus. We document that the expansion of Nat CS genes occurs as duplicated blocks distributed across 10 of the 19 poplar chromosomes, likely only as a result of segmental duplication events. Based on phylogenetic analysis, poplar Nat CS were assigned to six subgroups, which corresponded well to the Nat CS types (CS of Nat A–F, being consistent with previous reports in humans and yeast. In silico analysis of microarray data showed that in the process of normal development of the poplar, their Nat CS and AS genes are commonly expressed at one relatively low level but share distinct tissue-specific expression patterns. This exhaustive survey of Nat genes in poplar provides important information to assist future studies on their functional role in poplar.
Human Neural Stem Cells Overexpressing Choline Acetyltransferase Restore Unconditioned Fear in Rats with Amygdala Injury
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Kyungha Shin
Full Text Available Amygdala is involved in the fear memory that recognizes certain environmental cues predicting threatening events. Manipulation of neurotransmission within the amygdala affects the expression of conditioned and unconditioned emotional memories such as fear freezing behaviour. We previously demonstrated that F3.ChAT human neural stem cells (NSCs overexpressing choline acetyltransferase (ChAT improve cognitive function of Alzheimer's disease model rats with hippocampal or cholinergic nerve injuries by increasing acetylcholine (ACh level. In the present study, we examined the effect of F3.ChAT cells on the deficit of unconditioned fear freezing. Rats given N-methyl-d-aspartate (NMDA in their amygdala 2 weeks prior to cat odor exposure displayed very short resting (freezing time compared to normal animals. NMDA induced neuronal degeneration in the amygdala, leading to a decreased ACh concentration in cerebrospinal fluid. However, intracerebroventricular transplantation of F3.ChAT cells attenuated amygdala lesions 4 weeks after transplantation. The transplanted cells were found in the NMDA-injury sites and produced ChAT protein. In addition, F3.ChAT-receiving rats recuperated freezing time staying remote from the cat odor source, according to the recovery of brain ACh concentration. The results indicate that human NSCs overexpressing ChAT may facilitate retrieval of unconditioned fear memory by increasing ACh level.
Andrographolide: A potent antituberculosis compound that targets Aminoglycoside 2'-N-acetyltransferase in Mycobacterium tuberculosis.
Prabu, Amudha; Hassan, Sameer; Prabuseenivasan; Shainaba, A S; Hanna, L E; Kumar, Vanaja
Tuberculosis (TB) still remains a major challenging infectious disease. The increased rate of emergence of multi-drug resistant and extensively-drug resistant strains of the organism has further complicated the situation, resulting in an urgent need for new anti-TB drugs. Antimycobacterial activity of Andrographis paniculata was evaluated using a rapid LRP assay and the probable targets were identified by docking analysis. The methanolic extract of A. paniculata showed maximum antimycobacterial activity at 250μg/ml against all the tested strains of M. tuberculosis (H37Rv, MDR, and drug sensitive). Based on bioassay guided fractionation, andrographolide was identified as the potent molecule. With the docking analysis, both ICDH (Isocitrate Dehydrogenase) and AAC (Aminoglycoside 2'-N-acetyltransferase) were predicted as targets of andrographolide in M. tuberculosis. Molecular simulation revealed that, ICDH showed low binding affinity to andrographolide. However, for AAC, the andrographolide was observed to be well within the active site after 10ns of molecular simulation. This suggests that ACC (PDB ID 1M4I) could be the probable target for andrographolide. Copyright © 2015 Elsevier Inc. All rights reserved.
Novel ligands of Choline Acetyltransferase designed by in silico molecular docking, hologram QSAR and lead optimization
Kumar, Rajnish; Långström, Bengt; Darreh-Shori, Taher
Recent reports have brought back the acetylcholine synthesizing enzyme, choline acetyltransferase in the mainstream research in dementia and the cholinergic anti-inflammatory pathway. Here we report, a specific strategy for the design of novel ChAT ligands based on molecular docking, Hologram Quantitative Structure Activity Relationship (HQSAR) and lead optimization. Molecular docking was performed on a series of ChAT inhibitors to decipher the molecular fingerprint of their interaction with the active site of ChAT. Then robust statistical fragment HQSAR models were developed. A library of novel ligands was generated based on the pharmacophoric and shape similarity scoring function, and evaluated in silico for their molecular interactions with ChAT. Ten of the top scoring invented compounds are reported here. We confirmed the activity of α-NETA, the only commercially available ChAT inhibitor, and one of the seed compounds in our model, using a new simple colorimetric ChAT assay (IC50 ~ 88 nM). In contrast, α-NETA exhibited an IC50 of ~30 μM for the ACh-degrading cholinesterases. In conclusion, the overall results may provide useful insight for discovering novel ChAT ligands and potential positron emission tomography tracers as in vivo functional biomarkers of the health of central cholinergic system in neurodegenerative disorders, such as Alzheimer's disease.
In vitro inhibition of choline acetyltransferase by a series of 2-benzylidene-3-quinuclidinones
International Nuclear Information System (INIS)
Capacio, B.R.
Ten substituted 2-benzylidene-3-quinuclidinones were synthesized and evaluated for their relative potency as in vitro inhibitors of choline acetyltransferase (ChAT). Acetylcholine (ACh) synthesis was followed radiometrically by the incorporation of labeled acetate originating from 14 C-acetyl-CoA. Woolf-Augustinsson-Hofstee data analysis was used to calculate Vmax, Km, and Ki values. The inhibition was found to be noncompetitive or uncompetitive with respect to choline. Quantitative structure activity relationship correlations demonstrated a primary dependence on κ-σ, as well as steric properties of the substituted benzene ring. Additional radiometric and spectrophotometric were performed with 2-(3'-methyl)-benzylidene-3-quinuclidinone, one of the more potent analogs, to further elucidate the inhibitory mechanism. ChAT-mediated cleavage of ACh was measured spectrophotometrically by following the appearance of NADH at 340 nanometers in an enzyme coupled assay. Lineweaver-Burk analysis indicated mixed or uncompetitive inhibition with respect to both substrates of the forward reaction, suggesting interference with a rate limiting step
Distinct Localization of Peripheral and Central Types of Choline Acetyltransferase in the Rat Cochlea
International Nuclear Information System (INIS)
Kitanishi, Tsuyoshi; Aimi, Yoshinari; Kitano, Hiroya; Suzuki, Mikio; Kimura, Hiroshi; Saito, Atsushi; Shimizu, Takeshi; Tooyama, Ikuo
We previously discovered a splice variant of choline acetyltransferase (ChAT) mRNA, and designated the variant protein pChAT because of its preferential expression in peripheral neuronal structures. In this study, we examined the immunohistochemical localization of pChAT in rat cochlea and compared the distribution pattern to those of common ChAT (cChAT) and acetylcholinesterase. Some neuronal cell bodies and fibers in the spiral ganglia showed immunoreactivity for pChAT, predominantly the small spiral ganglion cells, indicating outer hair cell type II neurons. In contrast, cChAT- and acetylcholinesterase-positive structures were localized to fibers and not apparent in ganglion cells. After ablation of the cochlear nuclei, many pChAT-positive cochlear nerve fibers became clearly visible, whereas fibers immunopositive for cChAT and acetylcholine esterase disappeared. These results suggested that pChAT and cChAT are localized in different systems of the rat cochlea; pChAT in the afferent and cChAT in the efferent structures
Biogenic-amine acetylation: an additional function of the N-acetyltransferase from Fasciola hepatica.
Aisien, S O; Walter, R D
The previously described polyamine N-acetyltransferase from Fasciola hepatica has been observed to have an additional function, the acetylation of biogenic amines. The activities for biogenic amines, diamines and polyamines were in a constant ratio throughout the purification process. Biogenic amines found to be substrates for the enzyme included tyramine, tryptamine, beta-phenylethylamine and histamine, with Km values of 0.12 mM, 0.26 mM, 0.30 mM and 0.76 mM respectively. Octopamine, 5-hydroxytryptamine and alpha-phenylethylamine were also acceptable as substrates, though to a lesser degree. The optimum pH for biogenic-amine acetylation was 7.5, and CoA was inhibitory to the process, with a Ki of 5.5 microM. N-Acetylation appears to play a major role in the amine metabolism of this trematode. We presume that acetylation represents the process by which the parasite inactivates excess amines. PMID:8489501
Choline acetyltransferase in the hippocampus is associated with learning strategy preference in adult male rats.
Hawley, Wayne R; Witty, Christine F; Daniel, Jill M; Dohanich, Gary P
One principle of the multiple memory systems hypothesis posits that the hippocampus-based and striatum-based memory systems compete for control over learning. Consistent with this notion, previous research indicates that the cholinergic system of the hippocampus plays a role in modulating the preference for a hippocampus-based place learning strategy over a striatum-based stimulus--response learning strategy. Interestingly, in the hippocampus, greater activity and higher protein levels of choline acetyltransferase (ChAT), the enzyme that synthesizes acetylcholine, are associated with better performance on hippocampus-based learning and memory tasks. With this in mind, the primary aim of the current study was to determine if higher levels of ChAT and the high-affinity choline uptake transporter (CHT) in the hippocampus were associated with a preference for a hippocampus-based place learning strategy on a task that also could be solved by relying on a striatum-based stimulus--response learning strategy. Results confirmed that levels of ChAT in the dorsal region of the hippocampus were associated with a preference for a place learning strategy on a water maze task that could also be solved by adopting a stimulus-response learning strategy. Consistent with previous studies, the current results support the hypothesis that the cholinergic system of the hippocampus plays a role in balancing competition between memory systems that modulate learning strategy preference. Copyright © 2015 Elsevier B.V. All rights reserved.
Cloning and characterization of a serotonin N-acetyltransferase from a gymnosperm, loblolly pine (Pinus taeda).
Park, Sangkyu; Byeon, Yeong; Lee, Hyoung Yool; Kim, Young-Soon; Ahn, Taeho; Back, Kyoungwhan
Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis in both animals and plants. SNAT catalyzes serotonin into N-acetylserotonin, an immediate precursor for melatonin biosynthesis by N-acetylserotonin methyltransferase (ASMT). We cloned the SNAT gene from a gymnosperm loblolly pine (Pinus teada). The loblolly pine SNAT (PtSNAT) gene encodes 255 amino acids harboring a transit sequence with 67 amino acids and shows 67% amino acid identity with rice SNAT when comparing the mature polypeptide regions. Purified recombinant PtSNAT showed peak activity at 55°C with the K(m) (428 μM) and Vmax (3.9 nmol/min/mg protein) values. As predicted, PtSNAT localized to chloroplasts. The SNAT mRNA was constitutively expressed in all tissues, including leaf, bud, flower, and pinecone, whereas the corresponding protein was detected only in leaf. In accordance with the exclusive SNAT protein expression in leaf, melatonin was detected only in leaf at 0.45 ng per gram fresh weight. Sequence and phylogenetic analysis indicated that the gymnosperm PtSNAT had high homology with SNATs from all plant phyla (even with cyanobacteria), and formed a clade separated from the angiosperm SNATs, suggestive of direct gene transfer from cyanobacteria via endosymbiosis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
The chloramphenicol acetyltransferase vector as a tool for stable tagging of Neospora caninum.
Pereira, Luiz Miguel; Yatsuda, Ana Patrícia
Neospora caninum is an obligate intracellular Apicomplexa, a phylum where one of the current methods for functional studies relies on molecular genetic tools. For Toxoplasma gondii, the first method described, in 1993, was based on resistance against chloramphenicol. As in T. gondii, we developed a vector constituted of the chloramphenicol acetyltransferase gene (CAT) flanked by the N. caninum dihydrofolate reductase-thymidylate synthase (DHFR-TS) 5' coding sequence flanking region. Five weeks after transfection and under the selection of chloramphenicol the expression of CAT increased compared to the wild type and the resistance was retained for more than one year. Between the stop codon of CAT and the 3' UTR of DHFR, a Lac-Z gene controlled by the N. caninum tubulin 5' coding sequence flanking region was ligated, resulting in a vector with a reporter gene (Ncdhfr-CAT/NcTub-tetO/Lac-Z). The stability was maintained through an episomal pattern for 14 months when the tachyzoites succumbed, which was an unexpected phenomenon compared to T. gondii. Stable parasites expressing the Lac-Z gene allowed the detection of tachyzoites after invasion by enzymatic reaction (CPRG) and were visualised macro- and microscopically by X-Gal precipitation and fluorescence. This work developed the first vector for stable expression of proteins based on chloramphenicol resistance and controlled exclusively by N. caninum promoters. Copyright © 2014 Elsevier B.V. All rights reserved.
The deacetylase Sirt6 activates the acetyltransferase GCN5 and suppresses hepatic gluconeogenesis.
Dominy, John E; Lee, Yoonjin; Jedrychowski, Mark P; Chim, Helen; Jurczak, Michael J; Camporez, Joao Paulo; Ruan, Hai-Bin; Feldman, Jessica; Pierce, Kerry; Mostoslavsky, Raul; Denu, John M; Clish, Clary B; Yang, Xiaoyong; Shulman, Gerald I; Gygi, Steven P; Puigserver, Pere
Hepatic glucose production (HGP) maintains blood glucose levels during fasting but can also exacerbate diabetic hyperglycemia. HGP is dynamically controlled by a signaling/transcriptional network that regulates the expression/activity of gluconeogenic enzymes. A key mediator of gluconeogenic gene transcription is PGC-1α. PGC-1α's activation of gluconeogenic gene expression is dependent upon its acetylation state, which is controlled by the acetyltransferase GCN5 and the deacetylase Sirt1. Nevertheless, whether other chromatin modifiers-particularly other sirtuins-can modulate PGC-1α acetylation is currently unknown. Herein, we report that Sirt6 strongly controls PGC-1α acetylation. Surprisingly, Sirt6 induces PGC-1α acetylation and suppresses HGP. Sirt6 depletion decreases PGC-1α acetylation and promotes HGP. These acetylation effects are GCN5 dependent: Sirt6 interacts with and modifies GCN5, enhancing GCN5's activity. Lepr(db/db) mice, an obese/diabetic animal model, exhibit reduced Sirt6 levels; ectopic re-expression suppresses gluconeogenic genes and normalizes glycemia. Activation of hepatic Sirt6 may therefore be therapeutically useful for treating insulin-resistant diabetes. Copyright © 2012 Elsevier Inc. All rights reserved.
Arylamine N-acetyltransferase activity in bronchial epithelial cells and its inhibition by cellular oxidants
International Nuclear Information System (INIS)
Dairou, Julien; Petit, Emile; Ragunathan, Nilusha; Baeza-Squiban, Armelle; Marano, Francelyne; Dupret, Jean-Marie; Rodrigues-Lima, Fernando
Bronchial epithelial cells express xenobiotic-metabolizing enzymes (XMEs) that are involved in the biotransformation of inhaled toxic compounds. The activities of these XMEs in the lung may modulate respiratory toxicity and have been linked to several diseases of the airways. Arylamine N-acetyltransferases (NAT) are conjugating XMEs that play a key role in the biotransformation of aromatic amine pollutants such as the tobacco-smoke carcinogens 4-aminobiphenyl (4-ABP) and β-naphthylamine (β-NA). We show here that functional human NAT1 or its murine counterpart Nat2 are present in different lung epithelial cells i.e. Clara cells, type II alveolar cells and bronchial epithelial cells, thus indicating that inhaled aromatic amines may undergo NAT-dependent biotransformation in lung epithelium. Exposure of these cells to pathophysiologically relevant amounts of oxidants known to contribute to lung dysfunction, such as H 2 O 2 or peroxynitrite, was found to impair the NAT1/Nat2-dependent cellular biotransformation of aromatic amines. Genetic and non genetic impairment of intracellular NAT enzyme activities has been suggested to compromise the important detoxification pathway of aromatic amine N-acetylation and subsequently to contribute to an exacerbation of untoward effects of these pollutants on health. Our study suggests that oxidative/nitroxidative stress in lung epithelial cells, due to air pollution and/or inflammation, could contribute to local and/or systemic dysfunctions through the alteration of the functions of pulmonary NAT enzymes.
Saccharomyces cerevisiae Atf1p is an alcohol acetyltransferase and a thioesterase in vitro.
Nancolas, Bethany; Bull, Ian D; Stenner, Richard; Dufour, Virginie; Curnow, Paul
The alcohol-O-acyltransferases are bisubstrate enzymes that catalyse the transfer of acyl chains from an acyl-coenzyme A (CoA) donor to an acceptor alcohol. In the industrial yeast Saccharomyces cerevisiae this reaction produces acyl esters that are an important influence on the flavour of fermented beverages and foods. There is also a growing interest in using acyltransferases to produce bulk quantities of acyl esters in engineered microbial cell factories. However, the structure and function of the alcohol-O-acyltransferases remain only partly understood. Here, we recombinantly express, purify and characterize Atf1p, the major alcohol acetyltransferase from S. cerevisiae. We find that Atf1p is promiscuous with regard to the alcohol cosubstrate but that the acyltransfer activity is specific for acetyl-CoA. Additionally, we find that Atf1p is an efficient thioesterase in vitro with specificity towards medium-chain-length acyl-CoAs. Unexpectedly, we also find that mutating the supposed catalytic histidine (H191) within the conserved HXXXDG active site motif only moderately reduces the thioesterase activity of Atf1p. Our results imply a role for Atf1p in CoA homeostasis and suggest that engineering Atf1p to reduce the thioesterase activity could improve product yields of acetate esters from cellular factories. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.
Inference of Functionally-Relevant N-acetyltransferase Residues Based on Statistical Correlations.
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Andrew F Neuwald
Full Text Available Over evolutionary time, members of a superfamily of homologous proteins sharing a common structural core diverge into subgroups filling various functional niches. At the sequence level, such divergence appears as correlations that arise from residue patterns distinct to each subgroup. Such a superfamily may be viewed as a population of sequences corresponding to a complex, high-dimensional probability distribution. Here we model this distribution as hierarchical interrelated hidden Markov models (hiHMMs, which describe these sequence correlations implicitly. By characterizing such correlations one may hope to obtain information regarding functionally-relevant properties that have thus far evaded detection. To do so, we infer a hiHMM distribution from sequence data using Bayes' theorem and Markov chain Monte Carlo (MCMC sampling, which is widely recognized as the most effective approach for characterizing a complex, high dimensional distribution. Other routines then map correlated residue patterns to available structures with a view to hypothesis generation. When applied to N-acetyltransferases, this reveals sequence and structural features indicative of functionally important, yet generally unknown biochemical properties. Even for sets of proteins for which nothing is known beyond unannotated sequences and structures, this can lead to helpful insights. We describe, for example, a putative coenzyme-A-induced-fit substrate binding mechanism mediated by arginine residue switching between salt bridge and π-π stacking interactions. A suite of programs implementing this approach is available (psed.igs.umaryland.edu.
Inference of Functionally-Relevant N-acetyltransferase Residues Based on Statistical Correlations.
Neuwald, Andrew F; Altschul, Stephen F
Over evolutionary time, members of a superfamily of homologous proteins sharing a common structural core diverge into subgroups filling various functional niches. At the sequence level, such divergence appears as correlations that arise from residue patterns distinct to each subgroup. Such a superfamily may be viewed as a population of sequences corresponding to a complex, high-dimensional probability distribution. Here we model this distribution as hierarchical interrelated hidden Markov models (hiHMMs), which describe these sequence correlations implicitly. By characterizing such correlations one may hope to obtain information regarding functionally-relevant properties that have thus far evaded detection. To do so, we infer a hiHMM distribution from sequence data using Bayes' theorem and Markov chain Monte Carlo (MCMC) sampling, which is widely recognized as the most effective approach for characterizing a complex, high dimensional distribution. Other routines then map correlated residue patterns to available structures with a view to hypothesis generation. When applied to N-acetyltransferases, this reveals sequence and structural features indicative of functionally important, yet generally unknown biochemical properties. Even for sets of proteins for which nothing is known beyond unannotated sequences and structures, this can lead to helpful insights. We describe, for example, a putative coenzyme-A-induced-fit substrate binding mechanism mediated by arginine residue switching between salt bridge and π-π stacking interactions. A suite of programs implementing this approach is available (psed.igs.umaryland.edu).
Histone deacetylase inhibitors (HDACIs: multitargeted anticancer agents
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Ververis K
Full Text Available Katherine Ververis,1 Alison Hiong,1 Tom C Karagiannis,1,* Paul V Licciardi2,*1Epigenomic Medicine, Alfred Medical Research and Education Precinct, 2Allergy and Immune Disorders, Murdoch Childrens Research Institute, Melbourne, VIC, Australia*These authors contributed equally to this workAbstract: Histone deacetylase (HDAC inhibitors are an emerging class of therapeutics with potential as anticancer drugs. The rationale for developing HDAC inhibitors (and other chromatin-modifying agents as anticancer therapies arose from the understanding that in addition to genetic mutations, epigenetic changes such as dysregulation of HDAC enzymes can alter phenotype and gene expression, disturb homeostasis, and contribute to neoplastic growth. The family of HDAC inhibitors is large and diverse. It includes a range of naturally occurring and synthetic compounds that differ in terms of structure, function, and specificity. HDAC inhibitors have multiple cell type-specific effects in vitro and in vivo, such as growth arrest, cell differentiation, and apoptosis in malignant cells. HDAC inhibitors have the potential to be used as monotherapies or in combination with other anticancer therapies. Currently, there are two HDAC inhibitors that have received approval from the US FDA for the treatment of cutaneous T-cell lymphoma: vorinostat (suberoylanilide hydroxamic acid, Zolinza and depsipeptide (romidepsin, Istodax. More recently, depsipeptide has also gained FDA approval for the treatment of peripheral T-cell lymphoma. Many more clinical trials assessing the effects of various HDAC inhibitors on hematological and solid malignancies are currently being conducted. Despite the proven anticancer effects of particular HDAC inhibitors against certain cancers, many aspects of HDAC enzymes and HDAC inhibitors are still not fully understood. Increasing our understanding of the effects of HDAC inhibitors, their targets and mechanisms of action will be critical for the
histoneHMM: Differential analysis of histone modifications with broad genomic footprints
Czech Academy of Sciences Publication Activity Database
Heinig, M.; Colomé-Tatché, M.; Taudt, A.; Rintisch, C.; Schafer, S.; Pravenec, Michal; Hubner, N.; Vingron, M.; Johannes, F.
Roč. 16, Feb 22 (2015), s. 60 ISSN 1471-2105 R&D Projects: GA MŠk(CZ) 7E10067; GA ČR(CZ) GA13-04420S Institutional support: RVO:67985823 Keywords : ChIP - seq * histone modifications * Hidden Markov model * computational biology * differential analysis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.435, year: 2015
Aging and radiation induced alternations in liver histones
International Nuclear Information System (INIS)
Kozurkova, M.; Misurova, E.; Kropacova, K.
Age-related changes in histones in the liver of normal rats and in rats irradiated with 5.7 Gy gamma rays were examined. Quantitative histone changes in growing and aging rats (from 1 to 28 months of age) were found to be mild only. As they paralleled the DNA changes, the histone /DNA ratio remained stable with age. In total extracted histones there was a decrease in the H1 proportion in older groups with preceding increase in the H1 grad proportion. Thirty minutes after irradiation the amount of histones was reduced with age, probably due to an impaired extractability of histones. As the quantitative DNA changes were milder, the histone?DNA ratio decreased in aging liver after irradiation. Similar patterns of changes in proportion of the H1 fraction and H1 grad sub-fraction were observed in irradiated and nonirradiated animals in the former with an earlier onset. Irradiation, therefore, accelerated spontaneous age-related alternations. (author)
Novel chemokine-like activities of histones in tumor metastasis.
Chen, Ruochan; Xie, Yangchun; Zhong, Xiao; Fu, Yongmin; Huang, Yan; Zhen, Yixiang; Pan, Pinhua; Wang, Haichao; Bartlett, David L; Billiar, Timothy R; Lotze, Michael T; Zeh, Herbert J; Fan, Xue-Gong; Tang, Daolin; Kang, Rui
Histones are intracellular nucleosomal components and extracellular damage-associated molecular pattern molecules that modulate chromatin remodeling, as well as the immune response. However, their extracellular roles in cell migration and invasion remain undefined. Here, we demonstrate that histones are novel regulators of tumor metastasis with chemokine-like activities. Indeed, exogenous histones promote both hepatocellular carcinoma (HCC) cell migration and invasion through toll-like receptor (TLR)4, but not TLR2 or the receptor for advanced glycosylation end product. TLR4-mediated activation of nuclear factor-κB (NF-κB) by extracellular signal-regulated kinase (ERK) is required for histone-induced chemokine (e.g., C-C motif ligand 9/10) production. Pharmacological and genetic inhibition of TLR4-ERK-NF-κB signaling impairs histone-induced chemokine production and HCC cell migration. Additionally, TLR4 depletion (by using TLR4-/- mice and TLR4-shRNA) or inhibition of histone release/activity (by administration of heparin and H3 neutralizing antibody) attenuates lung metastasis of HCC cells injected via the tail vein of mice. Thus, histones promote tumor metastasis of HCC cells through the TLR4-NF-κB pathway and represent novel targets for treating patients with HCC.
Lateral Thinking: How Histone Modifications Regulate Gene Expression.
Lawrence, Moyra; Daujat, Sylvain; Schneider, Robert
The DNA of each cell is wrapped around histone octamers, forming so-called 'nucleosomal core particles'. These histone proteins have tails that project from the nucleosome and many residues in these tails can be post-translationally modified, influencing all DNA-based processes, including chromatin compaction, nucleosome dynamics, and transcription. In contrast to those present in histone tails, modifications in the core regions of the histones had remained largely uncharacterised until recently, when some of these modifications began to be analysed in detail. Overall, recent work has shown that histone core modifications can not only directly regulate transcription, but also influence processes such as DNA repair, replication, stemness, and changes in cell state. In this review, we focus on the most recent developments in our understanding of histone modifications, particularly those on the lateral surface of the nucleosome. This region is in direct contact with the DNA and is formed by the histone cores. We suggest that these lateral surface modifications represent a key insight into chromatin regulation in the cell. Therefore, lateral surface modifications form a key area of interest and a focal point of ongoing study in epigenetics. Copyright © 2015 Elsevier Ltd. All rights reserved.
Circulating Histones Are Mediators of Trauma-associated Lung Injury
Abrams, Simon T.; Zhang, Nan; Manson, Joanna; Liu, Tingting; Dart, Caroline; Baluwa, Florence; Wang, Susan Siyu; Brohi, Karim; Kipar, Anja; Yu, Weiping
Rationale: Acute lung injury is a common complication after severe trauma, which predisposes patients to multiple organ failure. This syndrome largely accounts for the late mortality that arises and despite many theories, the pathological mechanism is not fully understood. Discovery of histone-induced toxicity in mice presents a new dimension for elucidating the underlying pathophysiology. Objectives: To investigate the pathological roles of circulating histones in trauma-induced lung injury. Methods: Circulating histone levels in patients with severe trauma were determined and correlated with respiratory failure and Sequential Organ Failure Assessment (SOFA) scores. Their cause–effect relationship was studied using cells and mouse models. Measurements and Main Results: In a cohort of 52 patients with severe nonthoracic blunt trauma, circulating histones surged immediately after trauma to levels that were toxic to cultured endothelial cells. The high levels were significantly associated with the incidence of acute lung injury and SOFA scores, as well as markers of endothelial damage and coagulation activation. In in vitro systems, histones damaged endothelial cells, stimulated cytokine release, and induced neutrophil extracellular trap formation and myeloperoxidase release. Cellular toxicity resulted from their direct membrane interaction and resultant calcium influx. In mouse models, cytokines and markers for endothelial damage and coagulation activation significantly increased immediately after trauma or histone infusion. Pathological examinations showed that lungs were the predominantly affected organ with edema, hemorrhage, microvascular thrombosis, and neutrophil congestion. An anti-histone antibody could reduce these changes and protect mice from histone-induced lethality. Conclusions: This study elucidates a new mechanism for acute lung injury after severe trauma and proposes that circulating histones are viable therapeutic targets for improving survival
Treatment of Chronic Kidney Diseases with Histone Deacetylase Inhibitors
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Shougang eZhuang
Full Text Available Histone deacetylases (HDACs induce deacetylation of both histone and non-histone proteins and play a critical role in the modulation of physiological and pathological gene expression. Pharmacological inhibition of HDAC has been reported to attenuate progression of renal fibrogenesis in obstructed kidney and reduce cyst formation in polycystic kidney disease. HDAC inhibitors (HDACis are also able to ameliorate renal lesions in diabetes nephropathy, lupus nephritis, aristolochic acid nephropathy and transplant nephropathy. The beneficial effects of HDACis are associated with their anti-fibrosis, anti-inflammation, and immmunosuppressant effects. In this review, we summarize recent advances on the treatment of various chronic kidney diseases with HDACis in preclinical models.
DNA and factor VII–activating protease protect against the cytotoxicity of histones
Marsman, Gerben; von Richthofen, Helen; Bulder, Ingrid; Lupu, Florea; Hazelzet, Jan; Luken, Brenda M.
Circulating histones have been implicated as major mediators of inflammatory disease because of their strong cytotoxic effects. Histones form the protein core of nucleosomes; however, it is unclear whether histones and nucleosomes are equally cytotoxic. Several plasma proteins that neutralize histones are present in plasma. Importantly, factor VII–activating protease (FSAP) is activated upon contact with histones and subsequently proteolyzes histones. We aimed to determine the effect of FSAP on the cytotoxicity of both histones and nucleosomes. Indeed, FSAP protected against histone-induced cytotoxicity of cultured cells in vitro. Upon incubation of serum with histones, endogenous FSAP was activated and degraded histones, which also prevented cytotoxicity. Notably, histones as part of nucleosome complexes were not cytotoxic, whereas DNA digestion restored cytotoxicity. Histones in nucleosomes were inefficiently cleaved by FSAP, which resulted in limited cleavage of histone H3 and removal of the N-terminal tail. The specific isolation of either circulating nucleosomes or free histones from sera of Escherichia coli challenged baboons or patients with meningococcal sepsis revealed that histone H3 was present in the form of nucleosomes, whereas free histone H3 was not detected. All samples showed signs of FSAP activation. Markedly, we observed that all histone H3 in nucleosomes from the patients with sepsis, and most histone H3 from the baboons, was N-terminally truncated, giving rise to a similarly sized protein fragment as through cleavage by FSAP. Taken together, our results suggest that DNA and FSAP jointly limit histone cytotoxicity and that free histone H3 does not circulate in appreciable concentrations in sepsis. PMID:29296900
Chromatin Dynamics during Nucleotide Excision Repair: Histones on the Move
Adam, Salomé; Polo, Sophie E.
It has been a long-standing question how DNA damage repair proceeds in a nuclear environment where DNA is packaged into chromatin. Several decades of analysis combining in vitro and in vivo studies in various model organisms ranging from yeast to human have markedly increased our understanding of the mechanisms underlying chromatin disorganization upon damage detection and re-assembly after repair. Here, we review the methods that have been developed over the years to delineate chromatin alterations in response to DNA damage by focusing on the well-characterized Nucleotide Excision Repair (NER) pathway. We also highlight how these methods have provided key mechanistic insight into histone dynamics coupled to repair in mammals, raising new issues about the maintenance of chromatin integrity. In particular, we discuss how NER factors and central players in chromatin dynamics such as histone modifiers, nucleosome remodeling factors, and histone chaperones function to mobilize histones during repair. PMID:23109890
Histones and their phosphorylation during germination of rice seeds
International Nuclear Information System (INIS)
Iqbal Ahmed, C.M.; Padayatti, J.D.
Histones from nuclei of rice embryos were identified by their mobilities on 15% acid-urea polyacrylamide gel electrophoreogram, incorporation of ( 3 H)lysine and ( 14 C) arginine and lack of incorporation of ( 3 H)tryptophan. The ratio of histone to DNA in ungerminated embryos was 2.7 which decreased during germination reaching unity by 48 hr. There was preferential phosphorylation of lysine-rich histones, which paralleled the synthesis of DNA. In the presence of cytosine arabinoside and mitomycin-C, which inhibited the synthesis of DNA to the extend of 75-80% the phosphorylation of lysine-rich histone was reduced by 50-60% suggesting the dependence of phosphorylation on the ongoing synthesis of DNA. (auth.)
Physicochemical modifications of histones and their impact on epigenomics.
Andreoli, Federico; Del Rio, Alberto
The study of histone post-translational modifications (PTMs) has made extraordinary progress over the past few years and many epigenetic modifications have been identified and found to be associated with fundamental biological processes and pathological conditions. Most histone-modifying enzymes produce specific covalent modifications on histone tails that, taken together, elicit complex and concerted processes. An even higher level of complexity is generated by the action of small molecules that are able to modulate pharmacologically epigenetic enzymes and interfere with these biochemical mechanisms. In this article, we provide an overview of histone PTMs by reviewing and discussing them in terms of their physicochemical properties, emphasizing these concepts in view of recent research efforts to elucidate epigenetic mechanisms and devise future epigenetic drugs. Copyright © 2014 Elsevier Ltd. All rights reserved.
Benzodiazepines: rat pinealocyte binding sites and augmentation of norepinephrine-stimulated N-acetyltransferase activity
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Matthew, E.; Parfitt, A.G.; Sugden, D.; Engelhardt, D.L.; Zimmerman, E.A.; Klein, D.C.
Studies of (/sup 3/H)diazepam binding to intact rat pineal cells were carried out in tissue culture preparations. The binding was saturable, reversible and proportional to the number of cells used. Scatchard analysis resulted in a linear plot (Kd . 23 nM, maximum binding sites (Bmax) . 1.56 pmol/mg of protein for cells in monolayer culture; Kd . 7 nM, Bmax . 1.3 pmol/mg of protein for cells in suspension culture). Inhibition constants (Ki) for clonazepam (500 nM), flunitrazepam (38 nM) and Ro-5-4864 (5 nM) indicated that the binding sites were probably of the ''peripheral'' type. In addition, the effects of diazepam on norepinephrine-stimulated N-acetyltransferase (NAT) activity were studied in organ culture and dissociated cell culture. Diazepam (10-50 microM) both prolonged and increased the magnitude of the norepinephrine-induced increase in NAT activity but did not affect the initial rate of rise of enzyme activity. The effect was dose-dependent and was also seen with clonazepam, flunitrazepam and Ro-5-4864, but not with Ro-15-1788. Diazepam, by itself, at these concentrations, had no effect on NAT, but enzyme activity was increased by higher concentrations (0.1-1 mM). Although a relationship between the (/sup 3/H)diazepam binding sites described here and the effect of benzodiazepines on NAT cannot be established from these studies, the data suggest that the benzodiazepines may alter melatonin levels through their action on NAT.
Molecular and biochemical characterisation of a serine acetyltransferase of onion, Allium cepa (L.).
McManus, Michael T; Leung, Susanna; Lambert, Anya; Scott, Richard W; Pither-Joyce, Meeghan; Chen, Balance; McCallum, John
We have previously cloned a cDNA, designated SAT1, corresponding to a gene coding for a serine acetyltransferase (SAT) from onion (Allium cepa L.). The SAT1 locus was mapped to chromosome 7 of onion using a single-stranded conformation polymorphism (SSCP) in the 3' UTR of the gene. Northern analysis has demonstrated that expression of the SAT1 gene is induced in leaf tissue in response to low S-supply. Phylogenetic analysis has placed SAT1 in a strongly supported group (100% bootstrap) that comprises sequences that have been characterised biochemically, including Allium tuberosum, Spinacea oleracea, Glycine max, Citrullus vulgaris, and SAT5 (AT5g56760) of Arabidopsis thaliana. This group can be divided further with the SAT1 of A. cepa sequence grouping strongly with the A. tuberosum sequence. Translation of SAT1 from onion generates a protein of 289 amino acids with a calculated molecular mass of 30,573 Da and pI of 6.52. The conserved G277 and H282 residues that have been identified as critical for L-cysteine inhibition are observed at G272 and H277. SAT1 has been cloned into the pGEX plasmid, expressed in E. coli and SAT activity of the recombinant enzyme has been measured as acetyl-CoA hydrolysis detected at 232 nm. A Km of 0.72 mM was determined for l-serine as substrate, a Km of 92 microM was calculated with acetyl-CoA as substrate, and an inhibition curve for L-cysteine generated an IC50 value of 3.1 microM. Antibodies raised against the recombinant SAT1 protein recognised a protein of ca. 33 kDa in whole leaf onion extracts. These properties of the SAT1 enzyme from onion are compared with other SAT enzymes characterised from closely related species.
Arylamine N-acetyltransferase 2 (NAT2 genetic diversity and traditional subsistence: a worldwide population survey.
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Audrey Sabbagh
Full Text Available Arylamine N-acetyltransferase 2 (NAT2 is involved in human physiological responses to a variety of xenobiotic compounds, including common therapeutic drugs and exogenous chemicals present in the diet and the environment. Many questions remain about the evolutionary mechanisms that have led to the high prevalence of slow acetylators in the human species. Evidence from recent surveys of NAT2 gene variation suggests that NAT2 slow-causing variants might have become targets of positive selection as a consequence of the shift in modes of subsistence and lifestyle in human populations in the last 10,000 years. We aimed to test more extensively the hypothesis that slow acetylation prevalence in humans is related to the subsistence strategy adopted by the past populations. To this end, published frequency data on the most relevant genetic variants of NAT2 were collected from 128 population samples (14,679 individuals representing different subsistence modes and dietary habits, allowing a thorough analysis at both a worldwide and continent scale. A significantly higher prevalence of the slow acetylation phenotype was observed in populations practicing farming (45.4% and herding (48.2% as compared to populations mostly relying on hunting and gathering (22.4% (P = 0.0007. This was closely mirrored by the frequency of the slow 590A variant that was found to occur at a three-fold higher frequency in food producers (25% as compared to hunter-gatherers (8%. These findings are consistent with the hypothesis that the Neolithic transition to subsistence economies based on agricultural and pastoral resources modified the selective regime affecting the NAT2 acetylation pathway. Furthermore, the vast amount of data collected enabled us to provide a comprehensive and up-to-date description of NAT2 worldwide genetic diversity, thus building up a useful resource of frequency data for further studies interested in epidemiological or anthropological research
Choline acetyltransferase, acetylcholinesterase, and nicotinic acetylcholine receptors of human gingival and esophageal epithelia.
Nguyen, V T; Hall, L L; Gallacher, G; Ndoye, A; Jolkovsky, D L; Webber, R J; Buchli, R; Grando, S A
A non-neuronal cholinergic system that includes neuronal-like nicotinic acetylcholine receptors (nAChRs) has recently been described in epithelial cells that line the skin and the upper respiratory tract. Since the use of nicotine-containing products is associated with morbidity in the upper digestive tract, and since nicotine may alter cellular functions directly via nAChRs, we sought to identify and characterize a non-neuronal cholinergic system in the gingival and esophageal epithelia. mRNA transcripts for alpha3, alpha5, alpha7, and beta2 nAChR subunits, choline acetyltransferase, and the asymmetric and globular forms of acetylcholinesterase were amplified from gingival keratinocytes (KC) by means of polymerase chain-reactions. These proteins were visualized in the gingival and esophageal epithelia by means of specific antibodies. Variations in distribution and intensity of immunostaining were found, indicating that the repertoire of cholinergic enzymes and receptors expressed by the cells changes during epithelial maturation, and that an upward concentration gradient of free acetylcholine exists. Blocking of the nAChRs with mecamylamine resulted in reversible loss of cell-to-cell adhesion, and shrinking and rounding of cultured gingival KC. Activation of the receptors with acetylcholine or carbachol caused stretching and peripheral ruffling of the cytoplasmic aprons, and formation of new intercellular contacts. These results demonstrate that both the keratinizing epithelium of attached gingiva and the non-keratinizing epithelium lining the upper two-thirds of the esophageal mucosa possess a non-neuronal cholinergic system. The nAChRs expressed by these epithelia are coupled to regulation of cell adhesion and motility, and may provide a target for the deleterious effects of nicotine.
Polymorphisms of arylamine N-acetyltransferase2 and risk of lung and colorectal cancer
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Amjad Mahasneh
Full Text Available The arylamine N-acetyltransferase 2 (NAT2 enzymes detoxify a wide range of naturally occurring xenobiotics including carcinogens and drugs. Point mutations in the NAT2 gene result in the variant alleles M1 (NAT2 *5A, M2 (NAT2*6A, M3 (NAT2*7 and M4 (NAT2 *14A from the wild-type WT (NAT2 *4 allele. The current study was aimed at screening genetic polymorphisms of NAT2 gene in 49 lung cancer patients, 54 colorectal cancer patients and 99 cancer-free controls, using PCR-RFLP. There were significant differences in allele frequencies between lung cancer patients and controls in the WT, M2 and M3 alleles (p < 0.05. However, only M2 and M3 allele frequencies were different between colorectal cancer patients and controls (p < 0.05. There was a marginal significant difference in the distribution of rapid and slow acetylator genotypes between lung cancer patients and controls (p = 0.06 and p = 0.05, respectively, but not between colorectal cancer patients and controls (p = 1.0 and p = 0.95, respectively. Risk of lung cancer development was found to be lower in slow acetylators [odds ratio (OR: 0.51, 95% confidence interval (95% CI: 0.25, 1.02, p-value = 0.07]. No effect was observed in case of colorectal cancer. Our results showed that NAT2 genotypes and phenotypes might be involved in lung cancer but not colorectal cancer susceptibility in Jordan.
Selective production of deacetylated mannosylerythritol lipid, MEL-D, by acetyltransferase disruption mutant of Pseudozyma hubeiensis.
Konishi, Masaaki; Makino, Motoki
Mannosylerythritol lipids (MELs) are produced by several smut fungi of the Ustilaginaceae family; they are promising microbial biosurfactants and have excellent surface-active and self-assembling properties. Pseudozyma hubeiensis is a candidate for abundant MEL production and produces large amounts of 4-O-[(4'-mono-O-acetyl-2',3'-di-O-alkanoyl)-β-d-mannopyranosyl]-meso-erythritol (MEL-C). An acetyltransferase disruption mutant of P. hubeiensis, SY62-MM36, was obtained to selectively produce deacetylated 4-O-[(2',3'-di-O-alkanoyl)-β-d-mannopyranosyl]-meso-erythritol (MEL-D), and the structures of the products were determined. Lower mobility of major spots of the mutant on silica gel thin-layer chromatography verified its more hydrophilic nature than that of wild-type MEL-A, B, and C. Structural analyses confirmed the product to be MEL-D, which comprises acyl chains of caproic acid (C6:0), capric acid (C10:0), and lauric acid (C12:0). The critical micelle concentration (CMC) and the surface tension (γCMC) of the MEL-D were 2.0 × 10 -5 M and 29.7 mN/m, respectively. SY62-MM36 also produced a minor product that was estimated as triacylated MEL-D. The triacylated MEL-D had a CMC of 3.5 × 10 -5 M and a γCMC of 29.6 mN/m. In water, MEL-D formed a lamella liquid crystal phase over a broad range of concentrations. By fed-batch cultivation, the mutant produced 91.6 ± 6.3 g/L of MEL-D for 7 days. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
Immunohistochemical localization of choline acetyltransferase during development and in Chats mutants of Drosophila melanogaster.
Gorczyca, M G; Hall, J C
The distribution of choline acetyltransferase (CAT) in the nervous system of Drosophila melanogaster was determined by indirect immunohistochemical procedures using a monoclonal antibody specific to the enzyme. Immunoreactivity was first detected in the nervous system of 16 hr embryos, and increased considerably by the end of embryogenesis. Neuropil was preferentially stained, though cell bodies could also be observed. Staining was prominent in the CNS of all 3 larval instars but decreased substantially during the mid-pupal stage. Prior to eclosion, the level of immunoreactivity increased and the adult staining pattern became discernible. In the adult brain, staining was extensive, with numerous structures, such as the optic lobes and mushroom bodies, staining strongly. The adult thoracic ganglia were also moderately immunoreactive. These results imply a wide distribution of cholinergic neurons in the CNS of Drosophila. Immunoreactivity was also determined for 2 temperature-sensitive CAT mutants, Chats1 and Chats2. These files exhibit reduced CAT activity at permissive temperature, 18 degrees C, which eventually falls to undetectable levels after incubation at nonpermissive temperature, 30 degrees C. Chats2 mutants, after incubation at either 18 or 30 degrees C displayed virtually no staining. This result indicated that the immunoreactivity observed in wild-type flies was specifically associated with the enzyme encoded by the Cha gene. The intensity of staining in Chats1 mutants incubated at 18 degrees C appeared greater than in control flies, even though CAT enzyme activity in Chats1 is lower. This suggests that the enzyme molecule itself is structurally altered in Chats1 mutants. After incubation at 30 degrees C, staining in Chats1 flies decreased but did not disappear.
Experimental study of vascularized nerve graft: evaluation of nerve regeneration using choline acetyltransferase activity.
Iwai, M; Tamai, S; Yajima, H; Kawanishi, K
A comparative study of nerve regeneration was performed on vascularized nerve graft (VNG) and free nerve graft (FNG) in Fischer strain rats. A segment of the sciatic nerve with vascular pedicle of the femoral artery and vein was harvested from syngeneic donor rat for the VNG group and the sciatic nerve in the same length without vascular pedicle was harvested for the FNG group. They were transplanted to a nerve defect in the sciatic nerve of syngeneic recipient rats. At 2, 4, 6, 8, 12, 16, and 24 weeks after operation, the sciatic nerves were biopsied and processed for evaluation of choline acetyltransferase (CAT) activity, histological studies, and measurement of wet weight of the muscle innervated by the sciatic nerve. Electrophysiological evaluation of the grafted nerve was also performed before sacrifice. The average CAT activity in the distal to the distal suture site was 383 cpm in VNG and 361 cpm in FNG at 2 weeks; 6,189 cpm in VNG and 2,264 cpm in FNG at 4 weeks; and 11,299 cpm in VNG and 9,424 cpm in FNG at 6 weeks postoperatively. The value of the VNG group was statistically higher than that of the FNG group at 4 weeks postoperatively. Electrophysiological and histological findings also suggested that nerve regeneration in the VNG group was superior to that in the FNG group during the same period. However, there was no significant difference between the two groups after 6 weeks postoperatively in any of the evaluations. The CAT measurement was useful in the experiments, because it was highly sensitive and reproducible. Copyright 2001 Wiley-Liss, Inc.
The Spt-Ada-Gcn5 Acetyltransferase (SAGA complex in Aspergillus nidulans.
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Paraskevi Georgakopoulos
Full Text Available A mutation screen in Aspergillus nidulans uncovered mutations in the acdX gene that led to altered repression by acetate, but not by glucose. AcdX of A. nidulans is highly conserved with Spt8p of Saccharomyces cerevisiae, and since Spt8p is a component of the Spt-Ada-Gcn5 Acetyltransferase (SAGA complex, the SAGA complex may have a role in acetate repression in A. nidulans. We used a bioinformatic approach to identify genes encoding most members of the SAGA complex in A. nidulans, and a proteomic analysis to confirm that most protein components identified indeed exist as a complex in A. nidulans. No apparent compositional differences were detected in mycelia cultured in acetate compared to glucose medium. The methods used revealed apparent differences between Yeast and A. nidulans in the deubiquitination (DUB module of the complex, which in S. cerevisiae consists of Sgf11p, Sus1p, and Ubp8p. Although a convincing homologue of S. cerevisiae Ubp8p was identified in the A. nidulans genome, there were no apparent homologues for Sus1p and Sgf11p. In addition, when the SAGA complex was purified from A. nidulans, members of the DUB module were not co-purified with the complex, indicating that functional homologues of Sus1p and Sgf11p were not part of the complex. Thus, deubiquitination of H2B-Ub in stress conditions is likely to be regulated differently in A. nidulans compared to S. cerevisiae.
Histone variant innovation in a rapidly evolving chordate lineage
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Jansen Pascal WTC
Full Text Available Abstract Background Histone variants alter the composition of nucleosomes and play crucial roles in transcription, chromosome segregation, DNA repair, and sperm compaction. Modification of metazoan histone variant lineages occurs on a background of genome architecture that shows global similarities from sponges to vertebrates, but the urochordate, Oikopleura dioica, a member of the sister group to vertebrates, exhibits profound modification of this ancestral architecture. Results We show that a histone complement of 47 gene loci encodes 31 histone variants, grouped in distinct sets of developmental expression profiles throughout the life cycle. A particularly diverse array of 15 male-specific histone variants was uncovered, including a testes-specific H4t, the first metazoan H4 sequence variant reported. Universal histone variants H3.3, CenH3, and H2A.Z are present but O. dioica lacks homologs of macroH2A and H2AX. The genome encodes many H2A and H2B variants and the repertoire of H2A.Z isoforms is expanded through alternative splicing, incrementally regulating the number of acetylatable lysine residues in the functionally important N-terminal "charge patch". Mass spectrometry identified 40 acetylation, methylation and ubiquitylation posttranslational modifications (PTMs and showed that hallmark PTMs of "active" and "repressive" chromatin were present in O. dioica. No obvious reduction in silent heterochromatic marks was observed despite high gene density in this extraordinarily compacted chordate genome. Conclusions These results show that histone gene complements and their organization differ considerably even over modest phylogenetic distances. Substantial innovation among all core and linker histone variants has evolved in concert with adaptation of specific life history traits in this rapidly evolving chordate lineage.
Biochemical studies on histones of the central nervous system. 1
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Schmitt, M.; Matthies, H.
Rat brain histones were acetylated in vivo by intraventricular injection of [ 14 C]-acetate. More than 90% of the label is the result of a true acetylation. Enzymatic proteolysis of the labelled histone fraction and subsequent chromatographic investigation of the digestion products showed about 60% of the recovered radioactive material to be epsilon-acetyl lysine, whereas 22% of the radioactivity was found in an unidentified spot. (author)
Targeting histone abnormality in triple negative breast cancer
AWARD NUMBER: W81XWH-14-1-0238 TITLE: Targeting histone abnormality in triple negative breast cancer PRINCIPAL INVESTIGATOR: Steffi...4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Targeting histone abnormality in triple negative breast cancer 5b. GRANT NUMBER W81XWH-14-1-0238 5c...PDXs). In addition, two teams collaborated in studying the functional role of LSD2 in breast cancer progression. The interactive studies between two
15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) mediates repression of TNF-α by decreasing levels of acetylated histone H3 and H4 at its promoter
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Engdahl, Ryan; Monroy, M. Alexandra; Daly, John M.
Prostaglandin metabolite 15-Deoxy-Δ 12,14 -prostaglandin J2 (15d-PGJ2) is known to inhibit a number of pro-inflammatory cytokines as well as being a ligand for nuclear receptor PPARγ. We investigated the ability of 15d-PGJ2 to inhibit TNF-α gene expression through mechanisms that involve histone modification. Pretreatment with 15d-PGJ2 (10 μM) inhibited LPS-stimulated TNF-α mRNA in THP-1 monocytes or PMA-differentiated cells to nearly basal levels. A specific PPARγ ligand, GW1929, failed to inhibit LPS-induced TNF-α mRNA expression nor did a PPARγ antagonist, GW9662, alter the repression of TNF-α mRNA in LPS-stimulated cells pretreated with 15d-PGJ2 suggesting a PPARγ-independent inhibition of TNF-α mRNA in THP-1 cells. Transfection studies with a reporter construct and subsequent treatment with 15d-PGJ2 demonstrated a dose-dependent inhibition of the TNF-α promoter. Additional studies demonstrated that inhibition of histone deacetylases with trichostatin A (TSA) or overexpression of histone acetyltransferase CBP could overcome 15d-PGJ2-mediated repression of the TNF-α promoter, suggesting that an important mechanism whereby 15d-PGJ2 suppresses a cytokine is through factors that regulate histone modifications. To examine the endogenous TNF-α promoter, chromatin immunoprecipitations (ChIP) were performed. ChIP assays demonstrated that LPS stimulation induced an increase in histone H3 and H4 acetylation at the TNF-α promoter, which was reduced in cells pretreated with 15d-PGJ2. These results highlight the ability of acetylation and deacetylation factors to affect the TNF-α promoter and demonstrate that an additional important mechanism whereby 15d-PGJ2 mediates TNF-α transcriptional repression by altering levels of acetylated histone H3 and H4 at its promoter
Histone Acetylome-wide Association Study of Autism Spectrum Disorder.
Sun, Wenjie; Poschmann, Jeremie; Cruz-Herrera Del Rosario, Ricardo; Parikshak, Neelroop N; Hajan, Hajira Shreen; Kumar, Vibhor; Ramasamy, Ramalakshmi; Belgard, T Grant; Elanggovan, Bavani; Wong, Chloe Chung Yi; Mill, Jonathan; Geschwind, Daniel H; Prabhakar, Shyam
The association of histone modification changes with autism spectrum disorder (ASD) has not been systematically examined. We conducted a histone acetylome-wide association study (HAWAS) by performing H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) on 257 postmortem samples from ASD and matched control brains. Despite etiological heterogeneity, ≥68% of syndromic and idiopathic ASD cases shared a common acetylome signature at >5,000 cis-regulatory elements in prefrontal and temporal cortex. Similarly, multiple genes associated with rare genetic mutations in ASD showed common "epimutations." Acetylome aberrations in ASD were not attributable to genetic differentiation at cis-SNPs and highlighted genes involved in synaptic transmission, ion transport, epilepsy, behavioral abnormality, chemokinesis, histone deacetylation, and immunity. By correlating histone acetylation with genotype, we discovered >2,000 histone acetylation quantitative trait loci (haQTLs) in human brain regions, including four candidate causal variants for psychiatric diseases. Due to the relative stability of histone modifications postmortem, we anticipate that the HAWAS approach will be applicable to multiple diseases. Copyright © 2016 Elsevier Inc. All rights reserved.
Structural Mechanisms of Nucleosome Recognition by Linker Histones.
Zhou, Bing-Rui; Jiang, Jiansheng; Feng, Hanqiao; Ghirlando, Rodolfo; Xiao, T Sam; Bai, Yawen
Linker histones bind to the nucleosome and regulate the structure of chromatin and gene expression. Despite more than three decades of effort, the structural basis of nucleosome recognition by linker histones remains elusive. Here, we report the crystal structure of the globular domain of chicken linker histone H5 in complex with the nucleosome at 3.5 Å resolution, which is validated using nuclear magnetic resonance spectroscopy. The globular domain sits on the dyad of the nucleosome and interacts with both DNA linkers. Our structure integrates results from mutation analyses and previous cross-linking and fluorescence recovery after photobleach experiments, and it helps resolve the long debate on structural mechanisms of nucleosome recognition by linker histones. The on-dyad binding mode of the H5 globular domain is different from the recently reported off-dyad binding mode of Drosophila linker histone H1. We demonstrate that linker histones with different binding modes could fold chromatin to form distinct higher-order structures. Copyright © 2015 Elsevier Inc. All rights reserved.
Histone deacetylase inhibition abolishes stress-induced spatial memory impairment.
Vargas-López, Viviana; Lamprea, Marisol R; Múnera, Alejandro
Acute stress induced before spatial training impairs memory consolidation. Although non-epigenetic underpinning of such effect has been described, the epigenetic mechanisms involved have not yet been studied. Since spatial training and intense stress have opposite effects on histone acetylation balance, it is conceivable that disruption of such balance may underlie acute stress-induced spatial memory consolidation impairment and that inhibiting histone deacetylases prevents such effect. Trichostatin-A (TSA, a histone deacetylase inhibitor) was used to test its effectiveness in preventing stress' deleterious effect on memory. Male Wistar rats were trained in a spatial task in the Barnes maze; 1-h movement restraint was applied to half of them before training. Immediately after training, stressed and non-stressed animals were randomly assigned to receive either TSA (1mg/kg) or vehicle intraperitoneal injection. Twenty-four hours after training, long-term spatial memory was tested; plasma and brain tissue were collected immediately after the memory test to evaluate corticosterone levels and histone H3 acetylation in several brain areas. Stressed animals receiving vehicle displayed memory impairment, increased plasma corticosterone levels and markedly reduced histone H3 acetylation in prelimbic cortex and hippocampus. Such effects did not occur in stressed animals treated with TSA. The aforementioned results support the hypothesis that acute stress induced-memory impairment is related to histone deacetylation. Copyright © 2016 Elsevier Inc. All rights reserved.
Eukaryotic Replisome Components Cooperate to Process Histones During Chromosome Replication
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Magdalena Foltman
Full Text Available DNA unwinding at eukaryotic replication forks displaces parental histones, which must be redeposited onto nascent DNA in order to preserve chromatin structure. By screening systematically for replisome components that pick up histones released from chromatin into a yeast cell extract, we found that the Mcm2 helicase subunit binds histones cooperatively with the FACT (facilitiates chromatin transcription complex, which helps to re-establish chromatin during transcription. FACT does not associate with the Mcm2-7 helicase at replication origins during G1 phase but is subsequently incorporated into the replisome progression complex independently of histone binding and uniquely among histone chaperones. The amino terminal tail of Mcm2 binds histones via a conserved motif that is dispensable for DNA synthesis per se but helps preserve subtelomeric chromatin, retain the 2 micron minichromosome, and support growth in the absence of Ctf18-RFC. Our data indicate that the eukaryotic replication and transcription machineries use analogous assemblies of multiple chaperones to preserve chromatin integrity.
Dynamic alterations of linker histone variants during development.
Godde, James S; Ura, Kiyoe
The process of development can be viewed as a series of linker histone replacements which take place throughout spermatogenesis and oogenesis, as well as following fertilization or somatic nuclear transfer (SNT). Although few of the histone H1 variants in question have been shown to be essential for viability, the timing of their appearance as well as the affinity with which they are able to bind to chromatin seem to be important factors in their developmental role. A looser binding of linker histones to chromatin seems to correlate with the meiotic phases of gametogenesis and the establishment of a totipotent, as well as the maintenance of a pluripotent, state in early embryos, while tighter binding of linker histones to chromatin appears to be associated with the mitotic phases, as well as the increased levels of condensation that are required for the packaging of DNA into sperm. This latter process also involves the binding of certain basic non-histone proteins to DNA. While all proteins involved in chromatin compaction during development are highly basic in nature, in general they can be seen to change from lysine-rich variants to arginine-rich ones, and back again. The fact that linker histone transitions are conserved across diverse metazoan species speaks of their importance in packaging DNA in a variety of ways during this crucial period.
Open and closed: the roles of linker histones in plants and animals.
Over, Ryan S; Michaels, Scott D
Histones package DNA in all eukaryotes and play key roles in regulating gene expression. Approximately 150 base pairs of DNA wraps around an octamer of core histones to form the nucleosome, the basic unit of chromatin. Linker histones compact chromatin further by binding to and neutralizing the charge of the DNA between nucleosomes. It is well established that chromatin packing is regulated by a complex pattern of posttranslational modifications (PTMs) to core histones, but linker histone function is less well understood. In this review, we describe the current understanding of the many roles that linker histones play in cellular processes, including gene regulation, cell division, and development, while putting the linker histone in the context of other nuclear proteins. Although intriguing roles for plant linker histones are beginning to emerge, much of our current understanding comes from work in animal systems. Many unanswered questions remain and additional work is required to fully elucidate the complex processes mediated by linker histones in plants.
Histone as future drug target for malaria.
Rawat, D S; Lumb, V; Sharma, Y D; Pasha, S T; Singh, G
Malaria continues to be a major cause of mortality and morbidity in tropical countries and affecting around 100 countries of the world. As per WHO estimates, 300-500 million are being infected and 1-3 million deaths annually due to malaria. With the emerging knowledge about genome sequence of all the three counterparts involved in the disease of malaria, the parasite Plasmodium, vector Anopheles and host Homo sapien have helped the scientists to understand interactions between them. Simultaneous advancement in technology further improves the prospects to discover new targets for vaccines and drugs. Though the malaria vaccine is still far away in this situation there is need to develop a potent and affordable drug(s). Histones are the key protein of chromatin and play an important role in DNA packaging, replication and gene expression. They also show frequent post-translation modifications. The specific combinations of these posttranslational modifications are thought to alter chromatin structure by forming epigenetic bar codes that specify either transient or heritable patterns of genome function. Chromatin regulators and upstream pathways are therefore seen as promising targets for development of therapeutic drugs.
Interpreting clinical assays for histone deacetylase inhibitors
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Martinet, Nadine; Bertrand, Philippe
As opposed to genetics, dealing with gene expressions by direct DNA sequence modifications, the term epigenetics applies to all the external influences that target the chromatin structure of cells with impact on gene expression unrelated to the sequence coding of DNA itself. In normal cells, epigenetics modulates gene expression through all development steps. When "imprinted" early by the environment, epigenetic changes influence the organism at an early stage and can be transmitted to the progeny. Together with DNA sequence alterations, DNA aberrant cytosine methylation and microRNA deregulation, epigenetic modifications participate in the malignant transformation of cells. Their reversible nature has led to the emergence of the promising field of epigenetic therapy. The efforts made to inhibit in particular the epigenetic enzyme family called histone deacetylases (HDACs) are described. HDAC inhibitors (HDACi) have been proposed as a viable clinical therapeutic approach for the treatment of leukemia and solid tumors, but also to a lesser degree for noncancerous diseases. Three epigenetic drugs are already arriving at the patient's bedside, and more than 100 clinical assays for HDACi are registered on the National Cancer Institute website. They explore the eventual additive benefits of combined therapies. In the context of the pleiotropic effects of HDAC isoforms, more specific HDACi and more informative screening tests are being developed for the benefit of the patients
In vivo observation of a non-noradrenergic regulation of arylalkylamine N-acetyltransferase gene expression in the rat pineal complex.
Garidou, M L; Bartol, I; Calgari, C; Pévet, P; Simonneaux, V
The rodent pineal gland is the end point of several peripheral and central fibers innervating the superficial and deep parts of the gland. Up to now, only the sympathetic transmitter norepinephrine is thought to regulate melatonin synthesis, although numerous biochemical experiments have reported in vitro effects of various transmitters on melatonin synthesis. To find out whether there is non-noradrenergic regulation of in vivo pineal metabolism, the mRNA encoding the enzyme arylalkylamine N-acetyltransferase was studied using the highly sensitive technique of in situ hybridization. The existence of a marked nocturnal increase of arylalkylamine N-acetyltransferase mRNA in the superficial pineal gland was confirmed. Interestingly and for the first time, a similar daily variation was observed in the deep pineal. After removal of superior cervical ganglia, the daily rhythm in arylalkylamine N-acetyltransferase mRNA was abolished in both the superficial and deep pineal indicating that the rhythm is driven by sympathetic input in the entire pineal complex. Interestingly, the remaining arylalkylamine N-acetyltransferase mRNA level in the pineal of day- and night-time ganglionectomized rats was significantly higher than in the pineal of day-time intact animals. These data reveal a sympathetic-dependent day-time inhibition of arylalkylamine N-acetyltransferase gene expression. In addition, the day-time pineal arylalkylamine N-acetyltransferase mRNA expression in ganglionectomized rats persisted after adrenal gland removal but was reduced by 50% after propranolol injection. These results indicate that arylalkylamine N-acetyltransferase mRNA in ganglionectomized rats is not induced by circulating catecholamines and may be caused by both a centrally originated norepinephrine, as already suggested, and other non-adrenergic transmitter(s). In conclusion, this work shows that norepinephrine drives the nocturnal increase of arylalkylamine N-acetyltransferase gene expression both
Acetylations of Ftz-F1 and histone H4K5 are required for the fine-tuning of ecdysone biosynthesis during Drosophila metamorphosis.
Borsos, Barbara N; Pankotai, Tibor; Kovács, Dávid; Popescu, Christina; Páhi, Zoltán; Boros, Imre M
The molting during Drosophila development is tightly regulated by the ecdysone hormone. Several steps of the ecdysone biosynthesis have been already identified but the regulation of the entire process has not been clarified yet. We have previously reported that dATAC histone acetyltransferase complex is necessary for the steroid hormone biosynthesis process. To reveal possible mechanisms controlled by dATAC we made assumptions that either dATAC may influence directly the transcription of Halloween genes involved in steroid hormone biosynthesis or it may exert an indirect effect on it by acetylating the Ftz-F1 transcription factor which regulates the transcription of steroid converting genes. Here we show that the lack of dATAC complex results in increased mRNA level and decreased protein level of Ftz-F1. In this context, decreased mRNA and increased protein levels of Ftz-F1 were detected upon treatment of Drosophila S2 cells with histone deacetylase inhibitor trichostatin A. We showed that Ftz-F1, the transcriptional activator of Halloween genes, is acetylated in S2 cells. In addition, we found that ecdysone biosynthetic Halloween genes are transcribed in S2 cells and their expression can be influenced by deacetylase inhibitors. Furthermore, we could detect H4K5 acetylation at the regulatory regions of disembodied and shade Halloween genes, while H3K9 acetylation is absent on these genes. Based on our findings we conclude that the dATAC HAT complex might play a dual regulatory role in Drosophila steroid hormone biosynthesis through the acetylation of Ftz-F1 protein and the regulation of the H4K5 acetylation at the promoters of Halloween genes. Copyright © 2015 Elsevier Inc. All rights reserved.
Hexavalent Chromium (Cr(VI Down-Regulates Acetylation of Histone H4 at Lysine 16 through Induction of Stressor Protein Nupr1.
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Danqi Chen
Full Text Available The environmental and occupational carcinogen Hexavalent Chromium (Cr(VI has been shown to cause lung cancer in humans when inhaled. In spite of a considerable research effort, the mechanisms of Cr(VI-induced carcinogenesis remain largely unknown. Nupr1 (nuclear protein 1 is a small, highly basic, and unfolded protein with molecular weight of 8,800 daltons and is induced by a variety of stressors. Studies in animal models have suggested that Nupr1 is a key factor in the development of lung and pancreatic cancers, with little known about the underlying molecular mechanisms. Here we report that the level of Nupr1 is significantly increased in human bronchial epithelial BEAS2B cells following exposure to Cr(VI through epigenetic mechanisms. Interestingly, Cr(VI exposure also results in the loss of acetylation at histone H4K16, which is considered a 'hallmark' of human cancer. Cr(VI-induced reduction of H4K16 acetylation appears to be caused by the induction of Nupr1, since (a overexpression of Nupr1 decreased the levels of both H4K16 acetylation and the histone acetyltransferase MOF (male absent on the first; also known as Kat8, Myst 1, which specifically acetylates H4K16; (b the loss of acetylation of H4K16 upon Cr(VI exposure is greatly compromised by knockdown of Nupr1. Moreover, Nupr1-induced reduction of H4K16 acetylation correlates with the transcriptional down-regulation at several genomic loci. Notably, overexpression of Nupr1 induces anchorage-independent cell growth and knockdown of Nupr1 expression prevents Cr(VI-induced cell transformation. We propose that Cr(VI induces Nupr1 and rapidly perturbs gene expression by downregulating H4K16 acetylation, thereby contributing to Cr(VI-induced carcinogenesis.
Histone deacetylase 2 is decreased in peripheral blood pro-inflammatory CD8+ T and NKT-like lymphocytes following lung transplant.
Hodge, Greg; Hodge, Sandra; Holmes-Liew, Chien-Li; Reynolds, Paul N; Holmes, Mark
Immunosuppression therapy following lung transplantation fails to prevent chronic rejection in many patients, which is associated with lack of suppression of cytotoxic mediators and pro-inflammatory cytokines in peripheral blood T and natural killer T (NKT)-like cells. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) upregulate/downregulate pro-inflammatory gene expression, respectively; however, differences in the activity of these enzymes following lung transplant are unknown. We hypothesized decreased HDAC2 expression and increased HAT expression in pro-inflammatory lymphocytes following lung transplant. Blood was collected from 18 stable lung transplant patients and 10 healthy age-matched controls. Intracellular pro-inflammatory cytokines and HAT/HDAC2 expression were determined in lymphocyte subsets following culture using flow cytometry. A loss of HDAC2 in cluster of differentiation (CD) 8+ T and NKT-like cells in transplant patients compared with controls was noted (CD8+ T: 28 ± 10 (45 ± 10), CD8+NKT-like: 30 ± 13 (54 ± 16) (mean ± SD transplant) (control)). Loss of HDAC2 was associated with an increased percentage of CD8+ T and NKT-like cells expressing perforin, granzyme b, interferon gamma (IFN-γ) and TNF-α (no change in HAT expression in any lymphocyte subset). There was a negative correlation between loss of HDAC2 expression by CD8+ T cells with cumulative dose of prednisolone and time post-transplant. Treatment with 10 mg/L theophylline + 1 µmol/L prednisolone or 2.5 ng/mL cyclosporine A synergistically upregulated HDAC2 and inhibited IFN-γ and TNF-α production by CD8+ T and NKT-like lymphocytes. HDAC2 is decreased in CD8+ T and NKT-like pro-inflammatory lymphocytes following lung transplant. Treatment options that increase HDAC2 may improve graft survival. © 2016 Asian Pacific Society of Respirology.
Structural and Histone Binding Ability Characterizations of Human PWWP Domains
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Wu, Hong; Zeng, Hong; Lam, Robert; Tempel, Wolfram; Amaya, Maria F.; Xu, Chao; Dombrovski, Ludmila; Qiu, Wei; Wang, Yanming; Min, Jinrong (Toronto); (Penn)
The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently. The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members, implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3. PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical {beta}-barrel core, an insertion motif between the second and third {beta}-strands and a C-terminal {alpha}-helix bundle. Both the canonical {beta}-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones.
SUMOylated ORC2 Recruits a Histone Demethylase to Regulate Centromeric Histone Modification and Genomic Stability
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Chao Huang
Full Text Available Origin recognition complex 2 (ORC2, a subunit of the ORC, is essential for DNA replication initiation in eukaryotic cells. In addition to a role in DNA replication initiation at the G1/S phase, ORC2 has been shown to localize to the centromere during the G2/M phase. Here, we show that ORC2 is modified by small ubiquitin-like modifier 2 (SUMO2, but not SUMO1, at the G2/M phase of the cell cycle. SUMO2-modification of ORC2 is important for the recruitment of KDM5A in order to convert H3K4me3 to H3K4me2, a "permissive" histone marker for α-satellite transcription at the centromere. Persistent expression of SUMO-less ORC2 led to reduced α-satellite transcription and impaired pericentric heterochromatin silencing, which resulted in re-replication of heterochromatin DNA. DNA re-replication eventually activated the DNA damage response, causing the bypass of mitosis and the formation of polyploid cells. Thus, ORC2 sustains genomic stability by recruiting KDM5A to maintain centromere histone methylation in order to prevent DNA re-replication.
An Organellar Nα-Acetyltransferase, Naa60, Acetylates Cytosolic N Termini of Transmembrane Proteins and Maintains Golgi Integrity
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Henriette Aksnes
Full Text Available N-terminal acetylation is a major and vital protein modification catalyzed by N-terminal acetyltransferases (NATs. NatF, or Nα-acetyltransferase 60 (Naa60, was recently identified as a NAT in multicellular eukaryotes. Here, we find that Naa60 differs from all other known NATs by its Golgi localization. A new membrane topology assay named PROMPT and a selective membrane permeabilization assay established that Naa60 faces the cytosolic side of intracellular membranes. An Nt-acetylome analysis of NAA60-knockdown cells revealed that Naa60, as opposed to other NATs, specifically acetylates transmembrane proteins and has a preference for N termini facing the cytosol. Moreover, NAA60 knockdown causes Golgi fragmentation, indicating an important role in the maintenance of the Golgi's structural integrity. This work identifies a NAT associated with membranous compartments and establishes N-terminal acetylation as a common modification among transmembrane proteins, a thus-far poorly characterized part of the N-terminal acetylome.
Biochemical and Structural Analysis of an Eis Family Aminoglycoside Acetyltransferase from Bacillus anthracis
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Green, Keith D.; Biswas, Tapan; Chang, Changsoo; Wu, Ruiying; Chen, Wenjing; Janes, Brian K.; Chalupska, Dominika; Gornicki, Piotr; Hanna, Philip C.; Tsodikov, Oleg V.; Joachimiak, Andrzej; Garneau-Tsodikova, Sylvie
Proteins from the enhanced intracellular survival (Eis) family are versatile acetyltransferases that acetylate amines at multiple positions of several aminoglycosides (AGs). Their upregulation confers drug resistance. Homologues of Eis are present in diverse bacteria, including many pathogens. Eis from Mycobacterium tuberculosis (Eis_Mtb) has been well characterized. In this study, we explored the AG specificity and catalytic efficiency of the Eis family protein from Bacillus anthracis (Eis_Ban). Kinetic analysis of specificity and catalytic efficiency of acetylation of six AGs indicates that Eis_Ban displays significant differences from Eis_Mtb in both substrate binding and catalytic efficiency. The number of acetylated amines was also different for several AGs, indicating a distinct regiospecificity of Eis_Ban. Furthermore, most recently identified inhibitors of Eis_Mtb did not inhibit Eis_Ban, underscoring the differences between these two enzymes. To explain these differences, we determined an Eis_Ban crystal structure. The comparison of the crystal structures of Eis_Ban and Eis_Mtb demonstrates that critical residues lining their respective substrate binding pockets differ substantially, explaining their distinct specificities. Our results suggest that acetyltransferases of the Eis family evolved divergently to garner distinct specificities while conserving catalytic efficiency, possibly to counter distinct chemical challenges. The unique specificity features of these enzymes can be utilized as tools for developing AGs with novel modifications and help guide specific AG treatments to avoid Eis-mediated resistance.
Histone deacetylase 3 (HDAC 3) as emerging drug target in NF-kappa B-mediated inflammation
Leus, Niek G. J.; Zwinderman, Martijn R. H.; Dekker, Frank J.
Activation of inflammatory gene expression is regulated, among other factors, by post-translational modifications of histone proteins. The most investigated type of histone modifications is lysine acetylations. Histone deacetylases (HDACs) remove acetylations from lysines, thereby influencing
Nucleosome structure incorporated histone acetylation site prediction in Arabidopsis thaliana.
Zhao, Chen; Liu, Hui; Li, Jiang; Deng, Youping; Shi, Tieliu
Acetylation is a crucial post-translational modification for histones, and plays a key role in gene expression regulation. Due to limited data and lack of a clear acetylation consensus sequence, a few researches have focused on prediction of lysine acetylation sites. Several systematic prediction studies have been conducted for human and yeast, but less for Arabidopsis thaliana. Concerning the insufficient observation on acetylation site, we analyzed contributions of the peptide-alignment-based distance definition and 3D structure factors in acetylation prediction. We found that traditional structure contributes little to acetylation site prediction. Identified acetylation sites of histones in Arabidopsis thaliana are conserved and cross predictable with that of human by peptide based methods. However, the predicted specificity is overestimated, because of the existence of non-observed acetylable site. Here, by performing a complete exploration on the factors that affect the acetylability of lysines in histones, we focused on the relative position of lysine at nucleosome level, and defined a new structure feature to promote the performance in predicting the acetylability of all the histone lysines in A. thaliana. We found a new spacial correlated acetylation factor, and defined a ε-N spacial location based feature, which contains five core spacial ellipsoid wired areas. By incorporating the new feature, the performance of predicting the acetylability of all the histone lysines in A. Thaliana was promoted, in which the previous mispredicted acetylable lysines were corrected by comparing to the peptide-based prediction.
Extracellular DNA and histones: double-edged swords in immunothrombosis.
Gould, T J; Lysov, Z; Liaw, P C
The existence of extracellular DNA in human plasma, also known as cell-free DNA (cfDNA), was first described in the 1940s. In recent years, there has been a resurgence of interest in the functional significance of cfDNA, particularly in the context of neutrophil extracellular traps (NETs). cfDNA and histones are key components of NETs that aid in the host response to infection and inflammation. However, cfDNA and histones may also exert harmful effects by triggering coagulation, inflammation, and cell death and by impairing fibrinolysis. In this article, we will review the pathologic nature of cfDNA and histones in macrovascular and microvascular thrombosis, including venous thromboembolism, cancer, sepsis, and trauma. We will also discuss the prognostic value of cfDNA and histones in these disease states. Understanding the molecular and cellular pathways regulated by cfDNA and histones may provide novel insights to prevent pathological thrombus formation and vascular occlusion. © 2015 International Society on Thrombosis and Haemostasis.
Histones trigger sterile inflammation by activating the NLRP3 inflammasome.
Allam, Ramanjaneyulu; Darisipudi, Murthy Narayana; Tschopp, Jurg; Anders, Hans-Joachim
Sterile cell death mediated inflammation is linked to several pathological disorders and involves danger recognition of intracellular molecules released by necrotic cells that activate different groups of innate pattern recognition receptors. Toll-like receptors directly interact with their extrinsic or intrinsic agonists and induce multiple proinflammatory mediators. In contrast, the NLRP3 inflammasome is rather thought to represent a downstream element integrating various indirect stimuli into proteolytic cleavage of interleukin (IL)-1β and IL-18. Here, we report that histones released from necrotic cells induce IL-1β secretion in an NLRP3-ASC-caspase-1-dependent manner. Genetic deletion of NLRP3 in mice significantly attenuated histone-induced IL-1β production and neutrophil recruitment. Furthermore, necrotic cells induced neutrophil recruitment, which was significantly reduced by histone-neutralizing antibodies or depleting extracellular histones via enzymatic degradation. These results identify cytosolic uptake of necrotic cell-derived histones as a triggering mechanism of sterile inflammation, which involves NLRP3 inflammasome activation and IL-1β secretion via oxidative stress. © 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.
Selective Biological Responses of Phagocytes and Lungs to Purified Histones.
Fattahi, Fatemeh; Grailer, Jamison J; Lu, Hope; Dick, Rachel S; Parlett, Michella; Zetoune, Firas S; Nuñez, Gabriel; Ward, Peter A
Histones invoke strong proinflammatory responses in many different organs and cells. We assessed biological responses to purified or recombinant histones, using human and murine phagocytes and mouse lungs. H1 had the strongest ability in vitro to induce cell swelling independent of requirements for toll-like receptors (TLRs) 2 or 4. These responses were also associated with lactate dehydrogenase release. H3 and H2B were the strongest inducers of [Ca2+]i elevations in phagocytes. Cytokine and chemokine release from mouse and human phagocytes was predominately a function of H2A and H2B. Double TLR2 and TLR4 knockout (KO) mice had dramatically reduced cytokine release induced in macrophages exposed to individual histones. In contrast, macrophages from single TLR-KO mice showed few inhibitory effects on cytokine production. Using the NLRP3 inflammasome protocol, release of mature IL-1β was predominantly a feature of H1. Acute lung injury following the airway delivery of histones suggested that H1, H2A, and H2B were linked to alveolar leak of albumin and the buildup of polymorphonuclear neutrophils as well as the release of chemokines and cytokines into bronchoalveolar fluids. These results demonstrate distinct biological roles for individual histones in the context of inflammation biology and the requirement of both TLR2 and TLR4. © 2017 S. Karger AG, Basel.
The Histone Demethylase Jhdm1a Regulates Hepatic Gluconeogenesis
Zou, Tie; Yao, Annie Y.; Cooper, Marcus P.; Boyartchuk, Victor; Wang, Yong-Xu
Hepatic gluconeogenesis is required for maintaining blood glucose homeostasis; yet, in diabetes mellitus, this process is unrestrained and is a major contributor to fasting hyperglycemia. To date, the impacts of chromatin modifying enzymes and chromatin landscape on gluconeogenesis are poorly understood. Through catalyzing the removal of methyl groups from specific lysine residues in the histone tail, histone demethylases modulate chromatin structure and, hence, gene expression. Here we perform an RNA interference screen against the known histone demethylases and identify a histone H3 lysine 36 (H3K36) demethylase, Jhdm1a, as a key negative regulator of gluconeogenic gene expression. In vivo, silencing of Jhdm1a promotes liver glucose synthesis, while its exogenous expression reduces blood glucose level. Importantly, the regulation of gluconeogenesis by Jhdm1a requires its demethylation activity. Mechanistically, we find that Jhdm1a regulates the expression of a major gluconeogenic regulator, C/EBPα. This is achieved, at least in part, by its USF1-dependent association with the C/EBPα promoter and its subsequent demethylation of dimethylated H3K36 on the C/EBPα locus. Our work provides compelling evidence that links histone demethylation to transcriptional regulation of gluconeogenesis and has important implications for the treatment of diabetes. PMID:22719268
Histone demethylase UTX counteracts glucocorticoid deregulation of osteogenesis by modulating histone-dependent and -independent pathways.
Wang, Feng-Sheng; Lian, Wei-Shiung; Lee, Mel S; Weng, Wen-Tsan; Huang, Ying-Hsien; Chen, Yu-Shan; Sun, Yi-Chih; Wu, Shing-Long; Chuang, Pei-Chin; Ko, Jih-Yang
Excess glucocorticoid administration impairs osteogenic activities, which raises the risk of osteoporotic disorders. Epigenetic methylation of DNA and histone regulates the lineage commitment of progenitor cells. This study was undertaken to delineate the actions of histone lysine demethylase 6a (UTX) with regard to the glucocorticoid impediment of osteogenic differentiation. Osteogenic progenitor cells responded to supraphysiological glucocorticoid by elevating CpG dinucleotide methylation proximal to transcription start sites within Runx2 and osterix promoters and Wnt inhibitor Dickkopf-1 (Dkk1) expression concomitant with low UTX expression. 5'-Aza-deoxycystidine demethylation of Runx2 and osterix promoters abolished the glucocorticoid inhibition of mineralized matrix accumulation. Gain of UTX function attenuated the glucocorticoid-induced loss of osteogenic differentiation, whereas UTX silencing escalated adipogenic gene expression and adipocyte formation. UTX sustained osteogenic gene transcription through maintaining its occupancy to Runx2 and osterix promoters. It also mitigated the trimethylation of histone 3 at lysine 27 (H3K27me3), which reduced H3K27me3 enrichment to Dkk1 promoter and thereby lowered Dkk1 transcription. Modulation of β-catenin and Dkk1 actions restored UTX signaling in glucocorticoid-stressed cells. In vivo, UTX inhibition by exogenous methylprednisolone and GSK-J4 administration, an effect that disturbed H3K27me3, β-catenin, Dkk1, Runx2, and osterix levels, exacerbated trabecular microarchitecture loss and marrow adiposity. Taken together, glucocorticoid reduction of UTX function hindered osteogenic differentiation. Epigenetic hypomethylation of osteogenic transcription factor promoters and H3K27 contributed to the UXT alleviation of Dkk1 transcription and osteogenesis in glucocorticoid-stressed osteogenic progenitor cells. Control of UTX action has an epigenetic perspective of curtailing glucocorticoid impairment of osteogenic
Destabilization of the Outer and Inner Mitochondrial Membranes by Core and Linker Histones
Cascone, Annunziata; Bruelle, Celine; Lindholm, Dan; Bernardi, Paolo; Eriksson, Ove
Background Extensive DNA damage leads to apoptosis. Histones play a central role in DNA damage sensing and may mediate signals of genotoxic damage to cytosolic effectors including mitochondria. Methodology/Principal Findings We have investigated the effects of histones on mitochondrial function and membrane integrity. We demonstrate that both linker histone H1 and core histones H2A, H2B, H3, and H4 bind strongly to isolated mitochondria. All histones caused a rapid and massive release of the pro-apoptotic intermembrane space proteins cytochrome c and Smac/Diablo, indicating that they permeabilize the outer mitochondrial membrane. In addition, linker histone H1, but not core histones, permeabilized the inner membrane with a collapse of the membrane potential, release of pyridine nucleotides, and mitochondrial fragmentation. Conclusions We conclude that histones destabilize the mitochondrial membranes, a mechanism that may convey genotoxic signals to mitochondria and promote apoptosis following DNA damage. PMID:22523586
Two distinct modes for propagation of histone PTMs across the cell cycle
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Alabert, Constance; Barth, Teresa K; Reverón-Gómez, Nazaret
Epigenetic states defined by chromatin can be maintained through mitotic cell division. However, it remains unknown how histone-based information is transmitted. Here we combine nascent chromatin capture (NCC) and triple-SILAC (stable isotope labeling with amino acids in cell culture) labeling...... to track histone modifications and histone variants during DNA replication and across the cell cycle. We show that post-translational modifications (PTMs) are transmitted with parental histones to newly replicated DNA. Di- and trimethylation marks are diluted twofold upon DNA replication, as a consequence...... of new histone deposition. Importantly, within one cell cycle, all PTMs are restored. In general, new histones are modified to mirror the parental histones. However, H3K9 trimethylation (H3K9me3) and H3K27me3 are propagated by continuous modification of parental and new histones because the establishment...
Histone deacetylase inhibition regulates inflammation and enhances Tregs after allogeneic hematopoietic cell transplantation in humans
Choi, S.W.; Gatza, E.; Hou, G.; Sun, Y; Whitfield, J.; Song, Y.; Oravecz-Wilson, K.; Tawara, I.; Dinarello, C.A.; Reddy, P.
We examined immunological responses in patients receiving histone deacetylase (HDAC) inhibition (vorinostat) for graft-versus-host disease prophylaxis after allogeneic hematopoietic cell transplant. Vorinostat treatment increased histone acetylation in peripheral blood mononuclear cells (PBMCs) from
Low Proteolytic Clipping of Histone H3 in Cervical Cancer
Sandoval-Basilio, Jorge; Serafín-Higuera, Nicolás; Reyes-Hernandez, Octavio D.; Serafín-Higuera, Idanya; Leija-Montoya, Gabriela; Blanco-Morales, Magali; Sierra-Martínez, Monica; Ramos-Mondragon, Roberto; García, Silvia; López-Hernández, Luz Berenice; Yocupicio-Monroy, Martha; Alcaraz-Estrada, Sofia L.
Chromatin in cervical cancer (CC) undergoes chemical and structural changes that alter the expression pattern of genes. Recently, a potential mechanism, which regulates gene expression at transcriptional levels is the proteolytic clipping of histone H3. However, until now this process in CC has not been reported. Using HeLa cells as a model of CC and human samples from patients with CC, we identify that the H3 cleavage was lower in CC compared with control tissue. Additionally, the histone H3 clipping was performed by serine and aspartyl proteases in HeLa cells. These results suggest that histone H3 clipping operates as part of post-translational modification system in CC. PMID:27698925
Histone deacetylase inhibition as an alternative strategy against invasive aspergillosis
Directory of Open Access Journals (Sweden)
Frederic eLamoth
Full Text Available Invasive aspergillosis (IA is a life-threatening infection due to Aspergillus fumigatus and other Aspergillus spp. Drugs targeting the fungal cell membrane (triazoles, amphotericin B or cell wall (echinocandins are currently the sole therapeutic options against IA. Their limited efficacy and the emergence of resistance warrant the identification of new antifungal targets. Histone deacetylases (HDACs are enzymes responsible of the deacetylation of lysine residues of core histones, thus controlling chromatin remodeling and transcriptional activation. HDACs also control the acetylation and activation status of multiple non-histone proteins, including the heat shock protein 90 (Hsp90, an essential molecular chaperone for fungal virulence and antifungal resistance. This review provides an overview of the different HDACs in Aspergillus spp. as well as their respective contribution to total HDAC activity, fungal growth, stress responses, and virulence. The potential of HDAC inhibitors, currently under development for cancer therapy, as novel alternative antifungal agents against IA is discussed.
Diversification of histone H2A variants during plant evolution.
Kawashima, Tomokazu; Lorković, Zdravko J; Nishihama, Ryuichi; Ishizaki, Kimitsune; Axelsson, Elin; Yelagandula, Ramesh; Kohchi, Takayuki; Berger, Frederic
Among eukaryotes, the four core histones show an extremely high conservation of their structure and form nucleosomes that compact, protect, and regulate access to genetic information. Nevertheless, in multicellular eukaryotes the two families, histone H2A and histone H3, have diversified significantly in key residues. We present a phylogenetic analysis across the green plant lineage that reveals an early diversification of the H2A family in unicellular green algae and remarkable expansions of H2A variants in flowering plants. We define motifs and domains that differentiate plant H2A proteins into distinct variant classes. In non-flowering land plants, we identify a new class of H2A variants and propose their possible role in the emergence of the H2A.W variant class in flowering plants. Copyright © 2015 Elsevier Ltd. All rights reserved.
Post-Translational Modifications of Histones in Human Sperm.
Krejčí, Jana; Stixová, Lenka; Pagáčová, Eva; Legartová, Soňa; Kozubek, Stanislav; Lochmanová, Gabriela; Zdráhal, Zbyněk; Sehnalová, Petra; Dabravolski, Siarhei; Hejátko, Jan; Bártová, Eva
We examined the levels and distribution of post-translationally modified histones and protamines in human sperm. Using western blot immunoassay, immunofluorescence, mass spectrometry (MS), and FLIM-FRET approaches, we analyzed the status of histone modifications and the protamine P2. Among individual samples, we observed variability in the levels of H3K9me1, H3K9me2, H3K27me3, H3K36me3, and H3K79me1, but the level of acetylated (ac) histones H4 was relatively stable in the sperm head fractions, as demonstrated by western blot analysis. Sperm heads with lower levels of P2 exhibited lower levels of H3K9ac, H3K9me1, H3K27me3, H3K36me3, and H3K79me1. A very strong correlation was observed between the levels of P2 and H3K9me2. FLIM-FRET analysis additionally revealed that acetylated histones H4 are not only parts of sperm chromatin but also appear in a non-integrated form. Intriguingly, H4ac and H3K27me3 were detected in sperm tail fractions via western blot analysis. An appearance of specific histone H3 and H4 acetylation and H3 methylation in sperm tail fractions was also confirmed by both LC-MS/MS and MALDI-TOF MS analysis. Taken together, these data indicate that particular post-translational modifications of histones are uniquely distributed in human sperm, and this distribution varies among individuals and among the sperm of a single individual. © 2015 Wiley Periodicals, Inc.
Quantitative Histone Mass Spectrometry Identifies Elevated Histone H3 Lysine 27 (Lys27) Trimethylation in Melanoma.
Sengupta, Deepanwita; Byrum, Stephanie D; Avaritt, Nathan L; Davis, Lauren; Shields, Bradley; Mahmoud, Fade; Reynolds, Matthew; Orr, Lisa M; Mackintosh, Samuel G; Shalin, Sara C; Tackett, Alan J
Normal cell growth is characterized by a regulated epigenetic program that drives cellular activities such as gene transcription, DNA replication, and DNA damage repair. Perturbation of this epigenetic program can lead to events such as mis-regulation of gene transcription and diseases such as cancer. To begin to understand the epigenetic program correlated to the development of melanoma, we performed a novel quantitative mass spectrometric analysis of histone post-translational modifications mis-regulated in melanoma cell culture as well as patient tumors. Aggressive melanoma cell lines as well as metastatic melanoma were found to have elevated histone H3 Lys(27) trimethylation (H3K27me3) accompanied by overexpressed methyltransferase EZH2 that adds the specific modification. The altered epigenetic program that led to elevated H3K27me3 in melanoma cell culture was found to directly silence transcription of the tumor suppressor genes RUNX3 and E-cadherin. The EZH2-mediated silencing of RUNX3 and E-cadherin transcription was also validated in advanced stage human melanoma tissues. This is the first study focusing on the detailed epigenetic mechanisms leading to EZH2-mediated silencing of RUNX3 and E-cadherin tumor suppressors in melanoma. This study underscores the utility of using high resolution mass spectrometry to identify mis-regulated epigenetic programs in diseases such as cancer, which could ultimately lead to the identification of biological markers for diagnostic and prognostic applications. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Dissociation of histone and DNA synthesis in x-irradiated HeLa cells
International Nuclear Information System (INIS)
Bases, R.; Mendez, F.
Although histone synthesis and DNA synthesis are normally very well coordinated in HeLa cells, their histone synthesis proved relatively resistant to inhibition by ionizing radiation. During the first 24 h after 1,000 R the rate of cellular DNA synthesis progressively fell to small fractions of control values while histone synthesis with much less relative reduction. Acrylamide gel electropherograms of the acid soluble nuclear histones synthesized by irradiated HeLa cells were qualitatively normal
Inhibition of histone deacetylases sensitizes glioblastoma cells to lomustine
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Staberg, Mikkel; Michaelsen, Signe Regner; Rasmussen, Rikke Darling
are problems that call for a prompt development of novel therapeutic strategies. While only displaying modest efficacies as mono-therapy in pre-clinical settings, histone deacetylase inhibitors (HDACi) have shown promising sensitizing effects to a number of cytotoxic agents. Here, we sought to investigate...... the sensitizing effect of the HDACi trichostatin A (TSA) to the alkylating agent lomustine (CCNU), which is used in the clinic for the treatment of GBM. METHODS: Twelve primary GBM cell cultures grown as neurospheres were used in this study, as well as one established GBM-derived cell line (U87 MG). Histone...
Citrullination regulates pluripotency and histone H1 binding to chromatin
DEFF Research Database (Denmark)
Christophorou, Maria A; Castelo-Branco, Gonçalo; Halley-Stott, Richard P
citrullination of core histones has been linked to transcriptional regulation and the DNA damage response. PADI4 (also called PAD4 or PADV), the only PADI with a nuclear localization signal, was previously shown to act in myeloid cells where it mediates profound chromatin decondensation during the innate immune...... and activating their expression. Its inhibition lowers the percentage of pluripotent cells in the early mouse embryo and significantly reduces reprogramming efficiency. Using an unbiased proteomic approach we identify linker histone H1 variants, which are involved in the generation of compact chromatin, as novel...
Characterization of human papillomavirus type 16 pseudovirus containing histones.
Kim, Hyoung Jin; Kwag, Hye-Lim; Kim, Hong-Jin
Pseudoviruses (PsVs) that encapsidate a reporter plasmid DNA have been used as surrogates for native human papillomavirus (HPV), whose continuous production is technically difficult. HPV PsVs have been designed to form capsids made up of the major capsid protein L1 and the minor capsid proteins L2. HPV PsVs have been produced in 293TT cells transfected with plasmid expressing L1 and L2 protein and plasmid containing the reporter gene. Several studies have suggested that naturally occurring HPV virions contain cellular histones, and histones have also been identified in mature HPV PsVs. However, the effect of the histones on the properties of the PsVs has not been investigated. Using heparin chromatography, we separated mature HPV type 16 PsVs into three fractions (I, II, and III) according to their heparin-binding affinities. The amounts of cellular histone and cellular nucleotides per PsV were found to increase in the order fraction I, II and III. It appeared that PsVs in fraction I contains just small amount of cellular histone in Western blot analysis. The proportions of the three fractions in PsV preparations were 83.4, 7.5, and 9.1 % for fraction I, II, and III PsVs, respectively. In the electron microscope PsVs in fraction I appeared to have a more condensed structure than those in fractions II and III. Under the electron microscope fraction II and III PsVs appeared to be covered by substantial amounts of cellular histone while there was no visible histone covering PsVs of fraction I. Also the levels of reporter gene expression in infections of fraction II and III PsVs to 293TT cells were significantly lower than those in infections of fraction I PsV, and fraction II and III particles had significantly reduced immunogenicity. Our findings suggest that the involvement of large amounts of cellular histones during PsV formation interferes with the structural integrity of the PsVs and affects their immunogenicity. The fraction I particle therefore has the most
Histone signature of metanephric mesenchyme cell lines.
McLaughlin, Nathan; Yao, Xiao; Li, Yuwen; Saifudeen, Zubaida; El-Dahr, Samir S
The metanephric mesenchyme (MM) gives rise to nephrons, the filtering units of the mature kidney. The MM is composed of uninduced (Six2(high)/Lhx1(low)) and induced (Wnt-stimulated, Six2(low)/Lhx1(high)) cells. The global epigenetic state of MM cells is unknown, partly due to technical difficulty in isolating sufficient numbers of homogenous cell populations. We therefore took advantage of two mouse clonal cell lines representing the uninduced (mK3) and induced (mK4) metanephric mesenchyme (based on gene expression profiles and ability to induce branching of ureteric bud). ChIP-Seq revealed that whereas H3K4me3 active region "peaks" are enriched in metabolic genes, H3K27me3 peaks decorate mesenchyme and epithelial cell fate commitment genes. In uninduced mK3 cells, promoters of "stemness" genes (e.g., Six2, Osr1) are enriched with H3K4me3 peaks; these are lost in induced mK4 cells. ChIP-qPCR confirmed this finding and further demonstrated that G9a/H3K9me2 occupy the promoter region of Six2 in induced cells, consistent with the inactive state of transcription. Conversely, genes that mark the induced epithelialized state (e.g., Lhx1, Pax8), transition from a non-permissive to an active chromatin signature in mK3 vs. mK4 cells, respectively. Importantly, stimulation of Wnt signaling in uninduced mK3 cells provokes an active chromatin state (high H3K4me3, low H3K27me3), recruitment of β-catenin, and loss of pre-bound histone methyltransferase Ezh2 in silent induced genes followed by activation of transcription. We conclude that the chromatin signature of uninduced and induced cells correlates strongly with their gene expression states, suggesting a role of chromatin-based mechanisms in MM cell fate.
Histone deacetylase 1, 2, 6 and acetylated histone H4 in B- and T-cell lymphomas
DEFF Research Database (Denmark)
Marquard, L.; Poulsen, C.B.; Gjerdrum, L.M.
AIMS: Histone deacetylase (HDAC) inhibitors are novel therapeutics in the treatment of peripheral T-cell lymphoma, unspecified (PTCL) and diffuse large B-cell lymphoma (DLBCL), where, for unknown reasons, T-cell malignancies appear to be more sensitive than B-cell malignancies. The aim was to det......AIMS: Histone deacetylase (HDAC) inhibitors are novel therapeutics in the treatment of peripheral T-cell lymphoma, unspecified (PTCL) and diffuse large B-cell lymphoma (DLBCL), where, for unknown reasons, T-cell malignancies appear to be more sensitive than B-cell malignancies. The aim...... was to determine HDAC expression in DLBCL and PTCL which has not previously been investigated. METHODS AND RESULTS: The expression of HDAC1, HDAC2, HDAC6 and acetylated histone H4 was examined immunohistochemically in 31 DLBCL and 45 PTCL. All four markers showed high expression in both DLBCL and PTCL compared...
Further evidence for poly-ADP-ribosylated histones as DNA suppressors
International Nuclear Information System (INIS)
Yu, F.L.; Geronimo, I.H.; Bender, W.; Meginniss, K.E.
For many years histones have been considered to be the gene suppressors in eukaryotic cells. Recently, the authors have found strong evidence indicating that poly-ADP-ribosylated histones, rather than histones, are the potent inhibitors of DNA-dependent RNA synthesis. They now report additional evidence for this concept: 1) using histone inhibitor isolated directly from nuclei, the authors are able to confirm their earlier findings that the inhibitor substances are sensitive to pronase, snake venom phosphodiesterase digestion and 0.1N KOH hydrolysis, and are resistant to DNase I and RNase A digestion, 2) the O.D. 260/O.D.280 ratio of the histone inhibitor is between pure protein and nuclei acid, suggesting the inhibitor substance is a nucleoprotein hybrid. This result directly supports the fact that the isolated histone inhibitor is radioactive poly (ADP-ribose) labeled, 3) commercial histones show big differences in inhibitor activity. The authors believe this reflects the variation in poly-ADP-ribosylation among commercial histones, and 4) 0.1N KOH hydrolysis eliminates the poly (ADP-ribose) radioactivity from the acceptor proteins as well as histone inhibitor activity. Yet, on gel, the inhibitor shows identical histone bands and stain intensity before and after hydrolysis, indicating the histones per se are qualitatively and quantitatively unaffected by alkaline treatment. This result strongly suggests that histones themselves are not capable of inhibiting DNA-dependent RNA synthesis
Histone H3 Lysine Methylation in Cognition and Intellectual Disability Disorders
Parkel, Sven; Lopez-Atalaya, Jose P.; Barco, Angel
Recent research indicates that epigenetic mechanisms and, in particular, the post-translational modification (PTM) of histones may contribute to memory encoding and storage. Among the dozens of possible histone PTMs, the methylation/demethylation of lysines in the N-terminal tail of histone H3 exhibits particularly strong links with cognitive…
Neutrophil Extracellular Trap-Related Extracellular Histones Cause Vascular Necrosis in Severe GN.
Kumar, Santhosh V R; Kulkarni, Onkar P; Mulay, Shrikant R; Darisipudi, Murthy N; Romoli, Simone; Thomasova, Dana; Scherbaum, Christina R; Hohenstein, Bernd; Hugo, Christian; Müller, Susanna; Liapis, Helen; Anders, Hans-Joachim
Severe GN involves local neutrophil extracellular trap (NET) formation. We hypothesized a local cytotoxic effect of NET-related histone release in necrotizing GN. In vitro, histones from calf thymus or histones released by neutrophils undergoing NETosis killed glomerular endothelial cells, podocytes, and parietal epithelial cells in a dose-dependent manner. Histone-neutralizing agents such as antihistone IgG, activated protein C, or heparin prevented this effect. Histone toxicity on glomeruli ex vivo was Toll-like receptor 2/4 dependent, and lack of TLR2/4 attenuated histone-induced renal thrombotic microangiopathy and glomerular necrosis in mice. Anti-glomerular basement membrane GN involved NET formation and vascular necrosis, whereas blocking NET formation by peptidylarginine inhibition or preemptive anti-histone IgG injection significantly reduced all aspects of GN (i.e., vascular necrosis, podocyte loss, albuminuria, cytokine induction, recruitment or activation of glomerular leukocytes, and glomerular crescent formation). To evaluate histones as a therapeutic target, mice with established GN were treated with three different histone-neutralizing agents. Anti-histone IgG, recombinant activated protein C, and heparin were equally effective in abrogating severe GN, whereas combination therapy had no additive effects. Together, these results indicate that NET-related histone release during GN elicits cytotoxic and immunostimulatory effects. Furthermore, neutralizing extracellular histones is still therapeutic when initiated in established GN. Copyright © 2015 by the American Society of Nephrology.
Quantitative analysis of histone modifications: formaldehyde is a source of pathological n(6-formyllysine that is refractory to histone deacetylases.
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Bahar Edrissi
Full Text Available Aberrant protein modifications play an important role in the pathophysiology of many human diseases, in terms of both dysfunction of physiological modifications and the formation of pathological modifications by reaction of proteins with endogenous electrophiles. Recent studies have identified a chemical homolog of lysine acetylation, N(6-formyllysine, as an abundant modification of histone and chromatin proteins, one possible source of which is the reaction of lysine with 3'-formylphosphate residues from DNA oxidation. Using a new liquid chromatography-coupled to tandem mass spectrometry method to quantify all N(6-methyl-, -acetyl- and -formyl-lysine modifications, we now report that endogenous formaldehyde is a major source of N(6-formyllysine and that this adduct is widespread among cellular proteins in all compartments. N(6-formyllysine was evenly distributed among different classes of histone proteins from human TK6 cells at 1-4 modifications per 10(4 lysines, which contrasted strongly with lysine acetylation and mono-, di-, and tri-methylation levels of 1.5-380, 5-870, 0-1400, and 0-390 per 10(4 lysines, respectively. While isotope labeling studies revealed that lysine demethylation is not a source of N(6-formyllysine in histones, formaldehyde exposure was observed to cause a dose-dependent increase in N(6-formyllysine, with use of [(13C,(2H2]-formaldehyde revealing unchanged levels of adducts derived from endogenous sources. Inhibitors of class I and class II histone deacetylases did not affect the levels of N(6-formyllysine in TK6 cells, and the class III histone deacetylase, SIRT1, had minimal activity (<10% with a peptide substrate containing the formyl adduct. These data suggest that N(6-formyllysine is refractory to removal by histone deacetylases, which supports the idea that this abundant protein modification could interfere with normal regulation of gene expression if it arises at conserved sites of physiological protein secondary
Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant.
Earnshaw, W C; Allshire, R C; Black, B E; Bloom, K; Brinkley, B R; Brown, W; Cheeseman, I M; Choo, K H A; Copenhaver, G P; Deluca, J G; Desai, A; Diekmann, S; Erhardt, S; Fitzgerald-Hayes, M; Foltz, D; Fukagawa, T; Gassmann, R; Gerlich, D W; Glover, D M; Gorbsky, G J; Harrison, S C; Heun, P; Hirota, T; Jansen, L E T; Karpen, G; Kops, G J P L; Lampson, M A; Lens, S M; Losada, A; Luger, K; Maiato, H; Maddox, P S; Margolis, R L; Masumoto, H; McAinsh, A D; Mellone, B G; Meraldi, P; Musacchio, A; Oegema, K; O'Neill, R J; Salmon, E D; Scott, K C; Straight, A F; Stukenberg, P T; Sullivan, B A; Sullivan, K F; Sunkel, C E; Swedlow, J R; Walczak, C E; Warburton, P E; Westermann, S; Willard, H F; Wordeman, L; Yanagida, M; Yen, T J; Yoda, K; Cleveland, D W
The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.
Sulfonamide-Based Inhibitors of Aminoglycoside Acetyltransferase Eis Abolish Resistance to Kanamycin in Mycobacterium tuberculosis
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Garzan, Atefeh; Willby, Melisa J.; Green, Keith D.; Gajadeera, Chathurada S.; Hou, Caixia; Tsodikov, Oleg V.; Posey, James E.; Garneau-Tsodikova, Sylvie
A two-drug combination therapy where one drug targets an offending cell and the other targets a resistance mechanism to the first drug is a time-tested, yet underexploited approach to combat or prevent drug resistance. By high-throughput screening, we identified a sulfonamide scaffold that served as a pharmacophore to generate inhibitors of Mycobacterium tuberculosis acetyltransferase Eis, whose upregulation causes resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN) in Mycobacterium tuberculosis. Rational systematic derivatization of this scaffold to maximize Eis inhibition and abolish the Eis-mediated KAN resistance of M. tuberculosis yielded several highly potent agents. A crystal structure of Eis in complex with one of the most potent inhibitors revealed that the inhibitor bound Eis in the AG-binding pocket held by a conformationally malleable region of Eis (residues 28–37) bearing key hydrophobic residues. These Eis inhibitors are promising leads for preclinical development of innovative AG combination therapies against resistant TB.
Identification of the polyamine N8-acetyltransferase involved in the pathway of 1,3-diaminopropane production in Acanthamoeba culbertsoni.
Shukla, O P; Aisien, S O; Bergmann, B; Hellmund, C; Walter, R D
A cytosolic polyamine N-acetyltransferase that preferentially catalyzes the acetylation of spermidine in the N8-position was identified in the free-living pathogenic amoeba Acanthamoeba culbertsoni. In addition to spermidine, the enzyme also catalyzed the acetylation of spermine and putrescine with Michaelis constants (Km values) of 97, 12, and 10 microM, respectively. The Km value for acetylcoenzyme A (acetyl-CoA) was estimated to be 11 microM, whereas CoA had an inhibitory constant of 6 microM. The N-acetylase has a molecular mass of approximately 45 kDa. That the enzyme preferentially catalyzed the acetylation of spermidine at the N8-position, resulting in N8-acetylspermidine, the preferred substrate of the polyamine oxidase found in A. culbertsoni, indicates a role for the enzyme in the production of 1,3-diaminopropane, the major polyamine found in the Acanthamoeba.
Novel 6′-N-Aminoglycoside Acetyltransferase AAC(6′)-Iaj from a Clinical Isolate of Pseudomonas aeruginosa
Tada, Tatsuya; Miyoshi-Akiyama, Tohru; Shimada, Kayo; Shimojima, Masahiro
Pseudomonas aeruginosa NCGM1588 has a novel chromosomal class 1 integron, In151, which includes the aac(6′)-Iaj gene. The encoded protein, AAC(6′)-Iaj, was found to consist of 184 amino acids, with 70% identity to AAC(6′)-Ia. Escherichia coli transformed with a plasmid containing the aac(6′)-Iaj gene acquired resistance to all aminoglycosides tested except gentamicin. Of note, aac(6′)-Iaj contributed to the resistance to arbekacin. Thin-layer chromatography revealed that AAC(6′)-Iaj acetylated all aminoglycosides tested except gentamicin. These findings indicated that AAC(6′)-Iaj is a functional acetyltransferase that modifies the amino groups at the 6′ positions of aminoglycosides and contributes to aminoglycoside resistance of P. aeruginosa NCGM1588, including arbekacin. PMID:23070167
Genetic variants in microsomal epoxide hydrolase and N-acetyltransferase 2 in susceptibility of IBD in the Danish population
DEFF Research Database (Denmark)
Ernst, Anja; Andersen, Vibeke; Østergaard, Mette
, or severity of disease measured either as need for surgery or azathioprine treatment. Smoking was found to be a risk factor of CD (OR=1.8(1.4; 2.3) Pfactor regarding UC (0.7 (0.5-0.9) P=0.02) which is in agreement with previous findings in other study...... induce or sustain an immune response. Changes in detoxification of substances that causes epithelial damage may confer susceptibility to IBD. Hence, polymorphic enzymes involved in the detoxification processes may be risk factors of IBD. Methods. The two biotransformation enzymes microsomal epoxide...... hydrolase and N-acetyltransferase 2 were genotyped using TaqMan based Real-Time PCR in 388 patients with Crohn's disease (CD), 565 patients with ulcerative colitis (UC) and 796 healthy Danish controls. Results. No association was found between low microsomal epoxide hydrolase activity or slow N...
Prospective virtual screening with Ultrafast Shape Recognition: the identification of novel inhibitors of arylamine N-acetyltransferases
Ballester, Pedro J.; Westwood, Isaac; Laurieri, Nicola; Sim, Edith; Richards, W. Graham
There is currently a shortage of chemical molecules that can be used as bioactive probes to study molecular targets and potentially as starting points for drug discovery. One inexpensive way to address this problem is to use computational methods to screen a comprehensive database of small molecules to discover novel structures that could lead to alternative and better bioactive probes. Despite that pleasing logic the results have been somewhat mixed. Here we describe a virtual screening technique based on ligand–receptor shape complementarity, Ultrafast Shape Recognition (USR). USR is specifically applied to identify novel inhibitors of arylamine N-acetyltransferases by computationally screening almost 700 million molecular conformers in a time- and resource-efficient manner. A small number of the predicted active compounds were purchased and tested obtaining a confirmed hit rate of 40 per cent which is an outstanding result for a prospective virtual screening. PMID:19586957
Bifurcated Degradative Pathway of 3-Sulfolactate in Roseovarius nubinhibens ISM via Sulfoacetaldehyde Acetyltransferase and (S)-Cysteate Sulfolyase ▿ †
Denger, Karin; Mayer, Jutta; Buhmann, Matthias; Weinitschke, Sonja; Smits, Theo H. M.; Cook, Alasdair M.
Data from the genome sequence of the aerobic, marine bacterium Roseovarius nubinhibens ISM were interpreted such that 3-sulfolactate would be degraded as a sole source of carbon and energy for growth via a novel bifurcated pathway including two known desulfonative enzymes, sulfoacetaldehyde acetyltransferase (EC 220.127.116.11) (Xsc) and cysteate sulfo-lyase (EC 220.127.116.11) (CuyA). Strain ISM utilized sulfolactate quantitatively with stoichiometric excretion of the sulfonate sulfur as sulfate. A combination of enzyme assays, analytical chemistry, enzyme purification, peptide mass fingerprinting, and reverse transcription-PCR data supported the presence of an inducible, tripartite sulfolactate uptake system (SlcHFG), and a membrane-bound sulfolactate dehydrogenase (SlcD) which generated 3-sulfopyruvate, the point of bifurcation. 3-Sulfopyruvate was in part decarboxylated by 3-sulfopyruvate decarboxylase (EC 126.96.36.199) (ComDE), which was purified. The sulfoacetaldehyde that was formed was desulfonated by Xsc, which was identified, and the acetyl phosphate was converted to acetyl-coenzyme A by phosphate acetyltransferase (Pta). The other portion of the 3-sulfopyruvate was transaminated to (S)-cysteate, which was desulfonated by CuyA, which was identified. The sulfite that was formed was presumably exported by CuyZ (TC 9.B.7.1.1 in the transport classification system), and a periplasmic sulfite dehydrogenase is presumed. Bioinformatic analyses indicated that transporter SlcHFG is rare but that SlcD is involved in three different combinations of pathways, the bifurcated pathway shown here, via CuyA alone, and via Xsc alone. This novel pathway involves ComDE in biodegradation, whereas it was discovered in the biosynthesis of coenzyme M. The different pathways of desulfonation of sulfolactate presumably represent final steps in the biodegradation of sulfoquinovose (and exudates derived from it) in marine and aquatic environments. PMID:19581363
Thermodynamics of aminoglycoside and acyl-coenzyme A binding to the Salmonella enterica AAC(6')-Iy aminoglycoside N-acetyltransferase.
Hegde, Subray S; Dam, Tarun K; Brewer, C Fred; Blanchard, John S
Kinetic and mechanistic studies on the chromosomally encoded aminoglycoside 6'-N-acetyltransferase, AAC(6')-Iy, of Salmonella enterica that confers resistance toward aminoglycosides have been previously reported [Magnet et al. (2001) Biochemistry 40, 3700-3709]. In the present study, equilibrium binding and the thermodynamic parameters of binding of aminoglycosides and acyl-coenzyme A derivatives to AAC(6')-Iy and of two mutants, C109A and the C109A/C70A double mutant, have been studied using fluorescence spectroscopy and isothermal titration calorimetry (ITC). Association constants for different aminoglycosides varied greatly (4 x 10(4)-150 x 10(4)) while the association constants of several acyl-coenzyme A derivatives were similar (3.2 x 10(4)-4.5 x 10(4)). The association constants and van't Hoff enthalpy changes derived from intrinsic protein fluorescence changes were in agreement with independently measured values from isothermal titration calorimetry studies. Binding of both aminoglycosides and acyl-coenzyme A derivatives is strongly enthalpically driven and revealed opposing negative entropy changes, resulting in enthalpy-entropy compensation. The acetyltransferase exhibited a temperature-dependent binding of tobramycin with a negative heat capacity value of 410 cal mol(-1) K(-1). Isothermal titration studies of acetyl-coenzyme A and tobramycin binding to mutant forms of the enzyme indicated that completely conserved C109 does not play any direct role in the binding of either of the substrates, while C70 is directly involved in aminoglycoside binding. These results are discussed and compared with previous steady-state kinetic studies of the enzyme.
Toxic effects of extracellular histones and their neutralization by vitreous in retinal detachment.
Kawano, Hiroki; Ito, Takashi; Yamada, Shingo; Hashiguchi, Teruto; Maruyama, Ikuro; Hisatomi, Toshio; Nakamura, Makoto; Sakamoto, Taiji
Histones are DNA-binding proteins and are involved in chromatin remodeling and regulation of gene expression. Histones can be released after tissue injuries, and the extracellular histones cause cellular damage and organ dysfunction. Regardless of their clinical significance, the role and relevance of histones in ocular diseases are unknown. We studied the role of histones in eyes with retinal detachment (RD). Vitreous samples were collected during vitrectomy, and the concentration of histone H3 was measured by enzyme-linked immunosorbent assay. The location of the histones and related molecules was examined in a rat RD model. The release of histones and their effects on rat retinal progenitor cells R28 and ARPE-19 were evaluated in vitro. In addition, the protective role of the vitreous body against histones was tested. The intravitreal concentration of histones was higher in eyes with RD (mean, 30.9 ± 9.8 ng/ml) than in control eyes (below the limit of detection, Phistone H3 was observed on the outer side of the detached retina and was associated with photoreceptor death. Histone H3 was released from cultured R28 by oxidative stress. Histones at a concentration 10 μg/ml induced the production of interleukin-8 in ARPE-19 cells (2.5-fold increase, PHistones were toxic to cells at concentrations of ≥ 20 μg/ml. Vitreous body or hyaluronan decreased toxicity of histones by inhibiting diffusion of histones. These results indicate that histones are released from retinas with RD and may modulate the subretinal microenvironment by functioning as damage-associated molecular pattern molecules, thereby inducing proinflammatory cytokines or cell toxicity. In addition, the important role of the vitreous body and hyaluronan in protecting the retina from these toxic effects is suggested.
Hypothalamic leptin action is mediated by histone deacetylase 5
DEFF Research Database (Denmark)
Kabra, Dhiraj G; Pfuhlmann, Katrin; García-Cáceres, Cristina
Hypothalamic leptin signalling has a key role in food intake and energy-balance control and is often impaired in obese individuals. Here we identify histone deacetylase 5 (HDAC5) as a regulator of leptin signalling and organismal energy balance. Global HDAC5 KO mice have increased food intake and...
Histone Deacetylase Inhibition Protects Mice Against Lethal Postinfluenza Pneumococcal Infection.
Yagi, Kazuma; Ishii, Makoto; Namkoong, Ho; Fujii, Hideki; Asami, Takahiro; Suzuki, Shoji; Asakura, Takanori; Mizoguchi, Kosuke; Kamo, Tetsuro; Tasaka, Sadatomo; Iwata, Satoshi; Kunkel, Steven L; Hasegawa, Naoki; Betsuyaku, Tomoko
Secondary bacterial pneumonia following influenza virus infection is associated with high mortality, but the mechanism is largely unknown. Epigenetic gene regulation appears to play key roles in innate and adaptive immunity. We hypothesized that histone acetylation, a major epigenetic mechanism associated with transcriptionally active chromatin, might contribute to the poor outcome of postinfluenza pneumonia. Prospective experimental study. University research laboratory. C57BL/6 male mice. Mice were infected intranasally with 1.0 × 10 colony-forming units of Streptococcus pneumoniae, 7 days after intranasal inoculation with five plaque-forming units of influenza virus A/H1N1/PR8/34. The mice were intraperitoneally injected with the histone deacetylase inhibitor trichostatin A (1 mg/kg) or vehicle once a day from 1 hour after pneumococcal infection throughout the course of the experiment. The primary outcome was survival rate. Trichostatin A significantly suppressed histone deacetylase activity and significantly improved the survival rate of mice (56.3%) after postinfluenza pneumococcal infection when compared with vehicle-treated mice (20.0%), which was associated with a significant decrease in the total cell count of the bronchoalveolar lavage fluid. The interleukin-1β level in the serum and the number of natural killer cells in the lungs were significantly lower in the trichostatin A-treated group. The histone deacetylase inhibitor trichostatin A protects mice against postinfluenza pneumonia possibly through multiple factors, including decreasing local cell recruitment into the lungs and suppressing systemic inflammation.
acetyl (Lys14)-histone H3 predominantly mark the pericentromeric ...
Indian Academy of Sciences (India)
Epigenetic regulatory posttranslational histone modification marks not only function individually but also capable to act in combination as a unique pattern. A total of 16 plant species belonging to 11 genera of eight families (five dicots and threemonocots) including land plants, epiphytes (orchids) and the holokinetic taxa ...
histone H3 predominantly mark the pericentromeric chromatin ...
Indian Academy of Sciences (India)
2Department of Rehabilitation Sciences, Kansai University of Welfare Sciences, Kashiwara, Osaka 582-0026, Japan. Abstract. Epigenetic regulatory posttranslational histone modification marks not only function individually but also capable to act in combination as a unique pattern. A total of 16 plant species belonging to 11 ...
Cell shape regulates global histone acetylation in human mammaryepithelial cells
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Le Beyec, Johanne; Xu, Ren; Lee, Sun-Young; Nelson, Celeste M.; Rizki, Aylin; Alcaraz, Jordi; Bissell, Mina J.
Extracellular matrix (ECM) regulates cell morphology and gene expression in vivo; these relationships are maintained in three-dimensional (3D) cultures of mammary epithelial cells. In the presence of laminin-rich ECM (lrECM), mammary epithelial cells round up and undergo global histone deacetylation, a process critical for their functional differentiation. However, it remains unclear whether lrECM-dependent cell rounding and global histone deacetylation are indeed part of a common physical-biochemical pathway. Using 3D cultures as well as nonadhesive and micropatterned substrata, here we showed that the cell 'rounding' caused by lrECM was sufficient to induce deacetylation of histones H3 and H4 in the absence of biochemical cues. Microarray and confocal analysis demonstrated that this deacetylation in 3D culture is associated with a global increase in chromatin condensation and a reduction in gene expression. Whereas cells cultured on plastic substrata formed prominent stress fibers, cells grown in 3D lrECM or on micropatterns lacked these structures. Disruption of the actin cytoskeleton with cytochalasin D phenocopied the lrECM-induced cell rounding and histone deacetylation. These results reveal a novel link between ECM-controlled cell shape and chromatin structure, and suggest that this link is mediated by changes in the actin cytoskeleton.
Post-Translational Modifications of Histones in Human Sperm
Czech Academy of Sciences Publication Activity Database
Krejčí, Jana; Stixová, Lenka; Legartová, Soňa; Kozubek, Stanislav; Lochmanová, G.; Zdráhal, Z.; Sehnalová, Petra; Dabravolski, S.; Hejatko, J.; Bártová, Eva
Roč. 116, č. 10 (2015), s. 2195-2209 ISSN 0730-2312 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0068 Institutional support: RVO:68081707 Keywords : HUMAN SPERM * HISTONES * PROTAMINE P2 Subject RIV: BO - Biophysics Impact factor: 3.446, year: 2015
Differential patterns of histone acetylation in inflammatory bowel diseases
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Adcock Ian M
Full Text Available Abstract Post-translational modifications of histones, particularly acetylation, are associated with the regulation of inflammatory gene expression. We used two animal models of inflammation of the bowel and biopsy samples from patients with Crohn's disease (CD to study the expression of acetylated histones (H 3 and 4 in inflamed mucosa. Acetylation of histone H4 was significantly elevated in the inflamed mucosa in the trinitrobenzene sulfonic acid model of colitis particularly on lysine residues (K 8 and 12 in contrast to non-inflamed tissue. In addition, acetylated H4 was localised to inflamed tissue and to Peyer's patches (PP in dextran sulfate sodium (DSS-treated rat models. Within the PP, H3 acetylation was detected in the mantle zone whereas H4 acetylation was seen in both the periphery and the germinal centre. Finally, acetylation of H4 was significantly upregulated in inflamed biopsies and PP from patients with CD. Enhanced acetylation of H4K5 and K16 was seen in the PP. These results demonstrate that histone acetylation is associated with inflammation and may provide a novel therapeutic target for mucosal inflammation.
Natural and Synthetic Macrocyclic Inhibitors of the Histone Deacetylase Enzymes
DEFF Research Database (Denmark)
Maolanon, Alex; Kristensen, Helle; Leman, Luke
Inhibition of histone deacetylase (HDAC) enzymes has emerged as a target for development of cancer chemotherapy. Four compounds have gained approval for clinical use by the Food and Drug Administration (FDA) in the US, and several are currently in clinical trials. However, none of these compounds...
Identification of catechols as histone-lysine demethylase inhibitors
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Nielsen, Anders L; Kristensen, Line H; Stephansen, Karen B
Identification of inhibitors of histone-lysine demethylase (HDM) enzymes is important because of their involvement in the development of cancer. An ELISA-based assay was developed for identification of inhibitors of the HDM KDM4C in a natural products library. Based on one of the hits with affinity...
Implication of Posttranslational Histone Modifications in Nucleotide Excision Repair
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Shisheng Li
Full Text Available Histones are highly alkaline proteins that package and order the DNA into chromatin in eukaryotic cells. Nucleotide excision repair (NER is a conserved multistep reaction that removes a wide range of generally bulky and/or helix-distorting DNA lesions. Although the core biochemical mechanism of NER is relatively well known, how cells detect and repair lesions in diverse chromatin environments is still under intensive research. As with all DNA-related processes, the NER machinery must deal with the presence of organized chromatin and the physical obstacles it presents. A huge catalogue of posttranslational histone modifications has been documented. Although a comprehensive understanding of most of these modifications is still lacking, they are believed to be important regulatory elements for many biological processes, including DNA replication and repair, transcription and cell cycle control. Some of these modifications, including acetylation, methylation, phosphorylation and ubiquitination on the four core histones (H2A, H2B, H3 and H4 or the histone H2A variant H2AX, have been found to be implicated in different stages of the NER process. This review will summarize our recent understanding in this area.
Effect of histone deacetylase inhibitor, trichostatin A, on cartilage ...
African Journals Online (AJOL)
Purpose: To evaluate the effect of histone deacetylase (HDAC) inhibitor, trichostatin A (TCA), on cartilage regeneration in a rabbit perichondrial graft model. Methods: Perichondrial grafts (20 × 20 mm2) were derived from the ears of New Zealand rabbits and transplanted onto the paravertebral muscle of the face of each ...
Sepsis and ARDS: The Dark Side of Histones
Xu, Zhiheng; Huang, Yongbo; Mao, Pu; Zhang, Jianrong; Li, Yimin
Despite advances in management over the last several decades, sepsis and acute respiratory distress syndrome (ARDS) still remain major clinical challenges and the leading causes of death for patients in intensive care units (ICUs) due to insufficient understanding of the pathophysiological mechanisms of these diseases. However, recent studies have shown that histones, also known as chromatin-basic structure proteins, could be released into the extracellular space during severe stress and physical challenges to the body (e.g., sepsis and ARDS). Due to their cytotoxic and proinflammatory effects, extracellular histones can lead to excessive and overwhelming cell damage and death, thus contributing to the pathogenesis of both sepsis and ARDS. In addition, antihistone-based treatments (e.g., neutralizing antibodies, activated protein C, and heparin) have shown protective effects and have significantly improved the outcomes of mice suffering from sepsis and ARDS. Here, we review researches related to the pathological role of histone in context of sepsis and ARDS and evaluate the potential value of histones as biomarkers and therapeutic targets of these diseases. PMID:26609197
Relationship of histone acetylation to DNA topology and transcription.
Krajewski, W A; Luchnik, A N
An autonomously replicating plasmid constructed from bovine papiloma virus (BPV) and pBR322 was stably maintained as a nuclear episome in a mouse cell culture. Addition to a cell culture of sodium butyrate (5 mM) induced an increase in plasmid DNA supercoiling of 3-5 turns, an increase in acetylation of cellular histones, and a decrease in plasmid transcription by 2- to 4-fold. After withdrawal of butyrate, DNA supercoiling began to fluctuate in a wave-like manner with an amplitude of up to 3 turns and a period of 3-4 h. These waves gradually faded by 24 h. The transcription of the plasmid and acetylation of cellular histones also oscillated with the same period. The wave-like alterations were not correlated with the cell cycle, for there was no resumption of DNA replication after butyrate withdrawal for at least 24 h. In vitro chemical acetylation of histones with acetyl adenylate also led to an increase in the superhelical density of plasmid DNA. The parallel changes in transcription, histone acetylation, and DNA supercoiling in vivo may indicate a functional innerconnection. Also, the observed in vivo variation in the level of DNA supercoiling directly indicates the possibility of its natural regulation in eukaryotic cells.
Structural and histone binding ability characterizations of human PWWP domains.
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Hong Wu
Full Text Available The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently.The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members, implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3.PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical β-barrel core, an insertion motif between the second and third β-strands and a C-terminal α-helix bundle. Both the canonical β-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones.This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the
Analysis of the histone protein tail and DNA in nucleosome using molecular dynamics simulation
Fujimori, R.; Komatsu, Y.; Fukuda, M.; Miyakawa, T.; Morikawa, R.; Takasu, M.
We study the effect of the tails of H3 and H4 histones in the nucleosomes, where DNA and histones are packed in the form of chromatin. We perform molecular dynamics simulations of the complex of DNA and histones and calculate the mean square displacement and the gyration radius of the complex of DNA and histones for the cases with tails intact and the cases with tails missing. Our results show that the H3 tails are important for the motion of the histones. We also find that the motion of one tail is affected by other tails, although the tails are distanced apart, suggesting the correlated motion in biological systems.
HiHiMap: single-cell quantitation of histones and histone posttranslational modifications across the cell cycle by high-throughput imaging.
Zane, Linda; Chapus, Fleur; Pegoraro, Gianluca; Misteli, Tom
We describe Hi gh-throughput Hi stone Map ping (HiHiMap), a high-throughput imaging method to measure histones and histone posttranslational modifications (PTMs) in single cells. HiHiMap uses imaging-based quantification of DNA and cyclin A to stage individual cells in the cell cycle to determine the levels of histones or histone PTMs in each stage of the cell cycle. As proof of principle, we apply HiHiMap to measure the level of 21 core histones, histone variants, and PTMs in primary, immortalized, and transformed cells. We identify several histone modifications associated with oncogenic transformation. HiHiMap allows the rapid, high-throughput study of histones and histone PTMs across the cell cycle and the study of subpopulations of cells. © 2017 Zane et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
Arginine-rich histones have strong antiviral activity for influenza A viruses.
Hoeksema, Marloes; Tripathi, Shweta; White, Mitchell; Qi, Li; Taubenberger, Jeffery; van Eijk, Martin; Haagsman, Henk; Hartshorn, Kevan L
While histones are best known for DNA binding and transcription-regulating properties, they also have antimicrobial activity against a broad range of potentially pathogenic organisms. Histones are abundant in neutrophil extracellular traps, where they play an important role in NET-mediated antimicrobial killing. Here, we show anti-influenza activity of histones against both seasonal H3N2 and H1N1, but not pandemic H1N1. The arginine rich histones, H3 and H4, had greater neutralizing and viral aggregating activity than the lysine rich histones, H2A and H2B. Of all core histones, histone H4 is most potent in neutralizing IAV, and incubation with IAV with histone H4 results in a decrease in uptake and viral replication by epithelial cells when measured by qRT-PCR. The antiviral activity of histone H4 is mediated principally by direct effects on viral particles. Histone H4 binds to IAV as assessed by ELISA and co-sedimentation of H4 with IAV. H4 also induces aggregation, as assessed by confocal microscopy and light transmission assays. Despite strong antiviral activity against the seasonal IAV strains, H4 was inactive against pandemic H1N1. These findings indicate a possible role for histones in the innate immune response against IAV. © The Author(s) 2015.
The relationship between DNA synthesis and incorporation of (14C) lysine into different histone fractions in Ehrlich ascites tumour cells
International Nuclear Information System (INIS)
Malec, J.; Kornacka, L.; Wojnarowska, M.; Moscicka, M.
The effect of inhibition of DNA synthesis by hydroxyurea on ( 14 C) lysine incorporation into the main four histone fractions in Ehrlich ascites tumor cells, was examined in vitro. The radioactivity of lysine-rich histones, especially of histone f1, was preferentially decreased. The smallest decrease was observed for histone f3. The incorporation into other cellular proteins was but slightly affected. (author)
Histone and Ribosomal RNA Repetitive Gene Clusters of the Boll Weevil are Linked in a Tandem Array
Histones are the major protein component of chromatin structure. The histone family is made up of a quintet of proteins, four core histones (H2A, H2B, H3 & H4) and the linker histones (H1). Spacers are found between the coding regions. Among insects this quintet of genes is usually clustered and ...
Substrate- and Cofactor-independent Inhibition of Histone Demethylase KDM4C
DEFF Research Database (Denmark)
Leurs, Ulrike; Lohse, Brian; Rand, Kasper Dyrberg
Inhibition of histone demethylases has within recent years advanced into a new strategy for treating cancer and other diseases. Targeting specific histone demethylases can be challenging as the active sites of KDM1A-B and KDM-4A-D histone demethylases, respectively, are highly conserved. Most...... inhibitors developed up-to-date target either the cofactor- or substrate-binding sites of these enzymes, resulting in a lack of selectivity and off-target effects. This study describes the discovery of the first peptide-based inhibitors of KDM4 histone demethylases that do not share the histone peptide...... sequence, or inhibit through substrate competition. Through screening of DNA-encoded peptide libraries against KDM1 and -4 histone demethylases by phage display, two cyclic peptides targeting the histone demethylase KDM4C were identified and developed as inhibitors by amino acid replacement, truncation...
Nuclear levels and patterns of histone H3 modification and HP1 proteins after inhibition of histone deacetylases
Czech Academy of Sciences Publication Activity Database
Bártová, Eva; Pacherník, J.; Harničarová, Andrea; Kovařík, Aleš; Kovaříková, Martina; Hofmanová, Jiřina; Skalníková, M.; Kozubek, Michal; Kozubek, Stanislav
Roč. 118, č. 21 (2005), s. 5035-5046 R&D Projects: GA AV ČR(CZ) 1QS500040508; GA ČR(CZ) GA202/04/0907 Institutional research plan: CEZ:AV0Z50040507 Keywords : histone H3 * HDAC inhibitors * HP1 proteins Subject RIV: BO - Biophysics
A histone H3K9M mutation traps histone methyltransferase Clr4 to prevent heterochromatin spreading
Energy Technology Data Exchange (ETDEWEB)
Shan, Chun-Min; Wang, Jiyong; Xu, Ke; Chen, Huijie; Yue, Jia-Xing; Andrews, Stuart; Moresco, James J.; Yates, John R.; Nagy, Peter L.; Tong, Liang; Jia, Songtao
Histone lysine-to-methionine (K-to-M) mutations are associated with multiple cancers, and they function in a dominant fashion to block the methylation of corresponding lysines on wild type histones. However, their mechanisms of function are controversial. Here we show that in fission yeast, introducing the K9M mutation into one of the three histone H3 genes dominantly blocks H3K9 methylation on wild type H3 across the genome. In addition, H3K9M enhances the interaction of histone H3 tail with the H3K9 methyltransferase Clr4 in a SAM (S-adenosyl-methionine)-dependent manner, and Clr4 is trapped at nucleation sites to prevent its spreading and the formation of large heterochromatin domains. We further determined the crystal structure of an H3K9M peptide in complex with human H3K9 methyltransferase G9a and SAM, which reveales that the methionine side chain had enhanced van der Waals interactions with G9a. Therefore, our results provide a detailed mechanism by which H3K9M regulates H3K9 methylation.
Accumulation of histone variant H3.3 with age is associated with profound changes in the histone methylation landscape
DEFF Research Database (Denmark)
Tvardovskiy, Andrey; Schwämmle, Veit; Kempf, Stefan J
Deposition of replication-independent histone variant H3.3 into chromatin is essential for many biological processes, including development and reproduction. Unlike replication-dependent H3.1/2 isoforms, H3.3 is expressed throughout the cell cycle and becomes enriched in postmitotic cells with age...
Histones induce phosphatidylserine exposure and a procoagulant phenotype in human red blood cells.
Semeraro, F; Ammollo, C T; Esmon, N L; Esmon, C T
Extracellular histones exert part of their prothrombotic activity through the stimulation of blood cells. Besides platelets, histones can bind to red blood cells (RBCs), which are important contributors to thrombogenesis, but little is known about the functional consequences of this interaction. To evaluate the effect of histones on the procoagulant potential of human RBCs with particular regard to the expression of surface phosphatidylserine (PS). PS exposure on human RBCs treated with a natural mixture of histones or recombinant individual histones was evaluated with fluorescein isothiocyanate-annexin-V binding and measured with flow cytometry. Calcium influx in RBCs loaded with the calcium-sensitive fluorophore Fluo-4 AM was assessed with flow cytometry. The procoagulant potential of histone-treated RBCs was evaluated with a purified prothrombinase assay and a one-stage plasma recalcification clotting test. Natural histones induced PS exposure on RBCs in a dose-dependent manner, and neutralization or cleavage of histones by heparin or activated protein C, respectively, abolished PS externalization. H4 was mainly responsible for the stimulating activity of histones, whereas the other subtypes were almost ineffective. Similarly, natural histones and H4 induced influx of calcium into RBCs, whereas the other individual histones did not. Histone-induced exposure of PS on RBCs translated into increased prothrombinase complex-mediated prothrombin activation and accelerated fibrin formation in plasma. Histones induce RBCs to express a procoagulant phenotype through the externalization of PS. This finding provides new insights into the prothrombotic activity of extracellular histones. © 2014 International Society on Thrombosis and Haemostasis.
Vorinostat, a histone deacetylase (HDAC) inhibitor, promotes cell cycle arrest and re-sensitizes rituximab- and chemo-resistant lymphoma cells to chemotherapy agents.
Xue, Kai; Gu, Juan J; Zhang, Qunling; Mavis, Cory; Hernandez-Ilizaliturri, Francisco J; Czuczman, Myron S; Guo, Ye
Preclinical models of chemotherapy resistance and clinical observations derived from the prospective multicenter phase III collaborative trial in relapsed aggressive lymphoma (CORAL) study demonstrated that primary refractory/relapsed B cell diffuse large B cell lymphoma has a poor clinical outcome with current available second-line treatments. Preclinically, we found that rituximab resistance is associated with a deregulation on the mitochondrial potential rendering lymphoma cells resistant to chemotherapy-induced apoptotic stimuli. There is a dire need to develop agents capable to execute alternative pathways of cell death in an attempt to overcome chemotherapy resistance. Posttranscriptional histone modification plays an important role in regulating gene transcription and is altered by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs regulate several key cellular functions, including cell proliferation, cell cycle, apoptosis, angiogenesis, migration, antigen presentation, and/or immune regulation. Given their influence in multiple regulatory pathways, HDAC inhibition is an attractive strategy to evaluate its anti-proliferation activity in cancer cells. To this end, we studied the anti-proliferation activity and mechanisms of action of suberoylanilide hydroxamic acid (SAHA, vorinostat) in rituximab-chemotherapy-resistant preclinical models. A panel of rituximab-chemotherapy-sensitive (RSCL) and rituximab-chemotherapy-resistant cell lines (RRCL) and primary tumor cells isolated from relapsed/refractory B cell lymphoma patients were exposed to escalating doses of vorinostat. Changes in mitochondrial potential, ATP synthesis, and cell cycle distribution were determined by Alamar blue reduction, Titer-Glo luminescent assays, and flow cytometric, respectively. Protein lysates were isolated from vorinostat-exposed cells, and changes in members of Bcl-2 family, cell cycle regulatory proteins, and the acetylation status of histone H3 were
High-resolution structure of the native histone octamer
Energy Technology Data Exchange (ETDEWEB)
Wood, Christopher M., E-mail: firstname.lastname@example.com [School of Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF (United Kingdom); Nicholson, James M. [College of Biology and Medicine, CCLRC Daresbury Laboratory, Warrington, Cheshire WA4 4AD (United Kingdom); Lambert, Stanley J. [School of Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF (United Kingdom); Chantalat, Laurent [Structural Biology, Galderma R& D, 635 Routes des Lucioles, BP 87F-06902 Sophia Antipolis CEDEX (France); Reynolds, Colin D. [School of Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF (United Kingdom); Baldwin, John P. [College of Biology and Medicine, CCLRC Daresbury Laboratory, Warrington, Cheshire WA4 4AD (United Kingdom); School of Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF (United Kingdom)
The high-resolution (1.90 Å) model of the native histone octamer allows structural comparisons to be made with the nucleosome-core particle, along with an identification of a likely core-histone binding site. Crystals of native histone octamers (H2A–H2B)–(H4–H3)–(H3′–H4′)–(H2B′–H2A′) from chick erythrocytes in 2 M KCl, 1.35 M potassium phosphate pH 6.9 diffract X-rays to 1.90 Å resolution, yielding a structure with an R{sub work} value of 18.7% and an R{sub free} of 22.2%. The crystal space group is P6{sub 5}, the asymmetric unit of which contains one complete octamer. This high-resolution model of the histone-core octamer allows further insight into intermolecular interactions, including water molecules, that dock the histone dimers to the tetramer in the nucleosome-core particle and have relevance to nucleosome remodelling. The three key areas analysed are the H2A′–H3–H4 molecular cluster (also H2A–H3′–H4′), the H4–H2B′ interaction (also H4′–H2B) and the H2A′–H4 β-sheet interaction (also H2A–H4′). The latter of these three regions is important to nucleosome remodelling by RNA polymerase II, as it is shown to be a likely core-histone binding site, and its disruption creates an instability in the nucleosome-core particle. A majority of the water molecules in the high-resolution octamer have positions that correlate to similar positions in the high-resolution nucleosome-core particle structure, suggesting that the high-resolution octamer model can be used for comparative studies with the high-resolution nucleosome-core particle.
High-resolution structure of the native histone octamer
International Nuclear Information System (INIS)
Wood, Christopher M.; Nicholson, James M.; Lambert, Stanley J.; Chantalat, Laurent; Reynolds, Colin D.; Baldwin, John P.
The high-resolution (1.90 Å) model of the native histone octamer allows structural comparisons to be made with the nucleosome-core particle, along with an identification of a likely core-histone binding site. Crystals of native histone octamers (H2A–H2B)–(H4–H3)–(H3′–H4′)–(H2B′–H2A′) from chick erythrocytes in 2 M KCl, 1.35 M potassium phosphate pH 6.9 diffract X-rays to 1.90 Å resolution, yielding a structure with an R work value of 18.7% and an R free of 22.2%. The crystal space group is P6 5 , the asymmetric unit of which contains one complete octamer. This high-resolution model of the histone-core octamer allows further insight into intermolecular interactions, including water molecules, that dock the histone dimers to the tetramer in the nucleosome-core particle and have relevance to nucleosome remodelling. The three key areas analysed are the H2A′–H3–H4 molecular cluster (also H2A–H3′–H4′), the H4–H2B′ interaction (also H4′–H2B) and the H2A′–H4 β-sheet interaction (also H2A–H4′). The latter of these three regions is important to nucleosome remodelling by RNA polymerase II, as it is shown to be a likely core-histone binding site, and its disruption creates an instability in the nucleosome-core particle. A majority of the water molecules in the high-resolution octamer have positions that correlate to similar positions in the high-resolution nucleosome-core particle structure, suggesting that the high-resolution octamer model can be used for comparative studies with the high-resolution nucleosome-core particle
Genome-wide analysis of regions similar to promoters of histone genes
Chowdhary, Rajesh
Background: The purpose of this study is to: i) develop a computational model of promoters of human histone-encoding genes (shortly histone genes), an important class of genes that participate in various critical cellular processes, ii) use the model so developed to identify regions across the human genome that have similar structure as promoters of histone genes; such regions could represent potential genomic regulatory regions, e.g. promoters, of genes that may be coregulated with histone genes, and iii/ identify in this way genes that have high likelihood of being coregulated with the histone genes.Results: We successfully developed a histone promoter model using a comprehensive collection of histone genes. Based on leave-one-out cross-validation test, the model produced good prediction accuracy (94.1% sensitivity, 92.6% specificity, and 92.8% positive predictive value). We used this model to predict across the genome a number of genes that shared similar promoter structures with the histone gene promoters. We thus hypothesize that these predicted genes could be coregulated with histone genes. This hypothesis matches well with the available gene expression, gene ontology, and pathways data. Jointly with promoters of the above-mentioned genes, we found a large number of intergenic regions with similar structure as histone promoters.Conclusions: This study represents one of the most comprehensive computational analyses conducted thus far on a genome-wide scale of promoters of human histone genes. Our analysis suggests a number of other human genes that share a high similarity of promoter structure with the histone genes and thus are highly likely to be coregulated, and consequently coexpressed, with the histone genes. We also found that there are a large number of intergenic regions across the genome with their structures similar to promoters of histone genes. These regions may be promoters of yet unidentified genes, or may represent remote control regions that
Circulating Extracellular Histones Are Clinically Relevant Mediators of Multiple Organ Injury.
Kawai, Chihiro; Kotani, Hirokazu; Miyao, Masashi; Ishida, Tokiko; Jemail, Leila; Abiru, Hitoshi; Tamaki, Keiji
Extracellular histones are a damage-associated molecular pattern (DAMP) involved in the pathogenesis of various diseases. The mechanisms of histone-mediated injury in certain organs have been extensively studied, but an understanding of the pathophysiological role of histone-mediated injury in multiple organ injury remains elusive. To elucidate this role, we systemically subjected C57BL/6 mice to various doses of histones and performed a chronological evaluation of the morphological and functional changes in the lungs, liver, and kidneys. Notably, histone administration ultimately led to death after a dose-dependent aggravation of multiple organ injury. In chronological studies, pulmonary and hepatic injuries occurred within 15 minutes, whereas renal injuries presented at a later phase, suggesting that susceptibility to extracellular histones varies among organs. Histones bound to pulmonary and hepatic endothelial cells immediately after administration, leading to endothelial damage, which could be ameliorated by pretreatment with heparin. Furthermore, release of another DAMP, high-mobility group protein box 1, followed the histone-induced tissue damage, and an antibody against the molecule ameliorated hepatic and renal failure in a late phase. These findings indicate that extracellular histones induce multiple organ injury in two progressive stages-direct injury to endothelial cells and the subsequent release of other DAMPs-and that combination therapies against extracellular histones and high-mobility group protein box 1 may be a promising strategy for treating multiple organ injury. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
Circulating histones for predicting prognosis after cardiac surgery: a prospective study.
Gao, Hongxiang; Zhang, Naipu; Lu, Fangfang; Yu, Xindi; Zhu, Limin; Mo, Xi; Wang, Wei
The objective of this study was to assess the perioperative changes in circulating histones and their relationships with other biomarkers and clinical outcomes after cardiac surgery with cardiopulmonary bypass (CPB) in patients. Forty-eight patients with congenital cardiac diseases undergoing corrective procedure with CPB were prospectively enrolled in this study. Circulating histones, N-terminal pro-brain natriuretic peptide (NT-proBNP), procalcitonin (PCT) and C-reactive protein (CRP) were measured preoperatively (T0) and at 0 (T1), 24 (T2), 48 (T3) and 72 (T4) h postoperatively. The relationships between biomarkers and clinical outcomes were analysed. Circulating histones, NT-proBNP, PCT and CRP increased significantly postoperatively, with histones reaching the peak value earliest at T1. Circulating histone levels were higher in patients with adverse events. Receiver operating characteristic curve analysis showed that peak histone levels had a better predictive value for adverse events postoperatively. Peak histone levels correlated with the peak level of NT-proBNP (r = 0.563, P histones reached peak levels faster than NT-proBNP, PCT and CRP. Furthermore, peak histone levels correlated with biomarkers and postoperative clinical outcomes. Circulating histones may be used as a prognostic indicator for patients after cardiac surgery with CPB. ClinicalTrials.gov (ID: NCT02325765). © The Author 2016. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.
Extracellular Histones Increase Tissue Factor Activity and Enhance Thrombin Generation by Human Blood Monocytes.
Gould, Travis J; Lysov, Zakhar; Swystun, Laura L; Dwivedi, Dhruva J; Zarychanski, Ryan; Fox-Robichaud, Alison E; Liaw, Patricia C
Sepsis is characterized by systemic activation of inflammatory and coagulation pathways in response to infection. Recently, it was demonstrated that histones released into the circulation by dying/activated cells may contribute to sepsis pathology. Although the ability of extracellular histones to modulate the procoagulant activities of several cell types has been investigated, the influence of histones on the hemostatic functions of circulating monocytes is unknown. To address this, we investigated the ability of histones to modulate the procoagulant potential of THP-1 cells and peripheral blood monocytes, and examined the effects of plasmas obtained from septic patients to induce a procoagulant phenotype on monocytic cells. Tissue factor (TF) activity assays were performed on histone-treated THP-1 cells and blood monocytes. Exposure of monocytic cells to histones resulted in increases in TF activity, TF antigen, and phosphatidylserine exposure. Histones modulate the procoagulant activity via engagement of Toll-like receptors 2 and 4, and this effect was abrogated with inhibitory antibodies. Increased TF activity of histone-treated cells corresponded to enhanced thrombin generation in plasma determined by calibrated automated thrombography. Finally, TF activity was increased on monocytes exposed to plasma from septic patients, an effect that was attenuated in plasma from patients receiving unfractionated heparin (UFH). Our studies suggest that increased levels of extracellular histones found in sepsis contribute to dysregulated coagulation by increasing TF activity of monocytes. These procoagulant effects can be partially ameliorated in sepsis patients receiving UFH, thereby identifying extracellular histones as a potential therapeutic target for sepsis treatment.
Intracellular distribution of histone mRNAs in human fibroblasts studied by in situ hybridization
International Nuclear Information System (INIS)
Lawrence, J.B.; Singer, R.H.; Villnave, C.A.; Stein, J.L.; Stein, G.S.
We have used in situ hybridization to study the intracellular distribution of mRNAs for cell cycle-dependent core and H1 histone proteins in human WI-38 fibroblasts. Because histones are abundant nuclear proteins and histone mRNA expression is tightly coupled to DNA synthesis, it was of interest to determine whether histone mRNAs are localized near the nucleus. Cells were hybridized with tritiated DNA probes specific for either histone H1, histone H4, actin, or poly(A)+ mRNA and were processed for autoradiography. In exponentially growing cultures, the fraction of histone mRNA-positive cells correlated well with the fraction of cells in S phase and was eliminated by hydroxyurea inhibition of DNA synthesis. Within individual cells the label for histone mRNA was widely distributed throughout the cytoplasm and did not appear to be more heavily concentrated near the nucleus. However, histone mRNA appeared to exhibit patchy, nonhomogeneous localization, and a quantitative evaluation confirmed that grain distributions were not as uniform as they were after hybridizations to poly(A)+ mRNA. Actin mRNA in WI-38 cells was also widely distributed throughout the cytoplasm but differed from histone mRNA in that label for actin mRNA was frequently most dense at the outermost region of narrow cell extensions. The localization of actin mRNA was less pronounced but qualitatively very similar to that previously described for chicken embryonic myoblasts and fibroblasts. We conclude that localization of histones in WI-38 cells is not facilitated by localization of histone protein synthesis near the nucleus and that there are subtle but discrete and potentially functional differences in the distributions of histone, actin, and poly(A)+ mRNAs
Development of histone deacetylase inhibitor in antitumor study
International Nuclear Information System (INIS)
Zhang Min; Li Biao; Zhang Yifan
Human genome project has made a great progress, but the study about the gene expression regulation is still at the beginning. It's found that many diseases such as cancer are related to the deactivation of some gene expressions, whose epigenetic mechanisms involve DNA methylation, histone acetylation and other structural changes in chromatin. That disturbs the normal gene expression regulation and cell multiplication, which finally induce the carcinogenesis. So the re-expression of inactive genes may play a part in inhibiting the carcinogenesis. On the basis of the view, histone deacetylase (HDAC) inhibitor has a variety of applications in the study of disease and tumor, including the thyroid carcinoma. Furthermore, it comtribute to the mew way of the redifferentication and radioidine therapy against poorly differentiated thyroid carcinoma. (authors)
Dual-Acting Histone Deacetylase-Topoisomerase I Inhibitors
Guerrant, William; Patil, Vishal; Canzoneri, Joshua C.; Yao, Li-Pan; Hood, Rebecca; Oyelere, Adegboyega K.
Current chemotherapy regimens are comprised mostly of single-target drugs which are often plagued by toxic side effects and resistance development. A pharmacological strategy for circumventing these drawbacks could involve designing multivalent ligands that can modulate multiple targets while avoiding the toxicity of a single-targeted agent. Two attractive targets, histone deacetylase (HDAC) and topoisomerase I (Topo I), are cellular modulators that can broadly arrest cancer proliferation through a range of downstream effects. Both are clinically validated targets with multiple inhibitors in therapeutic use. We describe herein the design and synthesis of dual-acting histone deacetylase-topoisomerase I inhibitors. We also show that these dual-acting agents retain activity against HDAC and Topo I, and potently arrest cancer proliferation. PMID:23622981
The chromatin regulatory code: Beyond a histone code
Lesne, A.
In this commentary on the contribution by Arndt Benecke in this issue, I discuss why the notion of "chromatin code" introduced and elaborated in this paper is to be preferred to that of "histone code". Speaking of a code as regards nucleosome conformation and histone tail post-translational modifications only makes sense within the chromatin fiber, where their physico-chemical features can be translated into regulatory programs at the genome level, by means of a complex, multi-level interplay with the fiber architecture and dynamics settled in the course of Evolution. In particular, this chromatin code presumably exploits allosteric transitions of the chromatin fiber. The chromatin structure dependence of its translation suggests two alternative modes of transcription initiation regulation, also proposed in the paper by A. Benecke in this issue for interpreting strikingly bimodal micro-array data.
Structural analysis of PseH, the Campylobacter jejuni N-acetyltransferase involved in bacterial O-linked glycosylation
Energy Technology Data Exchange (ETDEWEB)
Song, Wan Seok; Nam, Mi Sun; Namgung, Byeol [Department of Systems Immunology, College of Biomedical Science, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Yoon, Sung-il, E-mail: firstname.lastname@example.com [Department of Systems Immunology, College of Biomedical Science, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Institute of Bioscience and Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of)
Campylobacter jejuni is a bacterium that uses flagella for motility and causes worldwide acute gastroenteritis in humans. The C. jejuni N-acetyltransferase PseH (cjPseH) is responsible for the third step in flagellin O-linked glycosylation and plays a key role in flagellar formation and motility. cjPseH transfers an acetyl group from an acetyl donor, acetyl coenzyme A (AcCoA), to the amino group of UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine to produce UDP-2,4-diacetamido-2,4,6-trideoxy-β-L-altropyranose. To elucidate the catalytic mechanism of cjPseH, crystal structures of cjPseH alone and in complex with AcCoA were determined at 1.95 Å resolution. cjPseH folds into a single-domain structure of a central β-sheet decorated by four α-helices with two continuously connected grooves. A deep groove (groove-A) accommodates the AcCoA molecule. Interestingly, the acetyl end of AcCoA points toward an open space in a neighboring shallow groove (groove-S), which is occupied by extra electron density that potentially serves as a pseudosubstrate, suggesting that the groove-S may provide a substrate-binding site. Structure-based comparative analysis suggests that cjPseH utilizes a unique catalytic mechanism of acetylation that has not been observed in other glycosylation-associated acetyltransferases. Thus, our studies on cjPseH will provide valuable information for the design of new antibiotics to treat C. jejuni-induced gastroenteritis. - Highlights: • cjPseH adopts a single-domain structure of a central β-sheet decorated by α-helices. • cjPseH features two continuously connected grooves on the protein surface. • Acetyl coenzyme A (AcCoA) binds into a deep groove of cjPseH in an 'L' shape. • The acetyl end of AcCoA points to a wide groove, a potential substrate-binding site.
The acetyltransferase Tip60 is a critical regulator of the differentiation-dependent amplification of human papillomaviruses.
Hong, Shiyuan; Dutta, Anindya; Laimins, Laimonis A
The life cycle of human papillomaviruses (HPVs) is dependent upon differentiation of the infected host epithelial cell as well as activation of the ataxia telangiectasia mutated (ATM) DNA repair pathway that in normal cells acts to repair double-strand DNA breaks. In normal cells, following DNA damage the acetyltransferase Tip60 must acetylate ATM proteins prior to their full activation by autophosphorylation. E6 proteins have been shown to induce the degradation of Tip60, suggesting that Tip60 action may not be required for activation of the ATM pathway in HPV-positive cells. We investigated what role, if any, Tip60 plays in regulating the differentiation-dependent HPV life cycle. Our study indicates that Tip60 levels and activity are increased in cells that stably maintain complete HPV genomes as episomes, while low levels are seen in cells that express only HPV E6 and E7 proteins. Knockdown of Tip60 with short hairpin RNAs in cells that maintain HPV episomes blocked ATM induction and differentiation-dependent genome amplification, demonstrating the critical role of Tip60 in the viral life cycle. The JAK/STAT transcription factor STAT-5 has previously been shown to regulate the phosphorylation of ATM. Our studies demonstrate that STAT-5 regulates Tip60 activation and this occurs in part by targeting glycogen synthase kinase 3β (GSK3β). Inhibition of either STAT-5, Tip60, or GSK3β blocked differentiation-dependent genome amplification. Taken together, our findings identify Tip60 to be an important regulator of HPV genome amplification whose activity during the viral life cycle is controlled by STAT-5 and the kinase GSK3β. Human papillomaviruses (HPVs) are the etiological agents of cervical and other anogenital cancers. HPVs regulate their differentiation-dependent life cycle by activation of DNA damage pathways. This study demonstrates that HPVs regulate the ATM DNA damage pathway through the action of the acetyltransferase Tip60. Furthermore, the innate
Kinetic and mutagenic characterization of the chromosomally encoded Salmonella enterica AAC(6')-Iy aminoglycoside N-acetyltransferase.
Magnet, S; Lambert, T; Courvalin, P; Blanchard, J S
The chromosomally encoded aminoglycoside N-acetyltransferase, AAC(6')-Iy, from Salmonella enterica confers resistance toward a number of aminoglycoside antibiotics. The structural gene was cloned and expressed and the purified enzyme existed in solution as a dimer of ca. 17 000 Da monomers. Acetyl-CoA was the preferred acyl donor, and most therapeutically important aminoglycosides were substrates for acetylation. Exceptions are those aminoglycosides that possess a 6'-hydroxyl substituent (e.g., lividomycin). Thus, the enzyme exhibited regioselective and exclusive acetyltransferase activity to 6'-amine-containing aminoglycosides. The enzyme exhibited Michaelis-Menten kinetics for some aminoglycoside substrates but "substrate activation" with others. Kinetic studies supported a random kinetic mechanism for the enzyme. The enzyme was inactivated by iodoacetamide in a biphasic manner, with half of the activity being lost rapidly and the other half more slowly. Tobramycin, but not acetyl-CoA, protected against inactivation. Each of the three cysteine residues (C70, C109, C145) in the wild-type enzyme were carboxamidomethylated by iodoacetamide. Cysteine 109 in AAC(6')-Iy is conserved in 12 AAC(6') enzyme sequences of the major class I subfamily. Surprisingly, mutation of this residue to alanine neither abolished activity nor altered the biphasic inactivation by iodoacetamide. The maximum velocity and V/K values for a number of aminoglycosides were elevated in this single mutant, and the kinetic behavior of substrates exhibiting linear vs nonlinear kinetics was reversed. Cysteine 70 in AAC(6')-Iy is either a cysteine or a threonine residue in all 12 AAC(6') enzymes of the major class I subfamily. The double mutant, C109A/C70A, was not inactivated by iodoacetamide. The double mutant exhibited large increases in the K(m) values for both acetyl-CoA and aminoglycoside substrates, and all aminoglycoside substrates exhibited Michaelis-Menten kinetics. Solvent kinetic isotope
Dual-Acting Histone Deacetylase-Topoisomerase I Inhibitors
Guerrant, William; Patil, Vishal; Canzoneri, Joshua C.; Yao, Li-Pan; Hood, Rebecca; Oyelere, Adegboyega K.
Current chemotherapy regimens are comprised mostly of single-target drugs which are often plagued by toxic side effects and resistance development. A pharmacological strategy for circumventing these drawbacks could involve designing multivalent ligands that can modulate multiple targets while avoiding the toxicity of a single-targeted agent. Two attractive targets, histone deacetylase (HDAC) and topoisomerase I (Topo I), are cellular modulators that can broadly arrest cancer proliferation thr...
Histone arginine methylation in cocaine action in the nucleus accumbens
Sun, HaoSheng; Scobie, Kimberly N.; Shao, Ningyi; Rabkin, Jaclyn; Dias, Caroline; Calipari, Erin S.; Maze, Ian; Pena, Catherine J.; Walker, Deena M.; Cahill, Michael E.; Chandra, Ramesh; Gancarz, Amy; Landry, Joseph A.; Cates, Hannah; Lobo, Mary-Kay; Dietz, David; Allis, C. David; Guccione, Ernesto; Turecki, Gustavo; Defilippi, Paola; Neve, Rachael L.; Hurd, Yasmin L.; Shen, Li; Nestler, Eric J.
Repeated cocaine exposure regulates transcriptional regulation within the nucleus accumbens (NAc), and epigenetic mechanisms—such as histone acetylation and methylation on Lys residues—have been linked to these lasting actions of cocaine. In contrast to Lys methylation, the role of histone Arg (R) methylation remains underexplored in addiction models. Here we show that protein-R-methyltransferase-6 (PRMT6) and its associated histone mark, asymmetric dimethylation of R2 on histone H3 (H3R2me2a), are decreased in the NAc of mice and rats after repeated cocaine exposure, including self-administration, and in the NAc of cocaine-addicted humans. Such PRMT6 down-regulation occurs selectively in NAc medium spiny neurons (MSNs) expressing dopamine D2 receptors (D2-MSNs), with opposite regulation occurring in D1-MSNs, and serves to protect against cocaine-induced addictive-like behavioral abnormalities. Using ChIP-seq, we identified Src kinase signaling inhibitor 1 (Srcin1; also referred to as p140Cap) as a key gene target for reduced H3R2me2a binding, and found that consequent Srcin1 induction in the NAc decreases Src signaling, cocaine reward, and the motivation to self-administer cocaine. Taken together, these findings suggest that suppression of Src signaling in NAc D2-MSNs, via PRMT6 and H3R2me2a down-regulation, functions as a homeostatic brake to restrain cocaine action, and provide novel candidates for the development of treatments for cocaine addiction. PMID:27506785
Histone occurrence in chromatin from Peridinium balticum, a binucleate dinoflagellate.
Rizzo, P J; Cox, E R
Peridinium balticum is one of two dinoflagellates known to have dissimilar nuclei together in the same cell. One nucleus (dinokaryotic) has permanently condensed chromosomes, while the other (eukaryotic) does not have morphologically distinct chromosomes. Acid extracts of chromatin prepared from a mixture of dinokaryotic and eukaryotic nuclei and purified eukaryotic nuclei give four bands that co-migrate with four of the five histones from calf thymus when analyzed in urea-containing polyacrylamide gels.
Histone arginine methylation in cocaine action in the nucleus accumbens.
Damez-Werno, Diane M; Sun, HaoSheng; Scobie, Kimberly N; Shao, Ningyi; Rabkin, Jaclyn; Dias, Caroline; Calipari, Erin S; Maze, Ian; Pena, Catherine J; Walker, Deena M; Cahill, Michael E; Chandra, Ramesh; Gancarz, Amy; Mouzon, Ezekiell; Landry, Joseph A; Cates, Hannah; Lobo, Mary-Kay; Dietz, David; Allis, C David; Guccione, Ernesto; Turecki, Gustavo; Defilippi, Paola; Neve, Rachael L; Hurd, Yasmin L; Shen, Li; Nestler, Eric J
Repeated cocaine exposure regulates transcriptional regulation within the nucleus accumbens (NAc), and epigenetic mechanisms-such as histone acetylation and methylation on Lys residues-have been linked to these lasting actions of cocaine. In contrast to Lys methylation, the role of histone Arg (R) methylation remains underexplored in addiction models. Here we show that protein-R-methyltransferase-6 (PRMT6) and its associated histone mark, asymmetric dimethylation of R2 on histone H3 (H3R2me2a), are decreased in the NAc of mice and rats after repeated cocaine exposure, including self-administration, and in the NAc of cocaine-addicted humans. Such PRMT6 down-regulation occurs selectively in NAc medium spiny neurons (MSNs) expressing dopamine D2 receptors (D2-MSNs), with opposite regulation occurring in D1-MSNs, and serves to protect against cocaine-induced addictive-like behavioral abnormalities. Using ChIP-seq, we identified Src kinase signaling inhibitor 1 (Srcin1; also referred to as p140Cap) as a key gene target for reduced H3R2me2a binding, and found that consequent Srcin1 induction in the NAc decreases Src signaling, cocaine reward, and the motivation to self-administer cocaine. Taken together, these findings suggest that suppression of Src signaling in NAc D2-MSNs, via PRMT6 and H3R2me2a down-regulation, functions as a homeostatic brake to restrain cocaine action, and provide novel candidates for the development of treatments for cocaine addiction.
Histone demethylase JMJD5 is essential for embryonic development
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Oh, Sangphil [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104 (United States); Janknecht, Ralf, E-mail: email@example.com [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104 (United States)
Highlights: Black-Right-Pointing-Pointer Histone demethylase JMJD5 is essential for embryogenesis. Black-Right-Pointing-Pointer Transcription of tumor suppressor p53 is upregulated in JMJD5 knockout embryos. Black-Right-Pointing-Pointer JMJD5 may antagonize p53-dependent growth inhibition and apoptosis. Black-Right-Pointing-Pointer JMJD5 is overexpressed in leukemias and breast cancer. -- Abstract: Histone lysine methylation is pivotal in regulating chromatin structure and thus profoundly affects the transcriptome. JMJD5 (jumonji C domain-containing 5) is a histone demethylase that specifically removes methyl moieties from dimethylated lysine 36 on histone H3 and exerts a pro-proliferative effect on breast cancer cells. Here, we generated JMJD5 knockout mice in order to study the physiological significance of this enzyme. Whereas heterozygous knockout mice displayed no overt phenotype, homozygous JMJD5 knockouts died around day 10 of embryonal development. JMJD5{sup -/-} embryos showed delayed development already at E8.5 and were actively resorbed at E10.5. While strong JMJD5 expression was observed only in the yolk sac at E8.5, JMJD5 was robustly expressed in E10.5 embryos at several sites, including the heart and eye. Lack of JMJD5 resulted in transcriptional upregulation of the tumor suppressor p53. Concurrently, the cell cycle inhibitor p21 and the pro-apoptotic molecule Noxa, both of which are prominent p53 target genes, became strongly upregulated in JMJD5{sup -/-} embryos. Collectively, our data indicate that JMJD5 is essential during embryonal development and a repressor of p53 expression. The latter suggests that JMJD5 has oncogenic activity and accordingly JMJD5 is upregulated in leukemias and breast cancer.
Rabbit N-acetyltransferase 2 genotyping method to investigate role of acetylation polymorphism on N- and O-acetylation of aromatic and heterocyclic amine carcinogens.
Hein, David W; Doll, Mark A
The rabbit was the initial animal model to investigate the acetylation polymorphism expressed in humans. Use of the rabbit model is compromised by lack of a rapid non-invasive method for determining acetylator phenotype. Slow acetylator phenotype in the rabbit results from deletion of the N-acetyltransferase 2 (NAT2) gene. A relatively quick and non-invasive method for identifying the gene deletion was developed and acetylator phenotypes confirmed by measurement of N- and O-acetyltransferase activities in hepatic cytosols. Rabbit liver cytosols catalyzed the N-acetylation of sulfamethazine (p = 0.0014), benzidine (p = 0.0257), 4-aminobiphenyl (p = 0.0012), and the O-acetylation of N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP; p = 0.002) at rates significantly higher in rabbits possessing NAT2 gene than rabbits with NAT2 gene deleted. In contrast, hepatic cytosols catalyzed the N-acetylation of p-aminobenzoic acid (an N-acetyltransferase 1 selective substrate) at rates that did not differ significantly (p > 0.05) between rabbits positive and negative for NAT2. The new NAT2 genotyping method facilitates use of the rabbit model to investigate the role of acetylator polymorphism in the metabolism of aromatic and heterocyclic amine drugs and carcinogens.
A unified phylogeny-based nomenclature for histone variants
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Talbert Paul B
Full Text Available Abstract Histone variants are non-allelic protein isoforms that play key roles in diversifying chromatin structure. The known number of such variants has greatly increased in recent years, but the lack of naming conventions for them has led to a variety of naming styles, multiple synonyms and misleading homographs that obscure variant relationships and complicate database searches. We propose here a unified nomenclature for variants of all five classes of histones that uses consistent but flexible naming conventions to produce names that are informative and readily searchable. The nomenclature builds on historical usage and incorporates phylogenetic relationships, which are strong predictors of structure and function. A key feature is the consistent use of punctuation to represent phylogenetic divergence, making explicit the relationships among variant subtypes that have previously been implicit or unclear. We recommend that by default new histone variants be named with organism-specific paralog-number suffixes that lack phylogenetic implication, while letter suffixes be reserved for structurally distinct clades of variants. For clarity and searchability, we encourage the use of descriptors that are separate from the phylogeny-based variant name to indicate developmental and other properties of variants that may be independent of structure.
Nuclear lactate dehydrogenase modulates histone modification in human hepatocytes
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Castonguay, Zachary; Auger, Christopher; Thomas, Sean C.; Chahma, M'hamed; Appanna, Vasu D., E-mail: firstname.lastname@example.com
Highlights: • Nuclear LDH is up-regulated under oxidative stress. • SIRT1 is co-immunoprecipitated bound to nuclear LDH. • Nuclear LDH is involved in histone deacetylation and epigenetics. - Abstract: It is becoming increasingly apparent that the nucleus harbors metabolic enzymes that affect genetic transforming events. Here, we describe a nuclear isoform of lactate dehydrogenase (nLDH) and its ability to orchestrate histone deacetylation by controlling the availability of nicotinamide adenine dinucleotide (NAD{sup +}), a key ingredient of the sirtuin-1 (SIRT1) deacetylase system. There was an increase in the expression of nLDH concomitant with the presence of hydrogen peroxide (H{sub 2}O{sub 2}) in the culture medium. Under oxidative stress, the NAD{sup +} generated by nLDH resulted in the enhanced deacetylation of histones compared to the control hepatocytes despite no discernable change in the levels of SIRT1. There appeared to be an intimate association between nLDH and SIRT1 as these two enzymes co-immunoprecipitated. The ability of nLDH to regulate epigenetic modifications by manipulating NAD{sup +} reveals an intricate link between metabolism and the processing of genetic information.
Current evidence for histone deacetylase inhibitors in pancreatic cancer.
Koutsounas, Ioannis; Giaginis, Constantinos; Patsouris, Efstratios; Theocharis, Stamatios
Pancreatic cancer is one of the most aggressive human cancers, with more than 200 000 deaths worldwide every year. Despite recent efforts, conventional treatment approaches, such as surgery and classic chemotherapy, have only slightly improved patient outcomes. More effective and well-tolerated therapies are required to reverse the current poor prognosis of this type of neoplasm. Among new agents, histone deacetylase inhibitors (HDACIs) are now being tested. HDACIs have multiple biological effects related to acetylation of histones and many non-histone proteins that are involved in regulation of gene expression, apoptosis, cell cycle progression and angiogenesis. HDACIs induce cell cycle arrest and can activate the extrinsic and intrinsic pathways of apoptosis in different cancer cell lines. In the present review, the main mechanisms by which HDACIs act in pancreatic cancer cells in vitro, as well as their antiproliferative effects in animal models are presented. HDACIs constitute a promising treatment for pancreatic cancer with encouraging anti-tumor effects, at well-tolerated doses.
Tetrahydroisoquinolines as novel histone deacetylase inhibitors for treatment of cancer
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Danqi Chen
Full Text Available Histone acetylation is a critical process in the regulation of chromatin structure and gene expression. Histone deacetylases (HDACs remove the acetyl group, leading to chromatin condensation and transcriptional repression. HDAC inhibitors are considered a new class of anticancer agents and have been shown to alter gene transcription and exert antitumor effects. This paper describes our work on the structural determination and structure-activity relationship (SAR optimization of tetrahydroisoquinoline compounds as HDAC inhibitors. These compounds were tested for their ability to inhibit HDAC 1, 3, 6 and for their ability to inhibit the proliferation of a panel of cancer cell lines. Among these, compound 82 showed the greatest inhibitory activity toward HDAC 1, 3, 6 and strongly inhibited growth of the cancer cell lines, with results clearly superior to those of the reference compound, vorinostat (SAHA. Compound 82 increased the acetylation of histones H3, H4 and tubulin in a concentration-dependent manner, suggesting that it is a broad inhibitor of HDACs.
Histone H3 Acetyl K9 and Histone H3 Tri Methyl K4 as Prognostic Markers for Patients with Cervical Cancer.
Beyer, Susanne; Zhu, Junyan; Mayr, Doris; Kuhn, Christina; Schulze, Sandra; Hofmann, Simone; Dannecker, Christian; Jeschke, Udo; Kost, Bernd P
Chromatin remodeling alters gene expression in carcinoma tissue. Although cervical cancer is the fourth most common cancer in women worldwide, a systematic study about the prognostic value of specific changes in the chromatin structure, such as histone acetylation or histone methylation, is missing. In this study, the expression of histone H3 acetyl K9, which is known to denote active regions at enhancers and promoters, and histone H3 tri methyl K4, which preferentially identifies active gene promoters, were examined as both show high metastatic potential. A panel of patients with cervical cancer was selected and the importance of the histone modifications concerning survival-time (overall survival and relapse-free survival) was analyzed in 250 cases. Histone H3 acetyl K9 staining was correlated with low grading, low FIGO (TNM classification and the International Federation of Gynecology and Obstetrics) status, negative N-status and low T-status in cervical cancer, showing a higher expression in adenocarcinoma than in squamous cell carcinoma. Cytoplasmic expression of histone H3 tri methyl K4 in a cervical cancer specimen was correlated with advanced T-status and poor prognosis. While cytoplasmic H3K4me3 expression seemed to be a marker of relapse-free survival, nuclear expression showed a correlation to poor prognosis in overall survival. Within this study, we analyzed the chemical modification of two histone proteins that are connected to active gene expression. Histone H3 acetyl K9 was found to be an independent marker of overall survival. Histone H3 tri methyl K4 was correlated with poor prognosis and it was found to be an independent marker of relapse-free survival. Therefore, we could show that chromatin remodeling plays an important role in cervical cancer biology.
Histone H3 Acetyl K9 and Histone H3 Tri Methyl K4 as Prognostic Markers for Patients with Cervical Cancer
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Susanne Beyer
Full Text Available Chromatin remodeling alters gene expression in carcinoma tissue. Although cervical cancer is the fourth most common cancer in women worldwide, a systematic study about the prognostic value of specific changes in the chromatin structure, such as histone acetylation or histone methylation, is missing. In this study, the expression of histone H3 acetyl K9, which is known to denote active regions at enhancers and promoters, and histone H3 tri methyl K4, which preferentially identifies active gene promoters, were examined as both show high metastatic potential. A panel of patients with cervical cancer was selected and the importance of the histone modifications concerning survival-time (overall survival and relapse-free survival was analyzed in 250 cases. Histone H3 acetyl K9 staining was correlated with low grading, low FIGO (TNM classification and the International Federation of Gynecology and Obstetrics status, negative N-status and low T-status in cervical cancer, showing a higher expression in adenocarcinoma than in squamous cell carcinoma. Cytoplasmic expression of histone H3 tri methyl K4 in a cervical cancer specimen was correlated with advanced T-status and poor prognosis. While cytoplasmic H3K4me3 expression seemed to be a marker of relapse-free survival, nuclear expression showed a correlation to poor prognosis in overall survival. Within this study, we analyzed the chemical modification of two histone proteins that are connected to active gene expression. Histone H3 acetyl K9 was found to be an independent marker of overall survival. Histone H3 tri methyl K4 was correlated with poor prognosis and it was found to be an independent marker of relapse-free survival. Therefore, we could show that chromatin remodeling plays an important role in cervical cancer biology.
Novel E3 ubiquitin ligases that regulate histone protein levels in the budding yeast Saccharomyces cerevisiae.
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Rakesh Kumar Singh
Full Text Available Core histone proteins are essential for packaging the genomic DNA into chromatin in all eukaryotes. Since multiple genes encode these histone proteins, there is potential for generating more histones than what is required for chromatin assembly. The positively charged histones have a very high affinity for negatively charged molecules such as DNA, and any excess of histone proteins results in deleterious effects on genomic stability and cell viability. Hence, histone levels are known to be tightly regulated via transcriptional, posttranscriptional and posttranslational mechanisms. We have previously elucidated the posttranslational regulation of histone protein levels by the ubiquitin-proteasome pathway involving the E2 ubiquitin conjugating enzymes Ubc4/5 and the HECT (Homologous to E6-AP C-Terminus domain containing E3 ligase Tom1 in the budding yeast. Here we report the identification of four additional E3 ligases containing the RING (Really Interesting New Gene finger domains that are involved in the ubiquitylation and subsequent degradation of excess histones in yeast. These E3 ligases are Pep5, Snt2 as well as two previously uncharacterized Open Reading Frames (ORFs YKR017C and YDR266C that we have named Hel1 and Hel2 (for Histone E3 Ligases respectively. Mutants lacking these E3 ligases are sensitive to histone overexpression as they fail to degrade excess histones and accumulate high levels of endogenous histones on histone chaperones. Co-immunoprecipitation assays showed that these E3 ligases interact with the major E2 enzyme Ubc4 that is involved in the degradation related ubiquitylation of histones. Using mutagenesis we further demonstrate that the RING domains of Hel1, Hel2 and Snt2 are required for histone regulation. Lastly, mutants corresponding to Hel1, Hel2 and Pep5 are sensitive to replication inhibitors. Overall, our results highlight the importance of posttranslational histone regulatory mechanisms that employ multiple E3
A PHD in histone language: on the role of histone methylation in plant responses to phosphate deficiency.
Chandrika, Nulu Naga Prafulla; Sundaravelpandian, Kalaipandian; Schmidt, Wolfgang
Post-translational modifications of core histones are important for various DNA-templated processes such as transcription and repair. We recently reported that the ALFIN LIKE 6 (AL6) gene, identified in a forward genetic screen, is critical for phosphate deficiency-induced root hair formation and several other processes associated with the regulation of cellular phosphate homeostasis. AL6 contains a Plant Homeo Domain (PHD) finger that can bind to trimethylated lysine 4 of histone H3 (H3K4me3). Homozygous mutants defective in AL6 expression form very short root hairs under phosphate-deficient conditions, presumably caused by altered expression of putative primary and secondary down-stream targets of AL6. In this Addendum, we speculate about possible roles of AL6, H3K4 trimethylation and other chromatin modifications in the adaptation of plants to low phosphate availability.
Thermodynamical study of interaction of histone H1 chromosomal protein and mitoxantrone anticancer drug
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Jafargholizadeh, Naser [Department of Cell and Molecular Biology, School of Biology, College of Science, University of Tehran, Tehran (Iran, Islamic Republic of); Zargar, Seyed Jalal, E-mail: email@example.com [Department of Cell and Molecular Biology, School of Biology, College of Science, University of Tehran, Tehran (Iran, Islamic Republic of); Safarian, Shahrokh; Habibi-Rezaei, Mehran [Department of Cell and Molecular Biology, School of Biology, College of Science, University of Tehran, Tehran (Iran, Islamic Republic of)
Highlights: Black-Right-Pointing-Pointer For the first time, our results show mitoxantrone anticancer drug binds to histone H1, via hydrophobic, hydrogen, van der Waals and electrostatic interactions. Black-Right-Pointing-Pointer Binding of mitoxantrone molecules to histone H1 is positive cooperative. Black-Right-Pointing-Pointer Histone H1 may be considered as a new target for mitoxantrone at the chromatin level. - Using ultraviolet spectroscopy technique, we have investigated the interaction of anticancer drug, mitoxantrone with calf thymus histone H1 chromosomal protein in 100 mM phosphate buffer, pH 7.0, at temperatures 300 and 310 K. UV spectroscopy results show interactions between mitoxantrone and histone H1 with a positive cooperative binding process which was confirmed by Scatchard plot. According to the obtained results, it is concluded that histone H1 can be considered as a target for mitoxantrone binding at the chromatin level.
Quantitative proteomic analysis of post-translational modifications of human histones
DEFF Research Database (Denmark)
Beck, Hans Christian; Nielsen, Eva C; Matthiesen, Rune
Histone proteins are subject to a range of post-transcriptional modifications in living cells. The combinatorial nature of these modifications constitutes the "histone code" that dictates chromatin structure and function during development, growth, differentiation, and homeostasis of cells....... Deciphering of the histone code is hampered by the lack of analytical methods for monitoring the combinatorial complexity of reversible multisite modifications of histones, including acetylation and methylation. To address this problem, we used LC-MSMS technology and Virtual Expert Mass Spectrometrist...... software for qualitative and quantitative proteomic analysis of histones extracted from human small cell lung cancer cells. A total of 32 acetylations, methylations, and ubiquitinations were located in the human histones H2A, H2B, H3, and H4, including seven novel modifications. An LC-MSMS-based method...
Post-cardiac arrest level of free-plasma DNA and DNA-histone complexes
DEFF Research Database (Denmark)
Jeppesen, A N; Hvas, A-M; Grejs, A M
Background Plasma DNA-histone complexes and total free-plasma DNA have the potential to quantify the ischaemia-reperfusion damages occurring after cardiac arrest. Furthermore, DNA-histone complexes may have the potential of being a target for future treatment. The aim was to examine if plasma DNA-histone...... after 22, 46 and 70 h. Samples for DNA-histone complexes were quantified by Cell Death Detection ELISAplus. The total free-plasma DNA analyses were quantified with qPCR by analysing the Beta-2 microglobulin gene. The control group comprised 40 healthy individuals. Results We found no difference...... in the level of DNA-histone complexes between the 22-h sample and healthy individuals (P = 0.10). In the 46-h sample, there was an increased level of DNA-histone complexes in non-survivors compared with survivors 30 days after the cardiac arrest (P
Protein SIC Secreted fromForms Complexes with Extracellular Histones That Boost Cytokine Production
DEFF Research Database (Denmark)
Westman, Johannes; Chakrakodi, Bhavya; Snäll, Johanna
determine the amplitude of such responses and influence the outcome of the disease. Here, we report that protein SIC, Streptococcal Inhibitor of Complement, an abundant secreted protein fromStreptococcus pyogenes, binds to extracellular histones, a group of danger signals released during necrotizing tissue...... damage. This interaction leads to the formation of large aggregatesin vitro. Extracellular histones and SIC are abundantly expressed and seen colocalized in biopsies from patients with necrotizing soft-tissue infections caused byS. pyogenes. In addition, binding of SIC to histones neutralized...... their antimicrobial activity. Likewise, the ability of histones to induce hemolysis was inhibited in the presence of SIC. However, when added to whole blood, SIC was not able to block the pro-inflammatory effect of histones. Instead SIC boosted the histone-triggered release of a broad range of cytokines...
A cell cycle-regulated histone H3 gene of alfalfa with an atypical promoter structure.
Robertson, A J; Kapros, T; Waterborg, J H
The control of cell cycle expression of histone genes in plants is incompletely understood. A new histone H3 gene was cloned from alfalfa (Medicago sativa) that codes for the replication-dependent histone H3.1 variant protein. Despite lacking all promoter sequence motifs that have been associated with cell cycle-dependent histone gene expression in plants, northern analysis of synchronized cells clearly linked gene expression to DNA replication. TTAATNA was recognized as a new sequence element in the 3' untranslated regions of this and all other cell cycle-dependent histone H3 genes of dicotyledonous plants. It is not found in the replication-independent histone H3 genes.
Thermodynamical study of interaction of histone H1 chromosomal protein and mitoxantrone anticancer drug
International Nuclear Information System (INIS)
Jafargholizadeh, Naser; Zargar, Seyed Jalal; Safarian, Shahrokh; Habibi-Rezaei, Mehran
Highlights: ► For the first time, our results show mitoxantrone anticancer drug binds to histone H1, via hydrophobic, hydrogen, van der Waals and electrostatic interactions. ► Binding of mitoxantrone molecules to histone H1 is positive cooperative. ► Histone H1 may be considered as a new target for mitoxantrone at the chromatin level. - Using ultraviolet spectroscopy technique, we have investigated the interaction of anticancer drug, mitoxantrone with calf thymus histone H1 chromosomal protein in 100 mM phosphate buffer, pH 7.0, at temperatures 300 and 310 K. UV spectroscopy results show interactions between mitoxantrone and histone H1 with a positive cooperative binding process which was confirmed by Scatchard plot. According to the obtained results, it is concluded that histone H1 can be considered as a target for mitoxantrone binding at the chromatin level.
Neural restrictive silencer factor and choline acetyltransferase expression in cerebral tissue of Alzheimer's Disease patients: A pilot study
González-Castañeda, Rocío E.; Sánchez-González, Víctor J.; Flores-Soto, Mario; Vázquez-Camacho, Gonzalo; Macías-Islas, Miguel A.; Ortiz, Genaro G.
Decreased Choline Acetyltransferase (ChAT) brain level is one of the main biochemical disorders in Alzheimer's Disease (AD). In rodents, recent data show that the CHAT gene can be regulated by a neural restrictive silencer factor (NRSF). The aim of the present work was to evaluate the gene and protein expression of CHAT and NRSF in frontal, temporal, entorhinal and parietal cortices of AD patient brains. Four brains from patients with AD and four brains from subjects without dementia were studied. Cerebral tissues were obtained and processed by the guanidine isothiocyanate method for RNA extraction. CHAT and NRSF gene and protein expression were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. CHAT gene expression levels were 39% lower in AD patients as compared to the control group (p 0.05, U test) than in the control subjects. These findings suggest for the first time that in the brain of AD patients high NRSF protein levels are related to low CHAT gene expression levels. PMID:23569405
The Acetyl Group Buffering Action of Carnitine Acetyltransferase Offsets Macronutrient-Induced Lysine Acetylation of Mitochondrial Proteins
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Michael N. Davies
Full Text Available Lysine acetylation (AcK, a posttranslational modification wherein a two-carbon acetyl group binds covalently to a lysine residue, occurs prominently on mitochondrial proteins and has been linked to metabolic dysfunction. An emergent theory suggests mitochondrial AcK occurs via mass action rather than targeted catalysis. To test this hypothesis, we performed mass spectrometry-based acetylproteomic analyses of quadriceps muscles from mice with skeletal muscle-specific deficiency of carnitine acetyltransferase (CrAT, an enzyme that buffers the mitochondrial acetyl-CoA pool by converting short-chain acyl-CoAs to their membrane permeant acylcarnitine counterparts. CrAT deficiency increased tissue acetyl-CoA levels and susceptibility to diet-induced AcK of broad-ranging mitochondrial proteins, coincident with diminished whole body glucose control. Sub-compartment acetylproteome analyses of muscles from obese mice and humans showed remarkable overrepresentation of mitochondrial matrix proteins. These findings reveal roles for CrAT and L-carnitine in modulating the muscle acetylproteome and provide strong experimental evidence favoring the nonenzymatic carbon pressure model of mitochondrial AcK.
Expression of an engineered granule-bound Escherichia coli maltose acetyltransferase in wild-type and amf potato plants.
Nazarian Firouzabadi, Farhad; Vincken, Jean-Paul; Ji, Qin; Suurs, Luc C J M; Visser, Richard G F
Starch is used in many industrial applications, but often requires chemical derivatization to enhance its properties before use. In particular, the stability of starch polymers in solution is improved by acetylation. A drawback of this treatment is the use of pollutant chemicals. A biological alternative to chemical derivatization was investigated by the expression of an amyloplast-targeted Escherichia coli maltose acetyltransferase (MAT) gene in tubers of wild-type (Kardal) and mutant amylose-free (amf) potato plants. MAT was expressed as such, or fused to the N- or C-terminus of a non-catalytic starch-binding domain (SBD) to target the starch granule. Starch granules derived from transgenic plants were found to contain acetyl groups, although their content was low, opening up an avenue to move away from the post-harvest chemical derivatization of starch. MAT inside starch granules was found to be active post-harvest when supplied with acetyl-coenzyme A and glucose or maltose, but it did not acetylate starch polymers in vitro. Starch granules from transformants in which MAT alone was expressed also showed MAT activity, indicating that MAT is accumulated in starch granules, and has affinity for starch by itself. Furthermore, starch granule morphology was altered, and fusion proteins containing MAT and SBD seemed to have a higher affinity for starch granules than two appended SBDs. These results are discussed against the background of the quaternary structure of MAT.
Method to produce acetyldiacylglycerols (ac-TAGs) by expression of an acetyltransferase gene isolated from Euonymus alatus (burning bush)
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Durrett, Timothy; Ohlrogge, John; Pollard, Michael
The present invention relates to novel diacylglycerol acyltransferase genes and proteins, and methods of their use. In particular, the invention describes genes encoding proteins having diacylglycerol acetyltransferase activity, specifically for transferring an acetyl group to a diacylglycerol substrate to form acetyl-Triacylglycerols (ac-TAGS), for example, a 3-acetyl-1,2-diacyl-sn-glycerol. The present invention encompasses both native and recombinant wild-type forms of the transferase, as well as mutants and variant forms. The present invention also relates to methods of using novel diacylglycerol acyltransferase genes and proteins, including their expression in transgenic organisms at commercially viable levels, for increasing production of 3-acetyl-1,2-diacyl-sn-glycerols in plant oils and altering the composition of oils produced by microorganisms, such as yeast, by increasing ac-TAG production. Additionally, oils produced by methods of the present inventions comprising genes and proteins are contemplated for use as biodiesel fuel, in polymer production and as naturally produced food oils with reduced calories.
Depletion of the human N-terminal acetyltransferase hNaa30 disrupts Golgi integrity and ARFRP1 localization.
Starheim, Kristian K; Kalvik, Thomas V; Bjørkøy, Geir; Arnesen, Thomas
The organization of the Golgi apparatus (GA) is tightly regulated. Golgi stack scattering is observed in cellular processes such as apoptosis and mitosis, and has also been associated with disruption of cellular lipid metabolism and neurodegenerative diseases. Our studies show that depletion of the human N-α-acetyltransferase 30 (hNaa30) induces fragmentation of the Golgi stack in HeLa and CAL-62 cell lines. The GA associated GTPase ADP ribosylation factor related protein 1 (ARFRP1) was previously shown to require N-terminal acetylation for membrane association and based on its N-terminal sequence, it is likely to be a substrate of hNaa30. ARFRP1 is involved in endosome-to- trans -Golgi network (TGN) traffic. We observed that ARFRP1 shifted from a predominantly cis -Golgi and TGN localization to localizing both Golgi and non-Golgi vesicular structures in hNaa30-depleted cells. However, we did not observe loss of membrane association of ARFRP1. We conclude that hNaa30 depletion induces Golgi scattering and induces aberrant ARFRP1 Golgi localization. © 2017 The Author(s).
Comparison of N-acetyltransferase-2 enzyme genotype-phenotype and xanthine oxidase enzyme activity between Swedes and Koreans.
Djordjevic, Natasa; Carrillo, Juan Antonio; Roh, Hyung-Keun; Karlsson, Sara; Ueda, Nobuhisa; Bertilsson, Leif; Aklillu, Eleni
The aim of this study was to compare xanthine oxidase (XO) and N-acetyltransferase-2 (NAT2) genotype and phenotype between Swedes (n = 113) and Koreans (n = 150), as well as to investigate the effect of sex, smoking, age, and oral contraceptive (OC) use on enzyme activities, using caffeine as a probe. XO and NAT2 activities were estimated by 1U/(1U+1X) and AFMU/(AFMU+1X+1U) urinary ratios, respectively. Participants were genotyped for 191G>A, 341T>C, 590G>A, and 857G>A NAT2 polymorphisms. There was no significant difference in XO activity between Swedes and Koreans. In Swedes, higher XO activity was observed in women (P < .003). There were significant differences in NAT2 genotype and phenotype between Swedes and Koreans. Koreans display significantly higher frequency of NAT2 fast acetylator genotype (89%), whereas the slow acetylator genotype is predominant (62%) in Swedes (P < .0001). Significantly higher NAT2 activity was observed in Koreans compared to Swedes (P < .0001). Having the same NAT2 fast acetylator genotype, Koreans display higher enzyme activity than Swedes (P < .004). OC use significantly increased NAT2 activity in Swedish women. In conclusion, Koreans display higher NAT2 activity than Swedes regardless of NAT2 genotype. Ethnicity, OC use, and genotype determine NAT2 activity, whereas sex is the only determinant of XO activity.
Adolescent, but not adult, binge ethanol exposure leads to persistent global reductions of choline acetyltransferase expressing neurons in brain.
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Ryan P Vetreno
Full Text Available During the adolescent transition from childhood to adulthood, notable maturational changes occur in brain neurotransmitter systems. The cholinergic system is composed of several distinct nuclei that exert neuromodulatory control over cognition, arousal, and reward. Binge drinking and alcohol abuse are common during this stage, which might alter the developmental trajectory of this system leading to long-term changes in adult neurobiology. In Experiment 1, adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g., 2-day on/2-day off from postnatal day [P] 25 to P55 treatment led to persistent, global reductions of choline acetyltransferase (ChAT expression. Administration of the Toll-like receptor 4 agonist lipopolysaccharide to young adult rats (P70 produced a reduction in ChAT+IR that mimicked AIE. To determine if the binge ethanol-induced ChAT decline was unique to the adolescent, Experiment 2 examined ChAT+IR in the basal forebrain following adolescent (P28-P48 and adult (P70-P90 binge ethanol exposure. Twenty-five days later, ChAT expression was reduced in adolescent, but not adult, binge ethanol-exposed animals. In Experiment 3, expression of ChAT and vesicular acetylcholine transporter expression was found to be significantly reduced in the alcoholic basal forebrain relative to moderate drinking controls. Together, these data suggest that adolescent binge ethanol decreases adult ChAT expression, possibly through neuroimmune mechanisms, which might impact adult cognition, arousal, or reward sensitivity.
Structure function studies on the lipoate-acetyltransferase--component-X-core assembly of the ox heart pyruvate dehydrogenase complex.
Hodgson, J A; De Marcucci, O G; Lindsay, J G
Component X, the recently recognised subunit of mammalian pyruvate dehydrogenase complex, was shown by immune blotting to be present in all of nine tissues dissected from rat. This finding indicated that component X was not an isoenzyme of the lipoate acetyltransferase (E2) associated with one or a limited number of tissues. Native pyruvate dehydrogenase complex was shown to bind IgG raised to isolated component X, indicating that there were at least some regions of the X subunit exposed at the periphery of the complex. Lipoyl groups of ox heart pyruvate dehydrogenase complex were specifically cross-linked by reaction with phenylene-o-bismaleimide in the presence of pyruvate and the subunits contributing to the products of cross-linking were identified by immune blotting. Species with very high Mr containing both E2 and component X, were formed in high yield, as well as apparent E2/E2 and E2/X dimers and trimers and an X/X dimer. These results showed that acetylated lipoyl groups of different E2 and X subunits were able to interact in all possible combinations. The types of cross-linked E2 products formed suggested that two thiols, reactible with phenylene-o-bismaleimide, were rapidly generated in the presence of pyruvate. The results were most easily explained by the presence of two acetylatable lipoyl groups on each E2 polypeptide.
No Association between Variant -acetyltransferase Genes, Cigarette Smoking and Prostate Cancer Susceptibility Among Men of African Descent
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La Creis Renee Kidd
Full Text Available Objective We evaluated the individual and combination effects of NAT1, NAT2 and tobacco smoking in a case-control study of 219 incident prostate cancer (PCa cases and 555 disease-free men. Methods Allelic discriminations for 15 NAT1 and NAT2 loci were detected in germ-line DNA samples using Taqman polymerase chain reaction (PCR assays. Single gene, gene-gene and gene-smoking interactions were analyzed using logistic regression models and multi-factor dimensionality reduction (MDR adjusted for age and subpopulation stratification. MDR involves a rigorous algorithm that has ample statistical power to assess and visualize gene-gene and gene-environment interactions using relatively small samples sizes (i.e., 200 cases and 200 controls. Results Despite the relatively high prevalence of NAT1*10/*10 (40.1%, NAT2 slow (30.6%, and NAT2 very slow acetylator genotypes (10.1% among our study participants, these putative risk factors did not individually or jointly increase PCa risk among all subjects or a subset analysis restricted to tobacco smokers. Conclusion Our data do not support the use of N -acetyltransferase genetic susceptibilities as PCa risk factors among men of African descent; however, subsequent studies in larger sample populations are needed to confirm this finding.
The early response of pineal N-acetyltransferase activity, melatonin and catecholamine levels in rats irradiated with gamma rays
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Kassayova, M.; Ahlersova, E.; Ahlers, I.; Pastorova, B.
Male Wistar rats adapted to an artificial light-dark regimen were whole-body gamma-irradiated with a dose of 14.35 Gy. Irradiation, sham-irradiation and decapitation 30, 60 and 120 min after the exposure were performed between 2000 h and 0100 h in the darkness. The serotonin N-acetyltransferase activity (NAT), the concentration of melatonin and corticosterone were also determined. Ionizing radiation did not change the activity of NAT, the key enzyme of melatonin synthesis; however, it decreased the concentration of pineal melatonin. The concentration of pineal dopamine and norepinephrine decreased 30 and 120 min after exposure, while the concentration of epinephrine was elevated 30 min after irradiation, though later it was markedly decreased. The serum melatonin level was not changed but an increase in corticosterone level was observed. In the early period after exposure a decrease in pineal melatonin occurred, accompanied by a decrease in pineal catecholamines. On the contrary, in the phase of developed radiation injury the signs of increased melatonin synthesis were observed on days 3 and 4 after the exposure. (author) 6 figs., 25 refs
Co-expression of G2-EPSPS and glyphosate acetyltransferase GAT genes conferring high tolerance to glyphosate in soybean
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Bingfu eGuo
Full Text Available Glyphosate is a widely used non-selective herbicide with broad spectrum of weed control around the world. At present, most of the commercial glyphosate tolerant soybeans utilize glyphosate tolerant gene CP4-EPSPS or glyphosate acetyltransferase gene GAT separately. In this study, both glyphosate tolerant gene G2-EPSPS and glyphosate degraded gene GAT were co-transferred into soybean and transgenic plants showed high tolerance to glyphosate. Molecular analysis including PCR, Sothern blot, qRT-PCR and Western blot revealed that target genes have been integrated into genome and expressed effectively at both mRNA and protein levels. Furthermore, the glyphosate tolerance analysis showed that no typical symptom was observed when compared with a glyphosate tolerant line HJ06-698 derived from GR1 transgenic soybean even at four-fold labeled rate of Roundup. Chlorophyll and shikimic acid content analysis of transgenic plant also revealed that these two indexes were not significantly altered after glyphosate application. These results indicated that co-expression of G2-EPSPS and GAT conferred high tolerance to the herbicide glyphosate in soybean. Therefore, combination of tolerant and degraded genes provides a new strategy for developing glyphosate tolerant transgenic crops.
Histone Code Modulation by Oncogenic PWWP-Domain Protein in Breast Cancers
transcriptional machinery [1, 2]. Methylation of lysine and arginine histone side chains and core domains serves to modulate the epigene- tic...Transl Res. 4:247-56. 2012 4. Liu L, Kimball S, Liu H, Holowatyj A, and Yang Z-Q. Genetic alterations of histone lysine methyltransferases and their...Therapeutic Potential. Current Cancer Therapy Reviews. 9:78-85, 2013 6. Labbé RM, Holowatyj A, Yang Z-Q. Histone lysine demethylase (KDM) subfamily 4
Chromosomal mapping reveals a dynamic organization of the histone genes in aphids (Hemiptera: Aphididae
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M. Mandrioli
Full Text Available Despite their involvement in different processes, histone genes have been analysed in few insects. In order to improve the knowledge about this important gene family, genes coding for histones have been analysed in the aphid Acyrthosiphon pisum showing that at the amino acid level, aphid histones are highly conserved. In particular, data from A. pisum confirm that H1 is the most variable of the five histones, whereas histones H3 and H4 are highly conserved with the H3 almost identical from insects to vertebrates. A. pisum histone genes are organized in a quintet with the H1 gene followed by H2A and H2B genes that are adjacent and transcribed in same directions, in the opposite strand in respect to the H1 gene. At the 3' end of the histone cluster, genes H3 and H4 constitute an oppositely transcribed pair. The span of the aphid histone genes (more than 7 kb is greater than the average length of the histone cluster till now reported in insects (about 5 kb. Furthermore, spacers that separate the aphid histone genes vary in length. The histone genes have been mapped in A. pisum and successively in the aphids Myzus persicae and Rhopalosiphum padi showing that they are present in a single large cluster located in an interstitial position of autosomes 1, differently from what reported in the Russian wheat aphid Diuraphis noxia, where histone genes have been localized in a telomere of the two X chromosomes suggesting a dynamic organization of this multigene family in aphids.
Noncoordinate histone synthesis in heat-shocked Drosophila cells is regulated at multiple levels.
Farrell-Towt, J; Sanders, M M
Transferring Drosophila tissue culture cells from 25 to 37 degrees C (heat shock) causes histone protein synthesis to become noncoordinate. To determine the level at which this is controlled, the synthesis, degradation, and translation of individual histone mRNAs was studied under both heat shock and control conditions. The increased synthesis of histone H2b protein during heat shock appears to be controlled primarily at the level of translation. During heat shock, H2b mRNA is transcribed at ...
Somatic mutations of the histone H3K27 demethylase, UTX, in human cancer
van Haaften, Gijs; Dalgliesh, Gillian L; Davies, Helen; Chen, Lina; Bignell, Graham; Greenman, Chris; Edkins, Sarah; Hardy, Claire; O'Meara, Sarah; Teague, Jon; Butler, Adam; Hinton, Jonathan; Latimer, Calli; Andrews, Jenny; Barthorpe, Syd; Beare, Dave; Buck, Gemma; Campbell, Peter J; Cole, Jennifer; Dunmore, Rebecca; Forbes, Simon; Jia, Mingming; Jones, David; Kok, Chai Yin; Leroy, Catherine; Lin, Meng-Lay; McBride, David J; Maddison, Mark; Maquire, Simon; McLay, Kirsten; Menzies, Andrew; Mironenko, Tatiana; Lee, Mulderrig; Mudie, Laura; Pleasance, Erin; Shepherd, Rebecca; Smith, Raffaella; Stebbings, Lucy; Stephens, Philip; Tang, Gurpreet; Tarpey, Patrick S; Turner, Rachel; Turrell, Kelly; Varian, Jennifer; West, Sofie; Widaa, Sara; Wray, Paul; Collins, V Peter; Ichimura, Koichi; Law, Simon; Wong, John; Yuen, Siu Tsan; Leung, Suet Yi; Tonon, Giovanni; DePinho, Ronald A; Tai, Yu-Tzu; Anderson, Kenneth C; Kahnoski, Richard J.; Massie, Aaron; Khoo, Sok Kean; Teh, Bin Tean; Stratton, Michael R; Futreal, P Andrew
Somatically acquired epigenetic changes are present in many cancers. Epigenetic regulation is maintained via post-translational modifications of core histones. Here, we describe inactivating somatic mutations in the histone lysine demethylase, UTX, pointing to histone H3 lysine methylation deregulation in multiple tumour types. UTX reintroduction into cancer cells with inactivating UTX mutations resulted in slowing of proliferation and marked transcriptional changes. These data identify UTX as a new human cancer gene. PMID:19330029
Extracellular histones play an inflammatory role in acid aspiration-induced acute respiratory distress syndrome.
Zhang, Yanlin; Wen, Zongmei; Guan, Li; Jiang, Ping; Gu, Tao; Zhao, Jinyuan; Lv, Xin; Wen, Tao
Systemic inflammation is a key feature in acid aspiration-induced acute respiratory distress syndrome (ARDS), but the factors that trigger inflammation are unclear. The authors hypothesize that extracellular histones, a newly identified inflammatory mediator, play important roles in the pathogenesis of ARDS. The authors used a hydrochloric acid aspiration-induced ARDS model to investigate whether extracellular histones are pathogenic and whether targeting histones are protective. Exogenous histones and antihistone antibody were administered to mice. Heparin can bind to histones, so the authors studied whether heparin could protect from ARDS using cell and mouse models. Furthermore, the authors analyzed whether extracellular histones are clinically involved in ARDS patients caused by gastric aspiration. Extracellular histones in bronchoalveolar lavage fluid of acid-treated mice were significantly higher (1.832 ± 0.698) at 3 h after injury than in sham-treated group (0.63 ± 0.153; P = 0.0252, n = 5 per group). Elevated histones may originate from damaged lung cells and neutrophil infiltration. Exogenous histones aggravated lung injury, whereas antihistone antibody markedly attenuated the intensity of ARDS. Notably, heparin provided a similar protective effect against ARDS. Analysis of plasma from ARDS patients (n = 21) showed elevated histones were significantly correlated with the degree of ARDS and were higher in nonsurvivors (2.723 ± 0.2933, n = 7) than in survivors (1.725 ± 0.1787, P = 0.006, n = 14). Extracellular histones may play a contributory role toward ARDS by promoting tissue damage and systemic inflammation and may become a novel marker reflecting disease activity. Targeting histones by neutralizing antibody or heparin shows potent protective effects, suggesting a potentially therapeutic strategy.
Some physico-chemical characteristics of a modified histone H2b on acute radiation affection
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Khrapunov, S.N.; Mel'nik, G.G.; Blyum, Ya.B.; Tsudzevich, B.A.; Kucherenko, N.E.
A study was made of optical characteristics of histone H2b isolated from liver nuclei 12 h following irradiation in a dose of 0.21 C/kg. It was demonstrated that under similar conditions, the control and exposed histones H2b have different steric organization which correlates with radiation-induced modifications of lateral radicals in H2b histone molecules
Histones Differentially Modulate the Anticoagulant and Profibrinolytic Activities of Heparin, Heparin Derivatives, and Dabigatran.
Ammollo, Concetta Tiziana; Semeraro, Nicola; Carratù, Maria Rosaria; Colucci, Mario; Semeraro, Fabrizio
The antithrombin activity of unfractionated heparin (UFH) is offset by extracellular histones, which, along with DNA, represent a novel mediator of thrombosis and a structural component of thrombi. Here, we systematically evaluated the effect of histones, DNA, and histone-DNA complexes on the anticoagulant and profibrinolytic activities of UFH, its derivatives enoxaparin and fondaparinux, and the direct thrombin inhibitor dabigatran. Thrombin generation was assessed by calibrated automated thrombinography, inhibition of factor Xa and thrombin by synthetic substrates, tissue plasminogen activator-mediated clot lysis by turbidimetry, and thrombin-activatable fibrinolysis inhibitor (TAFI) activation by a functional assay. Histones alone delayed coagulation and slightly stimulated fibrinolysis. The anticoagulant activity of UFH and enoxaparin was markedly inhibited by histones, whereas that of fondaparinux was enhanced. Histones neutralized both the anti-Xa and anti-IIa activities of UFH and preferentially blocked the anti-IIa activity of enoxaparin. The anti-Xa activity of fondaparinux was not influenced by histones when analyzed by chromogenic substrates, but was potentiated in a plasma prothrombinase assay. Histones inhibited the profibrinolytic activity of UFH and enoxaparin and enhanced that of fondaparinux by acting on the modulation of TAFI activation by anticoagulants. Histone H1 was mainly responsible for these effects. Histone-DNA complexes, as well as intact neutrophil extracellular traps, impaired the activities of UFH, enoxaparin, and fondaparinux. Dabigatran was not noticeably affected by histones and/or DNA, whatever the assay performed. In conclusion, histones and DNA present in the forming clot may variably influence the antithrombotic activities of anticoagulants, suggesting a potential therapeutic advantage of dabigatran and fondaparinux over heparins. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.
Inhibition of class I histone deacetylases in non-small cell lung cancer by honokiol leads to suppression of cancer cell growth and induction of cell death in vitro and in vivo.
Singh, Tripti; Prasad, Ram; Katiyar, Santosh K
Non-small-cell lung cancer (NSCLC) represents approximately 80% of all types of lung cancer. Here, we report the chemotherapeutic effect of honokiol, a phytochemical from Magnolia grandiflora, on NSCLC cells and the molecular mechanisms underlying these effects using in vitro and in vivo models. Treatment of NSCLC cells (A549, H1299, H460 and H226) with honokiol (20, 40 and 60 µM) inhibited histone deacetylase (HDAC) activity, reduced the levels of class I HDAC proteins and enhanced histone acetyltransferase activity in a dose-dependent manner. These effects of honokiol were associated with a significant reduction in the viability of NSCLC cells. Concomitant treatment of cells with a proteasome inhibitor, MG132, prevented honokiol-induced degradation of class I HDACs, suggesting that honokiol reduced the levels of HDACs in NSCLC cells through proteasomal degradation. Valproic acid, an inhibitor of HDACs, exhibited a similar pattern of reduced viability and induction of death of NSCLC cells. Treatment of A549 and H1299 cells with honokiol resulted in an increase in G 1 phase arrest, and a decrease in the levels of cyclin D1, D2 and cyclin dependent kinases. Further, administration of honokiol by oral gavage significantly inhibited the growth of subcutaneous A549 and H1299 tumor xenografts in athymic nude mice, which was associated with the induction of apoptotic cell death and marked inhibition of class I HDACs proteins and HDAC activity in the tumor xenograft tissues. Together, our study provides new insights into the role of class I HDACs in the chemotherapeutic effects of honokiol on lung cancer cells.
Total levels of hippocampal histone acetylation predict normal variability in mouse behavior.
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Addie May I Nesbitt
Full Text Available Genetic, pharmacological, and environmental interventions that alter total levels of histone acetylation in specific brain regions can modulate behaviors and treatment responses. Efforts have been made to identify specific genes that are affected by alterations in total histone acetylation and to propose that such gene specific modulation could explain the effects of total histone acetylation levels on behavior - the implication being that under naturalistic conditions variability in histone acetylation occurs primarily around the promoters of specific genes.Here we challenge this hypothesis by demonstrating with a novel flow cytometry based technique that normal variability in open field exploration, a hippocampus-related behavior, was associated with total levels of histone acetylation in the hippocampus but not in other brain regions.Results suggest that modulation of total levels of histone acetylation may play a role in regulating biological processes. We speculate in the discussion that endogenous regulation of total levels of histone acetylation may be a mechanism through which organisms regulate cellular plasticity. Flow cytometry provides a useful approach to measure total levels of histone acetylation at the single cell level. Relating such information to behavioral measures and treatment responses could inform drug delivery strategies to target histone deacetylase inhibitors and other chromatin modulators to places where they may be of benefit while avoiding areas where correction is not needed and could be harmful.
Histone H1 vaccine therapy for overcoming acute rejection in experimental organ transplantation.
Nakano, T; Ono, K; Goto, S; Lai, C-Y; Hsu, L-W; Kawamoto, S; Lin, Y-C; Kao, Y-H; Chiang, K-C; Ohmori, N; Goto, T; Sato, S; Jawan, B; Cheng, Y-F; Chen, C-L
In a rat tolerogenic orthotopic liver transplantation (OLT) model, the recipient serum (post-OLT serum) shows strong immunosuppressive activity. In our previous reports, we suggested that autoreactive antibody (Ab) against histone H1 is a major immunosuppressive factor in this serum. The present study sought to determine whether up-regulation of anti-histone H1 Ab by histone H1 vaccination led to tolerance. Using mixed lymphocyte reactions (MLR) and heterotopic heart transplantations (HHT), the alloreactive T-cell responses and allograft survivals of histone H1-immunized rats were compared with those of control rats. Cytokine and cellular profiles were determined by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The alloreactive T-cell response of histone H1-immunized rats was significantly lower than that of control rats, although there was no difference in nonspecific T-cell activation between the 2 groups. The allograft survival of histone H1-immunized rats was significantly prolonged after HHT. The major histocompatibility complex (MHC) class II and CD25 molecules of histone H1-immunized rats were significantly down-regulated compared with those of control rats. Moreover, the serum cytokine profile was modified by the immunization with histone H1. These results suggest that histone H1 vaccination of transplant recipients leads to the production of immunosuppressive factors and the modification of cytokine/cellular profiles.
Investigation of the reactions of histone protein hydroperoxides and their role in DNA damage
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Luxford, C.; Dean, R.T.; Davies, M.J.
Free radical attack on DNA results in base changes, cross-linking and strand cleavage leading to mutations if unrepaired. Histone proteins are intimately involved in DNA packaging and are excellent candidates for investigating DNA damage arising from protein-OOH-derived radicals. This study aimed (i) to investigate the formation of hydroperoxide on the linker histone H1 via radical reactions in the presence of O 2 ; (ii) to examine the radicals formed from transition metal ion-catalyzed breakdown of histone H1-OOH and (iii) to determine whether histone H1-OOH-derived radicals can damage DNA and free bases. (i) Histone H1 solutions were γ-irradiated ( 60 Co source) in the presence of O 2 and histone H1-OOH concentrations determined using a manual iodometric assay. Formation ( histone H1-OOH was dose-dependent and, in the absence of light or transition metal ions these hydroperoxides were found to be very stable (half life of 24 hours at 4degC ). (ii) Electron Paramagnetic Resonance (EPR) spectroscopy and spin trapping was used t investigate the Cu + -catalyzed breakdown of histone H1-OOH to form histone H1 protein side chain and -backbone carbon-centred radicals. Further EPR/spin trapping experiments showed that histone H1-OOH-derived radicals can oxidise pyrimidine bases (eg. uridine with the resultant trapping of three radical species; two pyrimidine radicals, C5-yl and Ct yl adducts (via addition of histone H1-OOH-derived radicals to the C5-C6 double bond o the pyrimidine ring) and an acyl radical adduct, whose origin is currently unknown. (iii) Damage to DNA and 2'-deoxyguanosine after reaction of histone H1-OOH-derive radicals were detected and quantified using HPLC (with EC and UV detection). We have identified 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) as a significant product ( histone H1-OOH-derived oxidative DNA modification. Increasing histone H1-OOH concentrations resulted in a concomitant increase in the amount of 8-oxodG formed. Our studies show
Biophysical characterization of the association of histones with single-stranded DNA.
Wang, Ying; van Merwyk, Luis; Tönsing, Katja; Walhorn, Volker; Anselmetti, Dario; Fernàndez-Busquets, Xavier
Despite the profound current knowledge of the architecture and dynamics of nucleosomes, little is known about the structures generated by the interaction of histones with single-stranded DNA (ssDNA), which is widely present during replication and transcription. Non-denaturing gel electrophoresis, transmission electron microscopy, atomic force microscopy, magnetic tweezers. Histones have a high affinity for ssDNA in 0.15M NaCl ionic strength, with an apparent binding constant similar to that calculated for their association with double-stranded DNA (dsDNA). The length of DNA (number of nucleotides in ssDNA or base pairs in dsDNA) associated with a fixed core histone mass is the same for both ssDNA and dsDNA. Although histone-ssDNA complexes show a high tendency to aggregate, nucleosome-like structures are formed at physiological salt concentrations. Core histones are able to protect ssDNA from digestion by micrococcal nuclease, and a shortening of ssDNA occurs upon its interaction with histones. The purified (+) strand of a cloned DNA fragment of nucleosomal origin has a higher affinity for histones than the purified complementary (-) strand. At physiological ionic strength histones have high affinity for ssDNA, possibly associating with it into nucleosome-like structures. In the cell nucleus histones may spontaneously interact with ssDNA to facilitate their participation in the replication and transcription of chromatin. Copyright © 2017 Elsevier B.V. All rights reserved.
Deficit in DNA content relative to histones in X-irradiated HeLa cells
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Bases, R.; Mendez, F.; Neubort, S.
The DNA and histone content of HeLa S-3 cell cultures was measured by direct mass assays 21 hours after 1000 rad of X-irradiation, when the cells were arrested in G2 phase. The nuclear DNA content of such cultures was found to be deficient (73 per cent of control values). In contrast, the synthesis of nuclear histones persisted, and the total histone content was close to 100 per cent of control values. When synchronously-growing cultures were irradiated in mid-S phase and examined 3.5 hours later in G2 phase, both DNA and histone content were equal to control values. (author)
O-antigen protects gram-negative bacteria from histone killing.
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Catherine Chaput
Full Text Available Beyond their traditional role of wrapping DNA, histones display antibacterial activity to Gram-negative and -positive bacteria. To identify bacterial components that allow survival to a histone challenge, we selected resistant bacteria from homologous Escherichia coli libraries that harbor plasmids carrying pieces of the chromosome in different sizes. We identified genes required for exopolysaccharide production and for the synthesis of the polysaccharide domain of the lipopolysaccharide, called O-antigen. Indeed, O-antigen and exopolysaccharide conferred further resistance to histones. Notably, O-antigen also conferred resistance to histones in the pathogens Shigella flexneri and Klebsiella pneumoniae.
Global turnover of histone post-translational modifications and variants in human cells
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Zee Barry M
Full Text Available Abstract Background Post-translational modifications (PTMs on the N-terminal tails of histones and histone variants regulate distinct transcriptional states and nuclear events. Whereas the functional effects of specific PTMs are the current subject of intense investigation, most studies characterize histone PTMs/variants in a non-temporal fashion and very few studies have reported kinetic information about these histone forms. Previous studies have used radiolabeling, fluorescence microscopy and chromatin immunoprecipitation to determine rates of histone turnover, and have found interesting correlations between increased turnover and increased gene expression. Therefore, histone turnover is an understudied yet potentially important parameter that may contribute to epigenetic regulation. Understanding turnover in the context of histone modifications and sequence variants could provide valuable additional insight into the function of histone replacement. Results In this study, we measured the metabolic rate of labeled isotope incorporation into the histone proteins of HeLa cells by combining stable isotope labeling of amino acids in cell culture (SILAC pulse experiments with quantitative mass spectrometry-based proteomics. In general, we found that most core histones have similar turnover rates, with the exception of the H2A variants, which exhibit a wider range of rates, potentially consistent with their epigenetic function. In addition, acetylated histones have a significantly faster turnover compared with general histone protein and methylated histones, although these rates vary considerably, depending on the site and overall degree of methylation. Histones containing transcriptionally active marks have been consistently found to have faster turnover rates than histones containing silent marks. Interestingly, the presence of both active and silent marks on the same peptide resulted in a slower turnover rate than either mark alone on that same
New clinical developments in histone deacetylase inhibitors for epigenetic therapy of cancer
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Ma Yuehua
Full Text Available Abstract DNA methylation and histone acetylation are two well known epigenetic chromatin modifications. Epigenetic agents leading to DNA hypomethylation and histone hyperacetylation have been approved for treatment of hematological disorders. The first histone deacetylase inhibitor, vorinostat, has been licensed for cutaneous T cell lymphoma treatment. More than 11 new epigenetic agents are in various stages of clinical development for therapy of multiple cancer types. In this review we summarize novel histone deacetylase inhibitors and new regimens from clinical trials for epigenetic therapy of cancer.
Apoptotic phosphorylation of histone H3 on Ser-10 by protein kinase Cδ.
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Choon-Ho Park
Full Text Available Phosphorylation of histone H3 on Ser-10 is regarded as an epigenetic mitotic marker and is tightly correlated with chromosome condensation during both mitosis and meiosis. However, it was also reported that histone H3 Ser-10 phosphorylation occurs when cells are exposed to various death stimuli, suggesting a potential role in the regulation of apoptosis. Here we report that histone H3 Ser-10 phosphorylation is mediated by the pro-apoptotic kinase protein kinase C (PKC δ during apoptosis. We observed that PKCδ robustly phosphorylates histone H3 on Ser-10 both in vitro and in vivo. Ectopic expression of catalytically active PKCδ efficiently induces condensed chromatin structure in the nucleus. We also discovered that activation of PKCδ is required for histone H3 Ser-10 phosphorylation after treatment with DNA damaging agents during apoptosis. Collectively, these findings suggest that PKCδ is the kinase responsible for histone H3 Ser-10 phosphoryation during apoptosis and thus contributes to chromatin condensation together with other apoptosis-related histone modifications. As a result, histone H3 Ser-10 phosphorylation can be designated a new 'apoptotic histone code' mediated by PKCδ.
Posttranslational modifications of the histone 3 tail and their impact on the activity of histone lysine demethylases in vitro.
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Brian Lohse
Full Text Available Posttranslational modifications (PTMs of the histone H3 tail such as methylation, acetylation and phosphorylation play important roles in epigenetic signaling. Here we study the effect of some of these PTMs on the demethylation rates of methylated lysine 9 in vitro using peptide substrates mimicking histone H3. Various combinations with other PTMs were employed to study possible cross-talk effects by comparing enzyme kinetic characteristics. We compared the kinetics of histone tail substrates for truncated histone lysine demethylases KDM4A and KDM4C containing only the catalytic core (cc and some combinations were characterized on full length (FL KDM4A and KDM4C. We found that the substrates combining trimethylated K4 and K9 resulted in a significant increase in the catalytic activity for FL-KDM4A. For the truncated versions of KDM4A and KDM4C a two-fold increase in the catalytic activity toward bis-trimethylated substrates could be observed. Furthermore, a significant difference in the catalytic activity between dimethylated and trimethylated substrates was found for full length demethylases in line with what has been reported previously for truncated demethylases. Histone peptide substrates phosphorylated at T11 could not be demethylated by neither truncated nor full length KDM4A and KDM4C, suggesting that phosphorylation of threonine 11 prevents demethylation of the H3K9me3 mark on the same peptide. Acetylation of K14 was also found to influence demethylation rates significantly. Thus, for truncated KDM4A, acetylation on K14 of the substrate leads to an increase in enzymatic catalytic efficiency (k cat/K m, while for truncated KDM4C it induces a decrease, primarily caused by changes in K m. This study demonstrates that demethylation activities towards trimethylated H3K9 are significantly influenced by other PTMs on the same peptide, and emphasizes the importance of studying these interactions at the peptide level to get a more detailed
Phosphorylation of histone H3T6 by PKCbeta(I) controls demethylation at histone H3K4.
Metzger, Eric; Imhof, Axel; Patel, Dharmeshkumar; Kahl, Philip; Hoffmeyer, Katrin; Friedrichs, Nicolaus; Müller, Judith M; Greschik, Holger; Kirfel, Jutta; Ji, Sujuan; Kunowska, Natalia; Beisenherz-Huss, Christian; Günther, Thomas; Buettner, Reinhard; Schüle, Roland
Demethylation at distinct lysine residues in histone H3 by lysine-specific demethylase 1 (LSD1) causes either gene repression or activation. As a component of co-repressor complexes, LSD1 contributes to target gene repression by removing mono- and dimethyl marks from lysine 4 of histone H3 (H3K4). In contrast, during androgen receptor (AR)-activated gene expression, LSD1 removes mono- and dimethyl marks from lysine 9 of histone H3 (H3K9). Yet, the mechanisms that control this dual specificity of demethylation are unknown. Here we show that phosphorylation of histone H3 at threonine 6 (H3T6) by protein kinase C beta I (PKCbeta(I), also known as PRKCbeta) is the key event that prevents LSD1 from demethylating H3K4 during AR-dependent gene activation. In vitro, histone H3 peptides methylated at lysine 4 and phosphorylated at threonine 6 are no longer LSD1 substrates. In vivo, PKCbeta(I) co-localizes with AR and LSD1 on target gene promoters and phosphorylates H3T6 after androgen-induced gene expression. RNA interference (RNAi)-mediated knockdown of PKCbeta(I) abrogates H3T6 phosphorylation, enhances demethylation at H3K4, and inhibits AR-dependent transcription. Activation of PKCbeta(I) requires androgen-dependent recruitment of the gatekeeper kinase protein kinase C (PKC)-related kinase 1 (PRK1). Notably, increased levels of PKCbeta(I) and phosphorylated H3T6 (H3T6ph) positively correlate with high Gleason scores of prostate carcinomas, and inhibition of PKCbeta(I) blocks AR-induced tumour cell proliferation in vitro and cancer progression of tumour xenografts in vivo. Together, our data establish that androgen-dependent kinase signalling leads to the writing of the new chromatin mark H3T6ph, which in consequence prevents removal of active methyl marks from H3K4 during AR-stimulated gene expression.
Identification of novel post-translational modifications in linker histones from chicken erythrocytes.
Sarg, Bettina; Lopez, Rita; Lindner, Herbert; Ponte, Inma; Suau, Pedro; Roque, Alicia
Chicken erythrocyte nuclei were digested with micrococcal nuclease and fractionated by centrifugation in low-salt buffer into soluble and insoluble fractions. Post-translational modifications of the purified linker histones of both fractions were analyzed by LC-ESI-MS/MS. All six histone H1 subtypes (H1.01, H1.02, H1.03, H1.10, H1.1L and H1.1R) and histone H5 were identified. Mass spectrometry analysis enabled the identification of a wide range of PTMs, including N(α)-terminal acetylation, acetylation, formylation, phosphorylation and oxidation. A total of nine new modifications in chicken linker histones were mapped, most of them located in the N-terminal and globular domains. Relative quantification of the modified peptides showed that linker histone PTMs were differentially distributed among both chromatin fractions, suggesting their relevance in the regulation of chromatin structure. The analysis of our results combined with previously reported data for chicken and some mammalian species showed that most of the modified positions were conserved throughout evolution, highlighting their importance in specific linker histone functions and epigenetics. Post-translational modifications of linker histones could have a role in the regulation of gene expression through the modulation of chromatin higher-order structure and chromatin remodeling. Finding new PTMs in linker histones is the first step to elucidate their role in the histone code. In this manuscript we report nine new post-translational modifications of the linker histones from chicken erythrocytes, one in H5 and eight in the H1 subtypes. Chromatin fractionated by centrifugation in low-salt buffer resulted in two fractions with different contents and compositions of linker histones and enriched in specific core histone PTMs. Of particular interest is the fact that linker histone PTMs were differentially distributed in both chromatin fractions, suggesting specific functions. Future studies are needed to
Impact of plasma histones in human sepsis and their contribution to cellular injury and inflammation.
Ekaney, Michael Liembo; Otto, Gordon Philipp; Sossdorf, Maik; Sponholz, Christoph; Boehringer, Michael; Loesche, Wolfgang; Rittirsch, Daniel; Wilharm, Arne; Kurzai, Oliver; Bauer, Michael; Claus, Ralf Alexander
Circulating histones have been identified as mediators of damage in animal models of sepsis and in patients with trauma-associated lung injury. Despite existing controversies on actual histone concentrations, clinical implications and mechanism of action in various disease conditions, histone levels in human sepsis, association with disease progression and mediated effects on endothelial and immune cells remain unreported. This study aimed to determine histone levels and its clinical implication in septic patients and to elucidate histone-mediated effects ex-vivo. Histone levels, endogenous activated protein C (APC) levels and clinical data from two independent cohorts of septic patients were obtained. Histone levels were compared with various control groups including healthy individuals, intensive care unit (ICU) patients without sepsis, ICU patients with multiple organ failure and patients with minor or multiple trauma, all without infection. Endothelial and monocytic cells were stimulated with histones. Cellular integrity and sepsis prototypical cytokines were evaluated. The mechanism of action of histones via Toll-like receptor 4 (TLR4) was evaluated using a function blocking antibody. Histone degradation in plasma was studied by immunoblotting. Histone H4 levels were significantly elevated in patients with sepsis (cohort I; n = 15 and cohort II; n = 19) versus ICU controls (n = 12), patients with multiple organ failure (n = 12) or minor trauma (n = 7), associated with need for renal replacement therapy and decrease in platelet count during disease progression, and remarkably were significantly associated with increased mortality rates in septic patients (ICU-, 28 day- and 90 day mortality rates). There was an inverse correlation between plasma histones and endogenous APC levels. Histone stimulation induced the release of sepsis prototypic cytokines and decreased cell integrity indicated by a significant increase of lactate dehydrogenase (LDH) and propidium
Hyaluronan protection of corneal endothelial cells against extracellular histones after phacoemulsification.
Kawano, Hiroki; Sakamoto, Taiji; Ito, Takashi; Miyata, Kazunori; Hashiguchi, Teruto; Maruyama, Ikuro
To determine the effect of histones on corneal endothelial cells generated during cataract surgery. Kagoshima University Hospital, Kagoshima, Japan. Experimental study. Standard phacoemulsification was performed on enucleated pig eyes. Histones in the anterior segment of the eye were determined by immunohistochemistry. Cultured human corneal endothelial cells were exposed to histones for 18 hours, and cell viability was determined by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitro-phenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt assay. The concentration of interleukin-6 (IL-6) in the culture medium of human corneal endothelial cells was measured using enzyme-linked immunosorbent assay. The effects of signal inhibitors U0126, SB203580, and SP600125 were evaluated. The protective effect of hyaluronan against histones was evaluated in human corneal endothelial cells with and without hyaluronan. Cellular debris containing histones was observed in the anterior chamber of pig eyes after phacoemulsification. Exposure of human corneal endothelial cells to 50 μg/mL of histones or more led to cytotoxic effects. The IL-6 concentration was significantly increased dose dependently after exposure of human corneal endothelial cells to histones (Phistone-induced IL-6 production was significantly decreased by extracellular signal-regulated kinases 1/2 and p-38 mitogen-activated protein kinase inhibitors (Phistones caused formation of histone aggregates, decreased the cytotoxic effects of the histones, and blocked the increase in IL-6 (PHistones were released extracellularly during phacoemulsification and exposure of human corneal endothelial cells to histones increased the IL-6 secretion. The intraoperative use of hyaluronan may decrease the cytotoxic effects of histones released during cataract surgery. No author has a financial or proprietary interest in any material or method mentioned. Copyright © 2014 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.
Stage-specific histone modification profiles reveal global transitions in the Xenopus embryonic epigenome.
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Tobias D Schneider
Full Text Available Vertebrate embryos are derived from a transitory pool of pluripotent cells. By the process of embryonic induction, these precursor cells are assigned to specific fates and differentiation programs. Histone post-translational modifications are thought to play a key role in the establishment and maintenance of stable gene expression patterns underlying these processes. While on gene level histone modifications are known to change during differentiation, very little is known about the quantitative fluctuations in bulk histone modifications during development. To investigate this issue we analysed histones isolated from four different developmental stages of Xenopus laevis by mass spectrometry. In toto, we quantified 59 modification states on core histones H3 and H4 from blastula to tadpole stages. During this developmental period, we observed in general an increase in the unmodified states, and a shift from histone modifications associated with transcriptional activity to transcriptionally repressive histone marks. We also compared these naturally occurring patterns with the histone modifications of murine ES cells, detecting large differences in the methylation patterns of histone H3 lysines 27 and 36 between pluripotent ES cells and pluripotent cells from Xenopus blastulae. By combining all detected modification transitions we could cluster their patterns according to their embryonic origin, defining specific histone modification profiles (HMPs for each developmental stage. To our knowledge, this data set represents the first compendium of covalent histone modifications and their quantitative flux during normogenesis in a vertebrate model organism. The HMPs indicate a stepwise maturation of the embryonic epigenome, which may be causal to the progressing restriction of cellular potency during development.
Deposition of newly synthesized histones: new histones H2A and H2B do not deposit in the same nucleosome with new Histones H3 and H4
International Nuclear Information System (INIS)
Jackson, V.
The authors have developed procedures to study histone-histone interactions during the deposition of histones in replicating cells. Cells are labeled for 60 min with dense amino acids, and subsequently, the histones within the nucleosomes are cross-linked into an octameric complex with formaldehyde. These complexes are sedimented to equilibrium in density gradients and octamer and dioctamer complexes separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With reversal of the cross-link, the distribution of the individual density-labeled histones in the octamer is determined. Newly synthesized H3 and H4 deposits as a tetramer and are associated with old H2A and H2B. Newly synthesized H2A and H2B deposit as a dimer associated with old H2A, H2B, H3, and H4. The significance of these results with respect to the dynamics of histone interactions in the nucleus is discussed. Control experiments are presented to test for artifactual formation of these complexes during preparative procedures. In addition, reconstitution experiments were performed to demonstrate that the composition of these octameric complexes can be determined from their distribution of density gradients
Radiosensitization of colorectal carcinoma cell lines by histone deacetylase inhibition
International Nuclear Information System (INIS)
Flatmark, Kjersti; Nome, Ragnhild V; Folkvord, Sigurd; Bratland, Åse; Rasmussen, Heidi; Ellefsen, Mali Strand; Fodstad, Øystein; Ree, Anne Hansen
The tumor response to preoperative radiotherapy of locally advanced rectal cancer varies greatly, warranting the use of experimental models to assay the efficacy of molecular targeting agents in rectal cancer radiosensitization. Histone deacetylase (HDAC) inhibitors, agents that cause hyperacetylation of histone proteins and thereby remodeling of chromatin structure, may override cell cycle checkpoint responses to DNA damage and amplify radiation-induced tumor cell death. Human colorectal carcinoma cell lines were exposed to ionizing radiation and HDAC inhibitors, and cell cycle profiles and regulatory factors, as well as clonogenicity, were analyzed. In addition to G 2 /M phase arrest following irradiation, the cell lines displayed cell cycle responses typical for either intact or defective p53 function (the presence or absence, respectively, of radiation-induced expression of the cell cycle inhibitor p21 and subsequent accumulation of G 1 phase cells). In contrast, histone acetylation was associated with complete depletion of the G 1 population of cells with functional p53 but accumulation of both G 1 and G 2 /M populations of cells with defective p53. The cellular phenotypes upon HDAC inhibition were consistent with the observed repression of Polo-like kinase-1, a regulatory G 2 /M phase kinase. Following pre-treatment with HDAC inhibitors currently undergoing clinical investigation, the inhibitory effect of ionizing radiation on clonogenicity was significantly amplified. In these experimental models, HDAC inhibition sensitized the tumor cells to ionizing radiation, which is in accordance with the concept of increased probability of tumor cell death when chromatin structure is modified
PARP-1 Interaction with and Activation by Histones and Nucleosomes.
Thomas, Colin; Kotova, Elena; Tulin, Alexei V
Poly(ADP-ribose) Polymerase 1 (PARP-1) is an abundant chromatin associated protein, typical for most eukaryotic nuclei. The localization of PARP-1 in chromatin and its enzymatic activation involves multiple interactions of PARP-1 with nucleosomal histones, other proteins, and DNA. We report a set of methods designed to reconstitute PARP-1 regulation in vitro. These methods involve the expression of PARP-1 and PARP-1-regulating proteins using bacterial and eukaryotic systems, purification of these proteins using chromatography, testing of individual interactions in vitro, assembly of active complexes, and reconstitution of PARP-1 regulating reactions in vitro.
Choline nutrition programs brain development via DNA and histone methylation.
Blusztajn, Jan Krzysztof; Mellott, Tiffany J
Choline is an essential nutrient for humans. Metabolically choline is used for the synthesis of membrane phospholipids (e.g. phosphatidylcholine), as a precursor of the neurotransmitter acetylcholine, and, following oxidation to betaine, choline functions as a methyl group donor in a pathway that produces S-adenosylmethionine. As a methyl donor choline influences DNA and histone methylation--two central epigenomic processes that regulate gene expression. Because the fetus and neonate have high demands for choline, its dietary intake during pregnancy and lactation is particularly important for normal development of the offspring. Studies in rodents have shown that high choline intake during gestation improves cognitive function in adulthood and prevents memory decline associated with old age. These behavioral changes are accompanied by electrophysiological, neuroanatomical, and neurochemical changes and by altered patterns of expression of multiple cortical and hippocampal genes including those encoding key proteins that contribute to the biochemical mechanisms of learning and memory. These actions of choline are observed long after the exposure to the nutrient ended (months) and correlate with fetal hepatic and cerebral cortical choline-evoked changes in global- and gene-specific DNA cytosine methylation and with dramatic changes of the methylation pattern of lysine residues 4, 9 and 27 of histone H3. Moreover, gestational choline modulates the expression of DNA (Dnmt1, Dnmt3a) and histone (G9a/Ehmt2/Kmt1c, Suv39h1/Kmt1a) methyltransferases. In addition to the central role of DNA and histone methylation in brain development, these processes are highly dynamic in adult brain, modulate the expression of genes critical for synaptic plasticity, and are involved in mechanisms of learning and memory. A recent study documented that in a cohort of normal elderly people, verbal and visual memory function correlated positively with the amount of dietary choline consumption
Adult ciliary neurotrophic factor receptors help maintain facial motor neuron choline acetyltransferase expression in vivo following nerve crush.
Lee, Nancy; Rydyznski, Carolyn E; Rasch, Matthew S; Trinh, Dennis S; MacLennan, A John
Exogenous ciliary neurotrophic factor (CNTF) administration promotes the survival of motor neurons in a wide range of models. It also increases the expression of the critical neurotransmitter enzyme choline acetyltransferase (ChAT) by in vitro motor neurons, likely independent of its effects on their survival. We have used the adult mouse facial nerve crush model and adult-onset conditional disruption of the CNTF receptor α (CNTFRα) gene to directly examine the in vivo roles played by endogenous CNTF receptors in adult motor neuron survival and ChAT maintenance, independent of developmental functions. We have previously shown that adult activation of the CreER gene construct in floxed CNTFRα mice depletes this essential receptor subunit in a large subset of motor neurons (and all skeletal muscle, as shown in this study) but has no effect on the survival of intact or lesioned motor neurons, indicating that these adult CNTF receptors play no essential survival role in this model, in contrast to their essential role during embryonic development. Here we show that this same CNTFRα depletion does not affect ChAT labeling in nonlesioned motor neurons, but it significantly increases the loss of ChAT following nerve crush. The data suggest that, although neither motor neuron nor muscle CNTF receptors play a significant, nonredundant role in the maintenance of ChAT in intact adult motor neurons, the receptors become essential for ChAT maintenance when the motor neurons are challenged by nerve crush. Therefore, the data suggest that the receptors act as a critical component of an endogenous neuroprotective mechanism. J. Comp. Neurol. 525:1206-1215, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
Localization of choline acetyltransferase and tyrosine hydroxylase immunoreactivities in the superior colliculus of the microbat,Rhinolophus ferrumequinum.
Jeong, Se-Jin; Jeon, Chang-Jin
The purpose of this study was to determine whether the superior colliculus (SC) of the microbat has the same neurochemical makeup as that of other mammals. We examined the organization of choline acetyltransferase (ChAT)- and tyrosine hydroxylase-immunoreactive (TH-IR) fibers/cells using standard immunohistochemistry with antibodies against ChAT and TH. ChAT-IR fibers observed in the superficial layers were denser than those in the deeper layers, and these fibers were classified into two types: small varicose fibers and large varicose fibers. ChAT-IR cells were predominantly located in the superficial layers with diverse morphologies. Among the well-known sources of cholinergic fibers in the mammalian SC, pedunculopontine tegmental nucleus (PPTN) and laterodorsal tegmental nucleus (LDTN) contained strongly labeled ChAT-IR cells, while no cholinergic structures were found in the parabigeminal nucleus (PBG) in the microbat brain. TH-immunoreactivity was found within fibers but not within cells. The density of TH-IR fibers was high in the zonal layer, moderate in the superficial gray and optic layers, and low in the deeper layers. Well-labeled TH-IR cells were also observed within area 13 and the locus coeruleus, known as the sources of catecholaminergic fibers in other mammalian SC. Although there are some cytoarchitectural variations among species, our results clearly showed elaborately organized ChAT-IR and TH-IR fibers/cells in the microbat SC. Our findings will contribute significantly to the understanding of actively constructed microbat visual systems.
daf-31 encodes the catalytic subunit of N alpha-acetyltransferase that regulates Caenorhabditis elegans development, metabolism and adult lifespan.
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Di Chen
Full Text Available The Caenorhabditis elegans dauer larva is a facultative state of diapause. Mutations affecting dauer signal transduction and morphogenesis have been reported. Of these, most that result in constitutive formation of dauer larvae are temperature-sensitive (ts. The daf-31 mutant was isolated in genetic screens looking for novel and underrepresented classes of mutants that form dauer and dauer-like larvae non-conditionally. Dauer-like larvae are arrested in development and have some, but not all, of the normal dauer characteristics. We show here that daf-31 mutants form dauer-like larvae under starvation conditions but are sensitive to SDS treatment. Moreover, metabolism is shifted to fat accumulation in daf-31 mutants. We cloned the daf-31 gene and it encodes an ortholog of the arrest-defective-1 protein (ARD1 that is the catalytic subunit of the major N alpha-acetyltransferase (NatA. A daf-31 promoter::GFP reporter gene indicates daf-31 is expressed in multiple tissues including neurons, pharynx, intestine and hypodermal cells. Interestingly, overexpression of daf-31 enhances the longevity phenotype of daf-2 mutants, which is dependent on the forkhead transcription factor (FOXO DAF-16. We demonstrate that overexpression of daf-31 stimulates the transcriptional activity of DAF-16 without influencing its subcellular localization. These data reveal an essential role of NatA in controlling C. elegans life history and also a novel interaction between ARD1 and FOXO transcription factors, which may contribute to understanding the function of ARD1 in mammals.
Immunohistochemical localization of two types of choline acetyltransferase in neurons and sensory cells of the octopus arm.
Sakaue, Yuko; Bellier, Jean-Pierre; Kimura, Shin; D'Este, Loredana; Takeuchi, Yoshihiro; Kimura, Hiroshi
Cholinergic structures in the arm of the cephalopod Octopus vulgaris were studied by immunohistochemistry using specific antisera for two types (common and peripheral) of acetylcholine synthetic enzyme choline acetyltransferase (ChAT): antiserum raised against the rat common type ChAT (cChAT), which is cross-reactive with molluscan cChAT, and antiserum raised against the rat peripheral type ChAT (pChAT), which has been used to delineate peripheral cholinergic structures in vertebrates, but not previously in invertebrates. Western blot analysis of octopus extracts revealed a single pChAT-positive band, suggesting that pChAT antiserum is cross-reactive with an octopus counterpart of rat pChAT. In immunohistochemistry, only neuronal structures of the octopus arm were stained by cChAT and pChAT antisera, although the pattern of distribution clearly differed between the two antisera. cChAT-positive varicose nerve fibers were observed in both the cerebrobrachial tract and neuropil of the axial nerve cord, while pChAT-positive varicose fibers were detected only in the neuropil of the axial nerve cord. After epitope retrieval, pChAT-positive neuronal cells and their processes became visible in all ganglia of the arm, including the axial and intramuscular nerve cords, and in ganglia of suckers. Moreover, pChAT-positive structures also became detectable in nerve fibers connecting the different ganglia, in smooth nerve fibers among muscle layers and dermal connective tissues, and in sensory cells of the suckers. These results suggest that the octopus arm has two types of cholinergic nerves: cChAT-positive nerves from brain ganglia and pChAT-positive nerves that are intrinsic to the arm.
Spt-Ada-Gcn5-Acetyltransferase (SAGA Complex in Plants: Genome Wide Identification, Evolutionary Conservation and Functional Determination.
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Rakesh Srivastava
Full Text Available The recruitment of RNA polymerase II on a promoter is assisted by the assembly of basal transcriptional machinery in eukaryotes. The Spt-Ada-Gcn5-Acetyltransferase (SAGA complex plays an important role in transcription regulation in eukaryotes. However, even in the advent of genome sequencing of various plants, SAGA complex has been poorly defined for their components and roles in plant development and physiological functions. Computational analysis of Arabidopsis thaliana and Oryza sativa genomes for SAGA complex resulted in the identification of 17 to 18 potential candidates for SAGA subunits. We have further classified the SAGA complex based on the conserved domains. Phylogenetic analysis revealed that the SAGA complex proteins are evolutionary conserved between plants, yeast and mammals. Functional annotation showed that they participate not only in chromatin remodeling and gene regulation, but also in different biological processes, which could be indirect and possibly mediated via the regulation of gene expression. The in silico expression analysis of the SAGA components in Arabidopsis and O. sativa clearly indicates that its components have a distinct expression profile at different developmental stages. The co-expression analysis of the SAGA components suggests that many of these subunits co-express at different developmental stages, during hormonal interaction and in response to stress conditions. Quantitative real-time PCR analysis of SAGA component genes further confirmed their expression in different plant tissues and stresses. The expression of representative salt, heat and light inducible genes were affected in mutant lines of SAGA subunits in Arabidopsis. Altogether, the present study reveals expedient evidences of involvement of the SAGA complex in plant gene regulation and stress responses.
daf-31 encodes the catalytic subunit of N alpha-acetyltransferase that regulates Caenorhabditis elegans development, metabolism and adult lifespan.
Chen, Di; Zhang, Jiuli; Minnerly, Justin; Kaul, Tiffany; Riddle, Donald L; Jia, Kailiang
The Caenorhabditis elegans dauer larva is a facultative state of diapause. Mutations affecting dauer signal transduction and morphogenesis have been reported. Of these, most that result in constitutive formation of dauer larvae are temperature-sensitive (ts). The daf-31 mutant was isolated in genetic screens looking for novel and underrepresented classes of mutants that form dauer and dauer-like larvae non-conditionally. Dauer-like larvae are arrested in development and have some, but not all, of the normal dauer characteristics. We show here that daf-31 mutants form dauer-like larvae under starvation conditions but are sensitive to SDS treatment. Moreover, metabolism is shifted to fat accumulation in daf-31 mutants. We cloned the daf-31 gene and it encodes an ortholog of the arrest-defective-1 protein (ARD1) that is the catalytic subunit of the major N alpha-acetyltransferase (NatA). A daf-31 promoter::GFP reporter gene indicates daf-31 is expressed in multiple tissues including neurons, pharynx, intestine and hypodermal cells. Interestingly, overexpression of daf-31 enhances the longevity phenotype of daf-2 mutants, which is dependent on the forkhead transcription factor (FOXO) DAF-16. We demonstrate that overexpression of daf-31 stimulates the transcriptional activity of DAF-16 without influencing its subcellular localization. These data reveal an essential role of NatA in controlling C. elegans life history and also a novel interaction between ARD1 and FOXO transcription factors, which may contribute to understanding the function of ARD1 in mammals.
In vitro characterization of the antivirulence target of Gram-positive pathogens, peptidoglycan O-acetyltransferase A (OatA.
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David Sychantha
Full Text Available The O-acetylation of the essential cell wall polymer peptidoglycan occurs in most Gram-positive bacterial pathogens, including species of Staphylococcus, Streptococcus and Enterococcus. This modification to peptidoglycan protects these pathogens from the lytic action of the lysozymes of innate immunity systems and, as such, is recognized as a virulence factor. The key enzyme involved, peptidoglycan O-acetyltransferase A (OatA represents a particular challenge to biochemical study since it is a membrane associated protein whose substrate is the insoluble peptidoglycan cell wall polymer. OatA is predicted to be bimodular, being comprised of an N-terminal integral membrane domain linked to a C-terminal extracytoplasmic domain. We present herein the first biochemical and kinetic characterization of the C-terminal catalytic domain of OatA from two important human pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Using both pseudosubstrates and novel biosynthetically-prepared peptidoglycan polymers, we characterized distinct substrate specificities for the two enzymes. In addition, the high resolution crystal structure of the C-terminal domain reveals an SGNH/GDSL-like hydrolase fold with a catalytic triad of amino acids but with a non-canonical oxyanion hole structure. Site-specific replacements confirmed the identity of the catalytic and oxyanion hole residues. A model is presented for the O-acetylation of peptidoglycan whereby the translocation of acetyl groups from a cytoplasmic source across the cytoplasmic membrane is catalyzed by the N-terminal domain of OatA for their transfer to peptidoglycan by its C-terminal domain. This study on the structure-function relationship of OatA provides a molecular and mechanistic understanding of this bacterial resistance mechanism opening the prospect for novel chemotherapeutic exploration to enhance innate immunity protection against Gram-positive pathogens.
Characterization of N-acetyltransferase 1 and 2 polymorphisms and haplotype analysis for inflammatory bowel disease and sporadic colorectal carcinoma
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Cobbs Gary A
Full Text Available Abstract Background N-acetyltransferase 1 (NAT1 and 2 (NAT2 are polymorphic isoenzymes responsible for the metabolism of numerous drugs and carcinogens. Acetylation catalyzed by NAT1 and NAT2 are important in metabolic activation of arylamines to electrophilic intermediates that initiate carcinogenesis. Inflammatory bowel diseases (IBD consist of Crohn's disease (CD and ulcerative colitis (UC, both are associated with increased colorectal cancer (CRC risk. We hypothesized that NAT1 and/or NAT2 polymorphisms contribute to the increased cancer evident in IBD. Methods A case control study was performed with 729 Caucasian participants, 123 CRC, 201 CD, 167 UC, 15 IBD dysplasia/cancer and 223 controls. NAT1 and NAT2 genotyping were performed using Taqman based techniques. Eight single nucleotide polymorphisms (SNPs were characterized for NAT1 and 7 SNPs for NAT2. Haplotype frequencies were estimated using an Expectation-Maximization (EM method. Disease groups were compared to a control group for the frequencies at each individual SNP separately. The same groups were compared for the frequencies of NAT1 and NAT2 haplotypes and deduced NAT2 phenotypes. Results No statistically significant differences were found for any comparison. Strong linkage disequilibrium was present among both the NAT1 SNPs and the NAT2 SNPs. Conclusion This study did not demonstrate an association between NAT1 and NAT2 polymorphisms and IBD or sporadic CRC, although power calculations indicate this study had sufficient sample size to detect differences in frequency as small as 0.05 to 0.15 depending on SNP or haplotype.
Middle-down hybrid chromatography/tandem mass spectrometry workflow for characterization of combinatorial post-translational modifications in histones
DEFF Research Database (Denmark)
Sidoli, Simone; Schwämmle, Veit; Ruminowicz, Chrystian
chromatography (WCX-HILIC) interfaced directly to high mass accuracy ESI MS/MS using electron transfer dissociation (ETD). This enabled automated and efficient separation and sequencing of hypermodified histone N-terminal tails for unambiguous localization of combinatorial PTMs. We present Histone Coder and Iso......Scale software to extract, filter and analyze MS/MS data, including quantification of co-fragmenting isobaric polypeptide species. We characterized histone tails derived from murine embryonic stem cells (ESCs) knockout in Suppressor of Zeste (Suz12(-/-) ) and quantified 256 combinatorial histone marks...... in histones H3, H4 and H2A. Furthermore, a total of 713 different combinatorial histone marks were identified in purified histone H3. We measured a 7-fold reduction of H3K27me2/me3 in Suz12(-/-) cells and detected significant changes of the relative abundance of 16 other single PTMs of histone H3 and other...
Loading of Arabidopsis centromeric histone CENH3 occurs mainly during G2 and requires the presence of the histone fold domain
Czech Academy of Sciences Publication Activity Database
Lermontova, L.; Schubert, V.; Fuchs, J.; Klatte, J.; Macas, Jiří; Schubert, I.
Roč. 18, - (2006), s. 2443-2451 ISSN 1040-4651 Institutional research plan: CEZ:AV0Z50510513 Keywords : histone CENH3 * Arabidopsis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 9.868, year: 2006
Epigenetic Histone Marks of Extended Meta-Polycentric Centromeres of Lathyrus and Pisum Chromosomes
Czech Academy of Sciences Publication Activity Database
Neumann, Pavel; Schubert, V.; Vrbová, Iva; Manning, Jasper Eugene; Houben, A.; Macas, Jiří
Roč. 7, č. 234 (2016) ISSN 1664-462X R&D Projects: GA ČR(CZ) GAP501/11/1843 Institutional support: RVO:60077344 Keywords : Centromere structure * epigenetic modifications * histone phosphorylation * histone methylation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.298, year: 2016
The putative oncogene GASC1 demethylates tri- and dimethylated lysine 9 on histone H3
DEFF Research Database (Denmark)
Cloos, Paul A C; Christensen, Jesper; Agger, Karl
Methylation of lysine and arginine residues on histone tails affects chromatin structure and gene transcription. Tri- and dimethylation of lysine 9 on histone H3 (H3K9me3/me2) is required for the binding of the repressive protein HP1 and is associated with heterochromatin formation...
Inhibition of histone deacetylases stimulates HBV replication independent of protein X
van de Klundert, Maarten A. A.; Swart, Marjolein; Zaaijer, Hans L.; Kootstra, Neeltje A.
Aim: HBV expresses an accessory protein called X (HBx), which supports HBV replication by increasing transcription from episomal templates. Here, we investigate whether HBx augments HBV replication by interfering with the deacetylation of HBV DNA associated histones by histone deacetylases (HDACs).
Absence of somatic histone H1 in oocytes and preblastula embryos of Xenopus laevis
Hock, R.; Moorman, A.; Fischer, D.; Scheer, U.
Available data on the occurrence and expression of somatic histone H1 during oogenesis and early embryogenesis of Xenopus laevis are contradictory. In particular the reported presence of a large storage pool of histone H1A in oocytes is difficult to reconcile with the high transcriptional activity
Evaluation of proteomic search engines for the analysis of histone modifications.
Yuan, Zuo-Fei; Lin, Shu; Molden, Rosalynn C; Garcia, Benjamin A
Identification of histone post-translational modifications (PTMs) is challenging for proteomics search engines. Including many histone PTMs in one search increases the number of candidate peptides dramatically, leading to low search speed and fewer identified spectra. To evaluate database search engines on identifying histone PTMs, we present a method in which one kind of modification is searched each time, for example, unmodified, individually modified, and multimodified, each search result is filtered with false discovery rate less than 1%, and the identifications of multiple search engines are combined to obtain confident results. We apply this method for eight search engines on histone data sets. We find that two search engines, pFind and Mascot, identify most of the confident results at a reasonable speed, so we recommend using them to identify histone modifications. During the evaluation, we also find some important aspects for the analysis of histone modifications. Our evaluation of different search engines on identifying histone modifications will hopefully help those who are hoping to enter the histone proteomics field. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD001118.
Evaluation of Proteomic Search Engines for the Analysis of Histone Modifications
Identification of histone post-translational modifications (PTMs) is challenging for proteomics search engines. Including many histone PTMs in one search increases the number of candidate peptides dramatically, leading to low search speed and fewer identified spectra. To evaluate database search engines on identifying histone PTMs, we present a method in which one kind of modification is searched each time, for example, unmodified, individually modified, and multimodified, each search result is filtered with false discovery rate less than 1%, and the identifications of multiple search engines are combined to obtain confident results. We apply this method for eight search engines on histone data sets. We find that two search engines, pFind and Mascot, identify most of the confident results at a reasonable speed, so we recommend using them to identify histone modifications. During the evaluation, we also find some important aspects for the analysis of histone modifications. Our evaluation of different search engines on identifying histone modifications will hopefully help those who are hoping to enter the histone proteomics field. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD001118. PMID:25167464
ERK/MAPK Regulates Hippocampal Histone Phosphorylation Following Contextual Fear Conditioning
Levenson, Jonathan M.; Sweatt, J. David; Chwang, Wilson B.; O'Riordan, Kenneth J.
Long-term memory formation is regulated by many distinct molecular mechanisms that control gene expression. An emerging model for effecting a stable, coordinated pattern of gene transcription involves epigenetic tagging through modifications of histones or DNA. In this study, we investigated the regulation of histone phosphorylation in the…
Impact of vaccine therapy using nuclear histone H1 on allograft survival in experimental organ transplantation.
Nakano, Toshiaki; Goto, Shigeru; Lai, Chia-Yun; Hsu, Li-Wen; Ono, Kazuhisa; Kawamoto, Seiji; Lin, Yu-Chun; Kao, Ying-Hsien; Chiang, Kuei-Chen; Ohmori, Naoya; Goto, Takeshi; Sato, Shuji; Tu, Chieh-Hsien; Jawan, Bruno; Cheng, Yu-Fan; Chen, Chao-Long
We recently reported that autoreactive antibody (Ab) against nuclear histone H1 had been identified as an immunosuppressive factor in a rat tolerogenic orthotopic liver transplantation (OLT) model. The present study aimed to determine whether the up-regulation of antihistone H1 Ab by histone H1 vaccination leads to tolerance. Histone H1-immunized rats were established by intraperitoneal vaccination with histone H1 at every two-weekly interval. By using mixed lymphocyte reaction (MLR) and heterotopic heart transplantation (HHT), the alloreactive T cell response and allograft survival of histone H1-immunized rats were compared with those of control rats. Cytokine and cellular profiles in histone H1-immunized rats were determined by reverse transcriptase polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Immunization with histone H1 in Freund's adjuvant induced alloreactive T cell unresponsiveness and prolonged heterotopic heart allograft survival. It also down-regulated the expression of major histocompatibility complex (MHC) class II and CD25 on splenic cells, elevated the T helper cell type 2 (Th2) skewing index (Interleukin (IL)-4/interferon (IFN)-gamma ratio or IL-4/IL-2 ratio) and modified the serum cytokine profiles. The present results suggest that histone H1 vaccination of transplant recipients, which leads to the production of immunosuppressive factor and the modification of the cytokine/cellular profiles, has great potential as a tolerance therapy for prospective transplantation.
Dengue virus capsid protein binds core histones and inhibits nucleosome formation in human liver cells.
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Tonya M Colpitts
Full Text Available Dengue virus (DENV is a member of the Flaviviridae and a globally (reemerging pathogen that causes serious human disease. There is no specific antiviral or vaccine for dengue virus infection. Flavivirus capsid (C is a structural protein responsible for gathering the viral RNA into a nucleocapsid that forms the core of a mature virus particle. Flaviviral replication is known to occur in the cytoplasm yet a large portion of capsid protein localizes to the nucleus during infection. The reasons for the nuclear presences of capsid are not completely understood. Here, we expressed mature DENV C in a tandem affinity purification assay to identify potential binding partners in human liver cells. DENV C targeted the four core histones, H2A, H2B, H3 and H4. DENV C bound recombinant histones in solution and colocalized with histones in the nucleus and cytoplasm of liver cells during DENV infection. We show that DENV C acts as a histone mimic, forming heterodimers with core histones, binding DNA and disrupting nucleosome formation. We also demonstrate that DENV infection increases the amounts of core histones in livers cells, which may be a cellular response to C binding away the histone proteins. Infection with DENV additionally alters levels of H2A phosphorylation in a time-dependent manner. The interactions of C and histones add an interesting new role for the presence of C in the nucleus during DENV infection.
Dengue virus capsid protein binds core histones and inhibits nucleosome formation in human liver cells.
Colpitts, Tonya M; Barthel, Sebastian; Wang, Penghua; Fikrig, Erol
Dengue virus (DENV) is a member of the Flaviviridae and a globally (re)emerging pathogen that causes serious human disease. There is no specific antiviral or vaccine for dengue virus infection. Flavivirus capsid (C) is a structural protein responsible for gathering the viral RNA into a nucleocapsid that forms the core of a mature virus particle. Flaviviral replication is known to occur in the cytoplasm yet a large portion of capsid protein localizes to the nucleus during infection. The reasons for the nuclear presences of capsid are not completely understood. Here, we expressed mature DENV C in a tandem affinity purification assay to identify potential binding partners in human liver cells. DENV C targeted the four core histones, H2A, H2B, H3 and H4. DENV C bound recombinant histones in solution and colocalized with histones in the nucleus and cytoplasm of liver cells during DENV infection. We show that DENV C acts as a histone mimic, forming heterodimers with core histones, binding DNA and disrupting nucleosome formation. We also demonstrate that DENV infection increases the amounts of core histones in livers cells, which may be a cellular response to C binding away the histone proteins. Infection with DENV additionally alters levels of H2A phosphorylation in a time-dependent manner. The interactions of C and histones add an interesting new role for the presence of C in the nucleus during DENV infection.
Development of a new rapid HPLC method for the fractionation of histones
International Nuclear Information System (INIS)
Gurley, L.R.; Valdez, J.G.; Prentice, D.A.; Spall, W.D.
To study histone functions, it is necessary to fractionate the histones into their five classes (H1, H2A, H2B, H3 and H4) and then to subfractionate these classes into variants having slightly different primary structures and into different phosphorylated and acetylated forms. With the advent of high-performance liquid chromatography (HPLC), it was hoped that laborious and time-consuming conventional methods could be replaced by a simple, rapid, high-resolving HPLC method for fractionating histones. However, problems of irreversible adsorption of the histones to HPLC column packings discouraged this development. Our laboratory has now determined that the strong adsorption of histones to HPLC columns results from two different forces: (1) polar interactions between the histones and the silanol groups of silica-based HPLC column packing, and (2) hydrophobic interactions between the histones and the bound organic phase of the column packings. By minimizing these forces, we have succeeded in developing an HPLC method suitable for histone studies
Effect of gamma irradiation on rat thymus arginine-rich H3 histone in vitro
International Nuclear Information System (INIS)
Patil, M.S.; Narasimhan, Saroja; Sreenivasan, A.
Physicochemical properties of rat thymus H3 histone have been studied following gamma radiation (25-90 krad) in 0.2 N HCl. Polyacrylamide gel electrophoretic pattern (PGE) of H3 histone indicated that aggregates were formed in the histone fraction following gamma irradiation. The PGE pattern of the irradiated-histone fraction remained unaltered even after it was treated with 8.0 M urea to eliminate noncovalent bonding. On the other hand, the irradiated sample treated with β-mercaptoethanol exhibited the PGE pattern which was essentially similar to that of unirradiated sample. These results indicate that the aggregates seen in the PGE pattern of irradiated-H3 histone may be formed through interpolypeptide chain disulphide linkeges rather than by noncovalent bonding. This contention is also supported by the fact that irradiated-H3 histone exhibited hyperchromic shift at 240-250 nm region as well as increased disulphide content. Other results revealed that DNA-dependent RNA synthesis in vitro was inhibited to a greater extent by irradiated-H3 histone than by unirradiated-H3 histone. (author)
Mechanical Stability and Fibrinolytic Resistance of Clots Containing Fibrin, DNA, and Histones*
Longstaff, Colin; Varjú, Imre; Sótonyi, Péter; Szabó, László; Krumrey, Michael; Hoell, Armin; Bóta, Attila; Varga, Zoltán; Komorowicz, Erzsébet; Kolev, Krasimir
Neutrophil extracellular traps are networks of DNA and associated proteins produced by nucleosome release from activated neutrophils in response to infection stimuli and have recently been identified as key mediators between innate immunity, inflammation, and hemostasis. The interaction of DNA and histones with a number of hemostatic factors has been shown to promote clotting and is associated with increased thrombosis, but little is known about the effects of DNA and histones on the regulation of fibrin stability and fibrinolysis. Here we demonstrate that the addition of histone-DNA complexes to fibrin results in thicker fibers (increase in median diameter from 84 to 123 nm according to scanning electron microscopy data) accompanied by improved stability and rigidity (the critical shear stress causing loss of fibrin viscosity increases from 150 to 376 Pa whereas the storage modulus of the gel increases from 62 to 82 pascals according to oscillation rheometric data). The effects of DNA and histones alone are subtle and suggest that histones affect clot structure whereas DNA changes the way clots are lysed. The combination of histones + DNA significantly prolongs clot lysis. Isothermal titration and confocal microscopy studies suggest that histones and DNA bind large fibrin degradation products with 191 and 136 nm dissociation constants, respectively, interactions that inhibit clot lysis. Heparin, which is known to interfere with the formation of neutrophil extracellular traps, appears to prolong lysis time at a concentration favoring ternary histone-DNA-heparin complex formation, and DNase effectively promotes clot lysis in combination with tissue plasminogen activator. PMID:23293023
The histone deacetylase inhibitor Trichostatin A modulates CD4+ T cell responses
DEFF Research Database (Denmark)
Moreira, José Manuel Alfonso; Scheipers, Peter; Sørensen, Poul
Histone deacetylase inhibitors (HDACIs) induce hyperacetylation of core histones modulating chromatin structure and affecting gene expression. These compounds are also able to induce growth arrest, cell differentiation, and apoptotic cell death of tumor cells in vitro as well as in vivo. Even...
Progressive methylation of ageing histones by Dot1 functions as a timer
De Vos, Dirk; Frederiks, Floor; Terweij, Marit; van Welsem, Tibor; Verzijlbergen, Kitty F.; Iachina, Ekaterina; de Graaf, Erik L.; Altelaar, A. F. Maarten; Oudgenoeg, Gideon; Heck, Albert J. R.; Krijgsveldz, Jeroen; Bakker, Barbara M.; van Leeuwen, Fred
Post-translational modifications of histone proteins have a crucial role in regulating gene expression. If efficiently re-established after chromosome duplication, histone modifications could help propagate gene expression patterns in dividing cells by epigenetic mechanisms. We used an integrated
The histone methyltransferase SET8 is required for S-phase progression
DEFF Research Database (Denmark)
Jørgensen, Stine; Elvers, Ingegerd; Trelle, Morten Beck
Chromatin structure and function is influenced by histone posttranslational modifications. SET8 (also known as PR-Set7 and SETD8) is a histone methyltransferase that monomethylates histonfe H4-K20. However, a function for SET8 in mammalian cell proliferation has not been determined. We show...
Deposition of nucleosomal antigens (histones and DNA) in the epidermal basement membrane in human lupus nephritis.
Grootscholten, C.; Bruggen, M.C.J. van; Pijl, J.W. van der; Jong, E.M.G.J. de; Ligtenberg, G.; Derksen, R.H.W.M.; Berden, J.H.M.
OBJECTIVE: Antinuclear autoantibodies complexed to nucleosomes can bind to heparan sulfate (HS) in the glomerular basement membrane. This binding is due to the binding of the positively charged histones to the strongly anionic HS. Nucleosomes and histones have been identified in glomerular deposits
Dengue Virus Capsid Protein Binds Core Histones and Inhibits Nucleosome Formation in Human Liver Cells
Colpitts, Tonya M.; Barthel, Sebastian; Wang, Penghua; Fikrig, Erol
Dengue virus (DENV) is a member of the Flaviviridae and a globally (re)emerging pathogen that causes serious human disease. There is no specific antiviral or vaccine for dengue virus infection. Flavivirus capsid (C) is a structural protein responsible for gathering the viral RNA into a nucleocapsid that forms the core of a mature virus particle. Flaviviral replication is known to occur in the cytoplasm yet a large portion of capsid protein localizes to the nucleus during infection. The reasons for the nuclear presences of capsid are not completely understood. Here, we expressed mature DENV C in a tandem affinity purification assay to identify potential binding partners in human liver cells. DENV C targeted the four core histones, H2A, H2B, H3 and H4. DENV C bound recombinant histones in solution and colocalized with histones in the nucleus and cytoplasm of liver cells during DENV infection. We show that DENV C acts as a histone mimic, forming heterodimers with core histones, binding DNA and disrupting nucleosome formation. We also demonstrate that DENV infection increases the amounts of core histones in livers cells, which may be a cellular response to C binding away the histone proteins. Infection with DENV additionally alters levels of H2A phosphorylation in a time-dependent manner. The interactions of C and histones add an interesting new role for the presence of C in the nucleus during DENV infection. PMID:21909430
Presence of Cytotoxic Extracellular Histones in Machine Perfusate of Donation After Circulatory Death Kidneys.
van Smaalen, Tim C; Beurskens, Daniëlle M H; Hoogland, E R Pieter; Winkens, Bjorn; Christiaans, Maarten H L; Reutelingsperger, Chris P; van Heurn, L W Ernest; Nicolaes, Gerry A F
Extracellular histones are cytotoxic molecules that are related to cell stress and death. They have been shown to play a crucial role in multiple pathophysiologic processes like sepsis, inflammation, vascular dysfunction, and thrombosis. Their role in organ donation and graft function and survival is still unknown. The aim of this study was to assess whether an association exists between the presence of extracellular histones in machine perfusates and deceased donor kidney viability. Machine perfusates of 390 donations after circulatory death kidneys were analyzed for histone concentration, and corresponding graft function and survival were assessed. Extracellular histone concentrations were significantly higher in perfusates of kidneys with posttransplant graft dysfunction (primary nonfunction and delayed graft function) and were an independent risk factor for delayed graft function (odds ratio, 2.152; 95% confidence interval [95% CI], 1.199-3.863) and 1 year graft failure (hazard ratio, 1.386; 95% CI, 1.037-1.853), but not for primary nonfunction (odds ratio, 1.342; 95% CI, 0.900-2.002). One year graft survival was 12% higher in the group with low histone concentrations (P = 0.008) as compared with the group that contained higher histone concentrations. This study warrants future studies to probe for a possible role of cytotoxic extracellular histones in organ viability and suggests that quantitation of extracellular histones might contribute to assessment of posttransplant graft function and survival.
Acetylation-Mediated Proteasomal Degradation of Core Histones during DNA Repair and Spermatogenesis
Qian, Min-Xian; Pang, Ye; Liu, Cui Hua; Haratake, Kousuke; Du, Bo-Yu; Ji, Dan-Yang; Wang, Guang-Fei; Zhu, Qian-Qian; Song, Wei; Yu, Yadong; Zhang, Xiao-Xu; Huang, Hai-Tao; Miao, Shiying; Chen, Lian-Bin; Zhang, Zi-Hui; Liang, Ya-Nan; Liu, Shan; Cha, Hwangho; Yang, Dong; Zhai, Yonggong; Komatsu, Takuo; Tsuruta, Fuminori; Li, Haitao; Cao, Cheng; Li, Wei; Li, Guo-Hong; Cheng, Yifan; Chiba, Tomoki; Wang, Linfang; Goldberg, Alfred L.; Shen, Yan; Qiu, Xiao-Bo
SUMMARY Histone acetylation plays critical roles in chromatin remodeling, DNA repair, and epigenetic regulation of gene expression, but the underlying mechanisms are unclear. Proteasomes usually catalyze ATP- and polyubiquitin-dependent proteolysis. Here we show that the proteasomes containing the activator PA200 catalyze the polyubiquitin-independent degradation of histones. Most proteasomes in mammalian testes ("spermatoproteasomes") contain a spermatid/sperm-specific α-subunit α4s/PSMA8 and/or the catalytic β-subunits of immunoproteasomes in addition to PA200. Deletion of PA200 in mice abolishes acetylation-dependent degradation of somatic core histones during DNA double-strand breaks, and delays core histone disappearance in elongated spermatids. Purified PA200 greatly promotes ATP-independent proteasomal degradation of the acetylated core histones, but not polyubiquitinated proteins. Furthermore, acetylation on histones is required for their binding to the bromodomain-like regions in PA200 and its yeast ortholog, Blm10. Thus, PA200/Blm10 specifically targets the core histones for acetylation-mediated degradation by proteasomes, providing mechanisms by which acetylation regulates histone degradation, DNA repair, and spermatogenesis. PMID:23706739
Nucleosomes and histones are present in glomerular deposits in human lupus nephritis
vanBruggen, MCJ; Kramers, C; Walgreen, B; Elema, JD; Kallenberg, CGM; vandenBorn, J; Smeenk, RJT; Assmann, KJM; Muller, S; Monestier, M; Berden, JHM
Background. Recently we showed that antinuclear autoantibodies complexed to nucleosomes can bind to heparan sulphate (HS) in the glomerular basement membrane (GEM) via the histone part of the nucleosome. Histones have been identified in glomerular deposits in human and murine lupus nephritis. In
Automated setup for characterization of intact histone tails in Suz12-/- stem cells
DEFF Research Database (Denmark)
Sidoli, Simone; Schwämmle, Veit; Hansen, Thomas Aarup
Epigenetics is defined as the study of heritable changes that occur without modifying the DNA sequence. Histone proteins are crucial components of epigenetic mechanisms and regulation, since they are fundamental for chromatin structure. Mass spectrometry-based proteomics is already an integrated...... developed a high-resolving and automated LC-MS/MS setup to characterize intact histone tails (middle-down strategy)...
Characterization of Complete Histone Tail Proteoforms Using Differential Ion Mobility Spectrometry
DEFF Research Database (Denmark)
Shliaha, Pavel V; Baird, Matthew A; Nielsen, Mogens M
Histone proteins are subject to dynamic post-translational modifications (PTMs) that cooperatively modulate the chromatin structure and function. Nearly all functional PTMs are found on the N-terminal histone domains (tails) of ∼50 residues protruding from the nucleosome core. Using high...
Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity
DEFF Research Database (Denmark)
Pasini, Diego; Bracken, Adrian P; Jensen, Michael R
SUZ12 is a recently identified Polycomb group (PcG) protein, which together with EZH2 and EED forms different Polycomb repressive complexes (PRC2/3). These complexes contain histone H3 lysine (K) 27/9 and histone H1 K26 methyltransferase activity specified by the EZH2 SET domain. Here we show...
Extranuclear detection of histones and nucleosomes in activated human lymphoblasts as an early event in apoptosis.
Gabler, C.; Blank, N.; Hieronymus, T.; Schiller, M.; Berden, J.H.M.; Kalden, J.R.; Lorenz, H.M.
OBJECTIVE: To evaluate the presence of histones and nucleosomes in cell lysates of freshly isolated peripheral blood mononuclear cells (PBMC), fully activated lymphoblasts, or lymphoblasts after induction of apoptosis. METHODS: Each histone class (H1, H2A, H2B, H3, and H4) was detected by western
Role of peroxynitrite induced structural changes on H2B histone by physicochemical method.
Khan, M Asad; Alam, Khursheed; Dixit, Kiran; Rizvi, M Moshahid A
Histones are small highly conserved cationic proteins which bind DNA and remain confined in the nucleus. These histones are quite vulnerable to oxidizing and nitrating agents. Peroxynitrite is a powerful oxidant and nitrating agent present in the biological system. In this study, peroxynitrite-induced nitration and oxidation of H2B was assessed by various physicochemical techniques. The carbonyl content and dityrosine were markedly elevated in peroxynitrite-modified H2B histone as compared to the native histone. Cross-linking of H2B was evident on polyacrylamide gel electrophoresis. 3-Nitrotyrosine was present only in peroxynitrite-modified H2B revealed by HPLC. The results showed that peroxynitrite-mediated nitration and oxidation in H2B histone exhibited hyperchromicity, decrease of tyrosine fluorescence accompanied by increase in ANS-binding specific fluorescence, loss of β-sheet structure, appearance of new peak in FT-IR, increase in melting temperature and also loss of α-helix to produce a partially folded structure in comparison to intrinsically disordered structure of native H2B histone. We concluded that the H2B histone, a constituent of core histones, is highly sensitive to peroxynitrite and can adopt different structures under nitrosative and oxidative stress in order to protect the packaged DNA from the deleterious insult of peroxynitrite. Copyright © 2015 Elsevier B.V. All rights reserved.
Amino acid composition of sperm histones in the house cricket Acheta domesticus.
Pallotta, D; Tessier, A
Histones were isolated from late spermatids and spermatozoa of the house cricket Acheta domesticus, and the individual histone fractions were separated by electrophoresis on polyacrylamide-urea gels. The stained gels were cut so as to isolate the different histone fractions, and the amino acid compositions were determined using the technique of Houston (Houston, L.L.: Anal. Biochem. 44, 81-88 (1971). Five of the histones had amino acid compositions resembling those for the histones of calf thymus and were thus identified as fractions F1, F3, F2a2, F2b, and F2al. Another protein (SH) located exclusively in the late spermatids and spermatozoa was found to be basic and histone-like. It is a protein containing relatively high amounts of arginine (12.6%) and low amounts of lysine (7.6%), and, as a result, it has a low ratio of lysine-arginine (0.6). Other noteworthy features are its high contents of serine, glutamic acid, and glycine. It is arginine rich histone and in this regard resembles other such proteins, but it does contain unique features which distinguish it from all previously described histones.
Paraquat Induces Epigenetic Changes by Promoting Histone Acetylation in Cell Culture Models of Dopaminergic Degeneration
Song, C.; Kanthasamy, A.; Jin, H.; Anantharam, V.; Kanthasamy, A. G.
Environmental neurotoxic exposure to agrochemicals has been implicated in the etiopathogenesis of Parkinson's disease (PD). The widely used herbicide paraquat is among the few environmental chemicals potentially linked with PD. Since epigenetic changes are beginning to emerge as key mechanisms in neurodegenerative diseases, herein we examined the effects of paraquat on histone acetylation, a major epigenetic change in chromatin that can regulate gene expression, chromatin remodeling, cell survival and cell death. Exposure of N27 dopaminergic cells to paraquat induced histone H3 acetylation in a time-dependent manner. However, paraquat did not alter acetylation of another core histone H4. Paraquat-induced histone acetylation was associated with decreased total histone deacetylase (HDAC) activity and HDAC4 and 7 protein expression levels. To determine if histone acetylation plays a role in paraquat-induced apoptosis, the novel HAT inhibitor anacardic acid was used. Anacardic acid treatment significantly attenuated paraquat-induced caspase-3 enzyme activity, suppressed proteolytic activation and kinase activity of protein kinase C delta (PKCδ) and also blocked paraquat-induced cytotoxicity. Together, these results demonstrate that the neurotoxic agent paraquat induced acetylation of core histones in cell culture models of PD and that inhibition of HAT activity by anacardic acid protects against apoptotic cell death, indicating that histone acetylation may represent key epigenetic changes in dopaminergic neuronal cells during neurotoxic insults. PMID:21777615
UHRF1 Links the Histone Code and DNA Methylation to Ensure Faithful Epigenetic Memory Inheritance
Directory of Open Access Journals (Sweden)
Christian Bronner
Full Text Available Epigenetics is the study of the transmission of cell memory through mitosis or meiosis that is not based on the DNA sequence. At the molecular level the epigenetic memory of a cell is embedded in DNA methylation, histone post-translational modifications, RNA interference and histone isoform variation. There is a tight link between histone post-translational modifications (the histone code and DNA methylation, as modifications of histones contribute to the establishment of DNA methylation patterns and vice versa. Interestingly, proteins have recently been identified that can simultaneously read both methylated DNA and the histone code. UHRF1 fulfills these requirements by having unique structural domains that allow concurrent recognition of histone modifications and methylated DNA. Herein, we review our current knowledge of UHRF1 and discuss how this protein ensures the link between histone marks and DNA methylation. Understanding the molecular functions of this protein may reveal the physiological relevance of the linkage between these layers of epigenetic marks.
Integrative analysis of histone ChIP-seq and transcription data using Bayesian mixture models
DEFF Research Database (Denmark)
Klein, Hans-Ulrich; Schäfer, Martin; Porse, Bo T
Histone modifications are a key epigenetic mechanism to activate or repress the transcription of genes. Datasets of matched transcription data and histone modification data obtained by ChIP-seq exist, but methods for integrative analysis of both data types are still rare. Here, we present a novel...
Precision mapping of coexisting modifications in histone H3 tails from embryonic stem cells by ETD-MS/MS
DEFF Research Database (Denmark)
Jung, Hye Ryung; Sidoli, Simone; Haldbo, Simon
Post-translational modifications (PTMs) of histones play a major role in regulating chromatin dynamics and influence processes such as transcription and DNA replication. Here, we report 114 distinct combinations of coexisting PTMs of histone H3 obtained from mouse embryonic stem (ES) cells. Histone......, and K36, suggesting interdependence between histone methylation marks. We hypothesize that the most abundant coexisting PTMs may provide a signature for the permissive state of mouse ES cells....
Pulmonary endothelial activation caused by extracellular histones contributes to neutrophil activation in acute respiratory distress syndrome.
Zhang, Yanlin; Guan, Li; Yu, Jie; Zhao, Zanmei; Mao, Lijun; Li, Shuqiang; Zhao, Jinyuan
During the acute respiratory distress syndrome (ARDS), neutrophils play a central role in the pathogenesis, and their activation requires interaction with the endothelium. Extracellular histones have been recognized as pivotal inflammatory mediators. This study was to investigate the role of pulmonary endothelial activation during the extracellular histone-induced inflammatory response in ARDS. ARDS was induced in male C57BL/6 mice by intravenous injection with lipopolysaccharide (LPS) or exogenous histones. Concurrent with LPS administration, anti-histone H4 antibody (anti-H4) or non-specific IgG was administered to study the role of extracellular histones. The circulating von Willebrand factor (vWF) and soluble thrombomodulin (sTM) were measured with ELISA kits at the preset time points. Myeloperoxidase (MPO) activity in lung tissue was measured with a MPO detection kit. The translocation of P-selectin and neutrophil infiltration were measured by immunohistochemical detection. For in vitro studies, histone H4 in the supernatant of mouse lung vascular endothelial cells (MLVECs) was measured by Western blot. The binding of extracellular histones with endothelial membrane was examined by confocal laser microscopy. Endothelial P-selectin translocation was measured by cell surface ELISA. Adhesion of neutrophils to MLVECs was assessed with a color video digital camera. The results showed that during LPS-induced ARDS extracellular histones caused endothelial and neutrophil activation, as seen by P-selectin translocation, release of vWF, an increase of circulating sTM, lung neutrophil infiltration and increased MPO activity. Extracellular histones directly bound and activated MLVECs in a dose-dependent manner. On the contrary, the direct stimulatory effect of exogenous histones on neutrophils was very limited, as measured by neutrophil adhesion and MPO activity. With the contribution of activated endothelium, extracellular histones could effectively activating
Conversion of deoxynivalenol to 3-acetyldeoxynivalenol in barley-derived fuel ethanol co-products with yeast expressing trichothecene 3-O-acetyltransferases
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Brooks Wynse S
Full Text Available Abstract Background The trichothecene mycotoxin deoxynivalenol (DON may be concentrated in distillers dried grains with solubles (DDGS; a co-product of fuel ethanol fermentation when grain containing DON is used to produce fuel ethanol. Even low levels of DON (≤ 5 ppm in DDGS sold as feed pose a significant threat to the health of monogastric animals. New and improved strategies to reduce DON in DDGS need to be developed and implemented to address this problem. Enzymes known as trichothecene 3-O-acetyltransferases convert DON to 3-acetyldeoxynivalenol (3ADON, and may reduce its toxicity in plants and animals. Results Two Fusarium trichothecene 3-O-acetyltransferases (FgTRI101 and FfTRI201 were cloned and expressed in yeast (Saccharomyces cerevisiae during a series of small-scale ethanol fermentations using barley (Hordeum vulgare. DON was concentrated 1.6 to 8.2 times in DDGS compared with the starting ground grain. During the fermentation process, FgTRI101 converted 9.2% to 55.3% of the DON to 3ADON, resulting in DDGS with reductions in DON and increases in 3ADON in the Virginia winter barley cultivars Eve, Thoroughbred and Price, and the experimental line VA06H-25. Analysis of barley mashes prepared from the barley line VA04B-125 showed that yeast expressing FfTRI201 were more effective at acetylating DON than those expressing FgTRI101; DON conversion for FfTRI201 ranged from 26.1% to 28.3%, whereas DON conversion for FgTRI101 ranged from 18.3% to 21.8% in VA04B-125 mashes. Ethanol yields were highest with the industrial yeast strain Ethanol Red®, which also consumed galactose when present in the mash. Conclusions This study demonstrates the potential of using yeast expressing a trichothecene 3-O-acetyltransferase to modify DON during commercial fuel ethanol fermentation.
Characterisation of the histone methyltransferase SET8 in cell cycle progression and the DNA damage response
DEFF Research Database (Denmark)
Jørgensen, Stine
Histone modifications and their catalysing enzymes have within the last few years proven to be essential players in many biological processes. Due to their ability to modulate chromatin structure and affect signalling pathways they are found to affect diverse processes such as transcription, DNA...... cellular homeostasis, has a role in supporting the chromatin structure to facilitate DNA replication. However, when cells are challenged by DNA damage efficient repair may be dependent on rapid degradation of SET8 and a reduction of the monomethyl mark on histone H4 lysine 20....... recombination and repair. I therefore initiated a mass spectrometry based study to identify changes in histone modifications after DNA damage. By using SILAC labelling of cells to quantatively measure the changes in histone modifications, we observed a marked reduction in the level of monomethylated Histone H4...
Histone Modifications, Modifiers and Readers in Melanoma Resistance to Targeted and Immune Therapy
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Stuart J Gallagher
Full Text Available The treatment of melanoma has been revolutionized by new therapies targeting MAPK signaling or the immune system. Unfortunately these therapies are hindered by either primary resistance or the development of acquired resistance. Resistance mechanisms involving somatic mutations in genes associated with resistance have been identified in some cases of melanoma, however, the cause of resistance remains largely unexplained in other cases. The importance of epigenetic factors targeting histones and histone modifiers in driving the behavior of melanoma is only starting to be unraveled and provides significant opportunity to combat the problems of therapy resistance. There is also an increasing ability to target these epigenetic changes with new drugs that inhibit these modifications to either prevent or overcome resistance to both MAPK inhibitors and immunotherapy. This review focuses on changes in histones, histone reader proteins and histone positioning, which can mediate resistance to new therapeutics and that can be targeted for future therapies.
Prepatterning of developmental gene expression by modified histones before zygotic genome activation
DEFF Research Database (Denmark)
Lindeman, Leif C.; Andersen, Ingrid S.; Reiner, Andrew H.
A hallmark of anamniote vertebrate development is a window of embryonic transcription-independent cell divisions before onset of zygotic genome activation (ZGA). Chromatin determinants of ZGA are unexplored; however, marking of developmental genes by modified histones in sperm suggests a predictive...... role of histone marks for ZGA. In zebrafish, pre-ZGA development for ten cell cycles provides an opportunity to examine whether genomic enrichment in modified histones is present before initiation of transcription. By profiling histone H3 trimethylation on all zebrafish promoters before and after ZGA......, we demonstrate here an epigenetic prepatterning of developmental gene expression. This involves pre-ZGA marking of transcriptionally inactive genes involved in homeostatic and developmental regulation by permissive H3K4me3 with or without repressive H3K9me3 or H3K27me3. Our data suggest that histone...
Radicals derived from histone hydroperoxides damage nucleobases in RNA and DNA
DEFF Research Database (Denmark)
Luxford, C; Dean, R T; Davies, Michael Jonathan
Exposure of individual histone proteins (H1, H2A, H2B, H3, or H4) and histone octamers (consisting of two molecules each of H2A, H2B, H3, and H4) to hydroxyl radicals, generated by gamma-irradiation, in the presence of O(2) generates protein-bound hydroperoxides in a dose-dependent fashion......; this is in accord with previous studies with other proteins. These histone hydroperoxides are stable in the absence of exogenous catalysts (e.g., heat, light, and transition metal ions), but in the presence of these agents decompose rapidly to give a variety of radicals which have been identified by EPR spin...... trapping. Histone hydroperoxide-derived radicals generated on decomposition of the hydroperoxides with Cu(+) react with both pyrimidine and purine nucleobases. Thus, with uridine the histone hydroperoxide-derived radicals undergo addition across the C(5)-C(6) double bond of the pyrimidine ring to give...
Inhibition of mitotic-specific histone phophorylation by sodium arsenite
Energy Technology Data Exchange (ETDEWEB)
Cobo, J.M. [Universidad de Alcala de Henares, Madrid (Spain); Valdez, J.G.; Gurley, L.R. [Los Alamos National Lab., NM (United States)
Synchronized cultures of Chinese hamster cells (line CHO) were used to measure the effects of 10{mu}M sodium arsenite on histone phosphorylation. This treatment caused cell proliferation to be temporarily arrested, after which the cells spontaneously resumed cell proliferation in a radiomimetric manner. Immediately following treatment, it was found that sodium arsenite affected only mitotic-specific HI and H3 phosphorylations. Neither interphase, nor mitotic, H2A and H4 phosphorylations were affected, nor was interphase HI Phosphorylation affected. The phosphorylation of HI was inhibited only in mitosis, reducing HI phosphorylation to 38.1% of control levels, which was the level of interphase HI phosphorylation. The phosphorylation of both H3 variants was inhibited in mitosis, the less hydrophobic H3 to 19% and the more hydrophobic H3 to 24% of control levels. These results suggest that sodium arsenite may inhibite cell proliferation by interfering with the cyclin B/p34{sup cdc2} histone kinase activity which is thought to play a key role in regulating the cell cycle. It has been proposed by our laboratory that HI and H3 phosphorylations play a role in restructuring interphase chromatin into metaphase chromosomes. Interference of this process by sodium arsenite may lead to structurally damaged chromosomes resulting in the increased cancer risks known to be produced by arsenic exposure from the environment.
Reactivation of latent HIV by histone deacetylase inhibitors.
Shirakawa, Kotaro; Chavez, Leonard; Hakre, Shweta; Calvanese, Vincenzo; Verdin, Eric
Latent HIV persists in CD4(+) T cells in infected patients under antiretroviral therapy (ART). Latency is associated with transcriptional silencing of the integrated provirus and driven, at least in part, by histone deacetylases (HDACs), a family of chromatin-associated proteins that regulate histone acetylation and the accessibility of DNA to transcription factors. Remarkably, inhibition of HDACs is sufficient to reactivate a fraction of latent HIV in a variety of experimental systems. This basic observation led to the shock and kill idea that forcing the transcriptional activation of HIV might lead to virus expression, to virus- or host-induced cell death of the reactivated cells, and to the eradication of the pool of latently infected cells. Such intervention might possibly lead to a cure for HIV-infected patients. Here, we review the basic biology of HDACs and their inhibitors, the role of HDACs in HIV latency, and recent efforts to use HDAC inhibitors to reactivate latent HIV in vitro and in vivo. Copyright © 2013 Elsevier Ltd. All rights reserved.
Effects of sequence on DNA wrapping around histones
Ortiz, Vanessa
A central question in biophysics is whether the sequence of a DNA strand affects its mechanical properties. In epigenetics, these are thought to influence nucleosome positioning and gene expression. Theoretical and experimental attempts to answer this question have been hindered by an inability to directly resolve DNA structure and dynamics at the base-pair level. In our previous studies we used a detailed model of DNA to measure the effects of sequence on the stability of naked DNA under bending. Sequence was shown to influence DNA's ability to form kinks, which arise when certain motifs slide past others to form non-native contacts. Here, we have now included histone-DNA interactions to see if the results obtained for naked DNA are transferable to the problem of nucleosome positioning. Different DNA sequences interacting with the histone protein complex are studied, and their equilibrium and mechanical properties are compared among themselves and with the naked case. NLM training grant to the Computation and Informatics in Biology and Medicine Training Program (NLM T15LM007359).
Histone Deacetylase Inhibitor Therapy in Epithelial Ovarian Cancer
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Noriyuki Takai
Full Text Available Since epigenetic alterations are believed to be involved in the repression of tumor suppressor genes and promotion of tumorigenesis in ovarian cancers, novel compounds endowed with a histone deacetylase (HDAC inhibitory activity are an attractive therapeutic approach. In this review, we discuss the biologic and therapeutic effects of HDAC inhibitors (HDACIs in treating ovarian cancer. HDACIs were able to mediate inhibition of cell growth, cell cycle arrest, apoptosis, and expression of genes related to the malignant phenotype in a variety of ovarian cancer cell lines. Furthermore, HDACIs were able to induce the accumulation of acetylated histones in the chromatin of the p21WAF1 gene in human ovarian carcinoma cells. In xenograft models, some of HDACIs have demonstrated antitumor activity with only few side effects. Some clinical trials demonstrate that HDACI drugs provide an important class of new mechanism-based therapeutics for ovarian cancer. In this review, we discuss the biologic and therapeutic effects of HDACIs in treating ovarian cancer, especially focusing on preclinical studies and clinical trials.
SMYD2-Mediated Histone Methylation Contributes to HIV-1 Latency.
Boehm, Daniela; Jeng, Mark; Camus, Gregory; Gramatica, Andrea; Schwarzer, Roland; Johnson, Jeffrey R; Hull, Philip A; Montano, Mauricio; Sakane, Naoki; Pagans, Sara; Godin, Robert; Deeks, Steven G; Krogan, Nevan J; Greene, Warner C; Ott, Melanie
Transcriptional latency of HIV is a last barrier to viral eradication. Chromatin-remodeling complexes and post-translational histone modifications likely play key roles in HIV-1 reactivation, but the underlying mechanisms are incompletely understood. We performed an RNAi-based screen of human lysine methyltransferases and identified the SET and MYND domain-containing protein 2 (SMYD2) as an enzyme that regulates HIV-1 latency. Knockdown of SMYD2 or its pharmacological inhibition reactivated latent HIV-1 in T cell lines and in primary CD4 + T cells. SMYD2 associated with latent HIV-1 promoter chromatin, which was enriched in monomethylated lysine 20 at histone H4 (H4K20me1), a mark lost in cells lacking SMYD2. Further, we find that lethal 3 malignant brain tumor 1 (L3MBTL1), a reader protein with chromatin-compacting properties that recognizes H4K20me1, was recruited to the latent HIV-1 promoter in a SMYD2-dependent manner. We propose that a SMYD2-H4K20me1-L3MBTL1 axis contributes to HIV-1 latency and can be targeted with small-molecule SMYD2 inhibitors. Copyright © 2017 Elsevier Inc. All rights reserved.
ChIp-seq of bovine cells (MDBK) to study butyrate-induced histone modification with 10 datasets
Next-generation sequencing was combined with chromatin immunoprecipitation (ChIP) technology to analyze histone modification (acetylation) induced by butyrate and to map the epigenomic landscape of normal histone H3, H4 in rumen cells of the cow. Ten variants of histone H3 and H4 modification were m...
Identification of a Novel 6′-N-Aminoglycoside Acetyltransferase, AAC(6′)-Iak, from a Multidrug-Resistant Clinical Isolate of Stenotrophomonas maltophilia
Tada, Tatsuya; Miyoshi-Akiyama, Tohru; Dahal, Rajan K.; Mishra, Shyam K.; Shimada, Kayo; Ohara, Hiroshi; Pokhrel, Bharat M.
Stenotrophomonas maltophilia IOMTU250 has a novel 6′-N-aminoglycoside acetyltransferase-encoding gene, aac(6′)-Iak. The encoded protein, AAC(6′)-Iak, consists of 153 amino acids and has 86.3% identity to AAC(6′)-Iz. Escherichia coli transformed with a plasmid containing aac(6′)-Iak exhibited decreased susceptibility to arbekacin, dibekacin, neomycin, netilmicin, sisomicin, and tobramycin. Thin-layer chromatography showed that AAC(6′)-Iak acetylated amikacin, arbekacin, dibekacin, isepamicin, kanamycin, neomycin, netilmicin, sisomicin, and tobramycin but not apramycin, gentamicin, or lividomycin. PMID:25092711
Identification of a novel 6'-N-aminoglycoside acetyltransferase, AAC(6')-Iak, from a multidrug-resistant clinical isolate of Stenotrophomonas maltophilia.
Tada, Tatsuya; Miyoshi-Akiyama, Tohru; Dahal, Rajan K; Mishra, Shyam K; Shimada, Kayo; Ohara, Hiroshi; Kirikae, Teruo; Pokhrel, Bharat M
Stenotrophomonas maltophilia IOMTU250 has a novel 6'-N-aminoglycoside acetyltransferase-encoding gene, aac(6')-Iak. The encoded protein, AAC(6')-Iak, consists of 153 amino acids and has 86.3% identity to AAC(6')-Iz. Escherichia coli transformed with a plasmid containing aac(6')-Iak exhibited decreased susceptibility to arbekacin, dibekacin, neomycin, netilmicin, sisomicin, and tobramycin. Thin-layer chromatography showed that AAC(6')-Iak acetylated amikacin, arbekacin, dibekacin, isepamicin, kanamycin, neomycin, netilmicin, sisomicin, and tobramycin but not apramycin, gentamicin, or lividomycin. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
Circadian Dynamics of the Cone-Rod Homeobox (CRX) Transcription Factor in the Rat Pineal Gland and Its Role in Regulation of Arylalkylamine N-Acetyltransferase (AANAT)
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Rohde, Kristian; Rovsing, Louise; Ho, Anthony K
The cone-rod homeobox (Crx) gene encodes a transcription factor in the retina and pineal gland. Crx deficiency influences the pineal transcriptome, including a reduced expression of arylalkylamine N-acetyltransferase (Aanat), a key enzyme in nocturnal pineal melatonin production. However, previous...... that the rhythmic nature of pineal CRX protein may directly modulate the daily profile of Aanat expression by inducing nighttime expression of this enzyme, thus facilitating nocturnal melatonin synthesis in addition to its role in ensuring a correct tissue distribution of Aanat expression....
Histone fractionation by high-performance liquid chromatography on cyanoalkylsilane (CN) reverse-phase columns
International Nuclear Information System (INIS)
Gurley, L.R.; Prentice, D.A.; Valdez, J.G.; Spall, W.D.
Previous work described conditions for the rapid fractionation of histones by high-performance liquid chromatography (HPLC) using a reverse-phase μBondapak C 18 column. That procedure resolved the major classes of histones with one exception: the more hydrophobic H2A variant, (MHP)H2A, was not resolved from the H4 histone class. This report extends that work describing experiments using a μBondapak CN column which better resolves the classes of histones from each other including the resolution of (MHP)H2A from the H4. In addition, the less hydrophobic H2A variant, (LHP)H2A, is partially resolved from the (MHP)H2A, and the less hydrophobic H3 variant, (LHP)H3, is resolved from the more hydrophobic H3 variant, (MHP)H3. Lower trifluoroacetic acid (TFA) concentrations (0.1%) in the eluting water/acetonitrile solvent were used with the CN column than were used with the C 18 column which increased the sensitivity of histone detection by ultraviolet absorption at 206 nm. Greater than 95% of the total [ 3 H]lysine-labeled protein applied to the CN column was eluted from the column. Contaminating nonhistone proteins were found to chromatograph in the region of histone elution. These were greatly reduced by isolating nuclei prior to histone preparation. The fractionation of the histones appears to be based on the hydrophobic properties of the proteins. The histone fractions (identified by their electrophoretic mobilities) were eluted from the CN column in the following order: H1, H2B, (LHP)H2A, (MHP)H2A, H4, (LHP)H3, and (MHP)H3. Phosphorylated and acetylated histone species were not resolved from their unmodified parental species
Platelet-activating factor (PAF) stimulates the PAF-synthesizing enzyme acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine O2-acetyltransferase and PAF synthesis in neutrophils.
Doebber, T W; Wu, M S
Platelet activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) induced in isolated rat peritoneal and human peripheral neutrophils a rapid and potent activation of the PAF biosynthetic enzyme acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine O2-acetyltransferase (EC 22.214.171.124). The PAF-induced activation of the neutrophil acetyltransferase (8-10 times basal neutrophil activity) was maximal within 30 sec after PAF addition, as was the PAF-stimulated degranulation. After 1 min of PA...
Effects of single nucleotide polymorphisms on human N-acetyltransferase 2 structure and dynamics by molecular dynamics simulation.
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M Rajasekaran
Full Text Available BACKGROUND: Arylamine N-acetyltransferase 2 (NAT2 is an important catalytic enzyme that metabolizes the carcinogenic arylamines, hydrazine drugs and chemicals. This enzyme is highly polymorphic in different human populations. Several polymorphisms of NAT2, including the single amino acid substitutions R64Q, I114T, D122N, L137F, Q145P, R197Q, and G286E, are classified as slow acetylators, whereas the wild-type NAT2 is classified as a fast acetylator. The slow acetylators are often associated with drug toxicity and efficacy as well as cancer susceptibility. The biological functions of these 7 mutations have previously been characterized, but the structural basis behind the reduced catalytic activity and reduced protein level is not clear. METHODOLOGY/PRINCIPAL FINDINGS: We performed multiple molecular dynamics simulations of these mutants as well as NAT2 to investigate the structural and dynamical effects throughout the protein structure, specifically the catalytic triad, cofactor binding site, and the substrate binding pocket. None of these mutations induced unfolding; instead, their effects were confined to the inter-domain, domain 3 and 17-residue insert region, where the flexibility was significantly reduced relative to the wild-type. Structural effects of these mutations propagate through space and cause a change in catalytic triad conformation, cofactor binding site, substrate binding pocket size/shape and electrostatic potential. CONCLUSIONS/SIGNIFICANCE: Our results showed that the dynamical properties of all the mutant structures, especially in inter-domain, domain 3 and 17-residue insert region were affected in the same manner. Similarly, the electrostatic potential of all the mutants were altered and also the functionally important regions such as catalytic triad, cofactor binding site, and substrate binding pocket adopted different orientation and/or conformation relative to the wild-type that may affect the functions of the mutants
Effect of monochromatic light on circadian rhythmic expression of clock genes and arylalkylamine N-acetyltransferase in chick retina.
Cao, Jing; Bian, Jiang; Wang, Zixu; Dong, Yulan; Chen, Yaoxing
Birds have more developed visual function. They not only have the ability to detect light and darkness but also have the color vision. Previous study showed that monochromatic light influenced avian physiological processes, which were controlled by clock genes. Therefore, bird's eye is a good model to studying the impact of color of light on circadian rhythms. Avian retina is one of the most important central oscillations. The study was designed to investigate the effect of color of light on the expression of clock genes and arylalkylamine N-acetyltransferase (Aanat) mRNA expression in chick retina. A total of 240 post-hatching day (P) 0 broiler chickens were exposed to blue (BL), green (GL), red (RL) and white light (WL) from a LED system under a light-dark cycle 12L:12D for 14 d. The results show that the significant daily variations existed in the gene expression of cBmal1, cBmal2, cCry1, cCry2, cPer2 and cPer3, but not for cClock under four light treatments. The genes cBmal1, cCry1, cPer2 and cPer3 presented circadian rhythmic expression under the various monochromatic lights. When compared with WL, GL elevated the expression of positive regulators of cellular clock (cBmal1, cBmal2 and cClock) and cAanat mRNA level, whereas RL increased the mRNA levels of negative regulators of cellular clock (cCry1, cCry2, cPer2 and cPer3) and decreased the cAanat mRNA expression in the retina. These results demonstrated that monochromatic light affect the periodic expression levels of the biological clock mRNA by positive and negative feedback loop interactions, GL activated the transcription of cAanat; while RL suppressed the transcription of cAanat. Thereby, color of light regulates ocular cAanat expression by affecting on expression of cellular clock regulators.
Interaction between cysteine synthase and serine O-acetyltransferase proteins and their stage specific expression in Leishmania donovani.
Singh, Kuljit; Singh, Krishn Pratap; Equbal, Asif; Suman, Shashi S; Zaidi, Amir; Garg, Gaurav; Pandey, Krishna; Das, Pradeep; Ali, Vahab
Leishmania possess a unique trypanothione redox metabolism with undebated roles in protection from oxidative damage and drug resistance. The biosynthesis of trypanothione depends on l-cysteine bioavailability which is regulated by cysteine biosynthesis pathway. The de novo cysteine biosynthesis pathway is comprised of serine O-acetyltransferase (SAT) and cysteine synthase (CS) enzymes which sequentially mediate two consecutive steps of cysteine biosynthesis, and is absent in mammalian host. However, despite the apparent dependency of redox metabolism on cysteine biosynthesis pathway, the role of SAT and CS in redox homeostasis has been unexplored in Leishmania parasites. Herein, we have characterized CS and SAT to investigate their interaction and relative abundance of these proteins in promastigote vs. amastigote growth stages of L. donovani. CS and SAT genes of L. donovani (LdCS and LdSAT) were cloned, expressed, and fusion proteins purified to homogeneity with affinity column chromatography. Purified LdCS contains PLP as cofactor and showed optimum enzymatic activity at pH 7.5. Enzyme kinetics showed that LdCS catalyses the synthesis of cysteine using O-acetylserine and sulfide with a K m of 15.86 mM and 0.17 mM, respectively. Digitonin fractionation and indirect immunofluorescence microscopy showed that LdCS and LdSAT are localized in the cytoplasm of promastigotes. Size exclusion chromatography, co-purification, pull down and immuno-precipitation assays demonstrated a stable complex formation between LdCS and LdSAT proteins. Furthermore, LdCS and LdSAT proteins expression/activity was upregulated in amastigote growth stage of the parasite. Thus, the stage specific differential expression of LdCS and LdSAT suggests that it may have a role in the redox homeostasis of Leishmania. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
In Bacillus subtilis, the SatA (Formerly YyaR) Acetyltransferase Detoxifies Streptothricin via Lysine Acetylation.
Burckhardt, Rachel M; Escalante-Semerena, Jorge C
Soil is a complex niche, where survival of microorganisms is at risk due to the presence of antimicrobial agents. Many microbes chemically modify cytotoxic compounds to block their deleterious effects. Streptothricin is a broad-spectrum antibiotic produced by streptomycetes that affects Gram-positive and Gram-negative bacteria alike. Here we identify the SatA (for s treptothricin a ce t yltransferase A , formerly YyaR) enzyme of Bacillus subtilis as the mechanism used by this soil bacterium to detoxify streptothricin. B. subtilis strains lacking satA were susceptible to streptothricin. Ectopic expression of satA + restored streptothricin resistance to B. subtilis satA ( Bs SatA) strains. Purified Bs SatA acetylated streptothricin in vitro at the expense of acetyl-coenzyme A (acetyl-CoA). A single acetyl moiety transferred onto streptothricin by SatA blocked the toxic effects of the antibiotic. SatA bound streptothricin with high affinity ( K d [dissociation constant] = 1 μM), and did not bind acetyl-CoA in the absence of streptothricin. Expression of B. subtilis satA + in Salmonella enterica conferred streptothricin resistance, indicating that SatA was necessary and sufficient to detoxify streptothricin. Using this heterologous system, we showed that the SatA homologue from Bacillus anthracis also had streptothricin acetyltransferase activity. Our data highlight the physiological relevance of lysine acetylation for the survival of B. subtilis in the soil. IMPORTANCE Experimental support is provided for the functional assignment of gene products of the soil-dwelling bacilli Bacillus subtilis and Bacillus anthracis This study focuses on one enzyme that is necessary and sufficient to block the cytotoxic effects of a common soil antibiotic. The enzyme alluded to is a member of a family of proteins that are broadly distributed in all domains of life but poorly studied in B. subtilis and B. anthracis The initial characterization of the enzyme provides insights into its
Antifungal Activity of Phenyl Derivative of Pyranocoumarin from Psoralea corylifolia L. Seeds by Inhibition of Acetylation Activity of Trichothecene 3-O-Acetyltransferase (Tri101
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Sangeetha Srinivasan
Full Text Available Antifungal activity of petroleum ether extract of Psoralea corylifolia L. seed, tested against Fusarium sp. namely, Fusarium oxysporum, Fusarium moniliforme, and Fusarium graminearum, was evaluated by agar well diffusion assay. The chromatographic fractionation of the extract yielded a new phenyl derivative of pyranocoumarin (PDP. The structure of the PDP was confirmed using spectroscopic characterization (GC-MS, IR, and NMR, and a molecular mass of m/z 414 [M-2H]+ with molecular formula C27H28O4 was obtained. The PDP had a potent antifungal activity with a minimum inhibitory concentration of 1 mg/mL against Fusarium sp. Molecular docking using Grid-Based Ligand Docking with Energetics (GLIDE, Schrodinger was carried out with the Tri101, trichothecene 3-O-acetyltransferase, as target protein to propose a mechanism for the antifungal activity. The ligand PDP showed bifurcated hydrogen bond interaction with active site residues at TYR 413 and a single hydrogen bond interaction at ARG 402 with a docking score −7.19 and glide energy of −45.78 kcal/mol. This indicated a strong binding of the ligand with the trichothecene 3-O-acetyltransferase, preventing as a result the acetylation of the trichothecene mycotoxin and destruction of the "self-defense mechanism" of the Fusarium sp.
CSR-1 RNAi pathway positively regulates histone expression in C. elegans.
Avgousti, Daphne C; Palani, Santhosh; Sherman, Yekaterina; Grishok, Alla
Endogenous small interfering RNAs (endo-siRNAs) have been discovered in many organisms, including mammals. In C. elegans, depletion of germline-enriched endo-siRNAs found in complex with the CSR-1 Argonaute protein causes sterility and defects in chromosome segregation in early embryos. We discovered that knockdown of either csr-1, the RNA-dependent RNA polymerase (RdRP) ego-1, or the dicer-related helicase drh-3, leads to defects in histone mRNA processing, resulting in severe depletion of core histone proteins. The maturation of replication-dependent histone mRNAs, unlike that of other mRNAs, requires processing of their 3'UTRs through an endonucleolytic cleavage guided by the U7 snRNA, which is lacking in C. elegans. We found that CSR-1-bound antisense endo-siRNAs match histone mRNAs and mRNA precursors. Consistently, we demonstrate that CSR-1 directly binds to histone mRNA in an ego-1-dependent manner using biotinylated 2'-O-methyl RNA oligonucleotides. Moreover, we demonstrate that increasing the dosage of histone genes rescues the lethality associated with depletion of CSR-1 and EGO-1. These results support a positive and direct effect of RNAi on histone gene expression.
Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation
Lopez, Rita; Sarg, Bettina; Lindner, Herbert; Bartolomé, Salvador; Ponte, Inma; Suau, Pedro; Roque, Alicia
Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a decrease in α-helix/turns, with increasing linker histone phosphorylation. This conformational change could act as the first step in the phosphorylation-induced effects on chromatin condensation. A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation. Analysis of specific genes, combining nuclease digestion and qPCR, showed that phosphorylated samples were more accessible than unphosphorylated samples, suggesting local chromatin relaxation. Chromatin aggregation was induced by MgCl2 and analyzed by dynamic light scattering (DLS). Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation. These findings provide new insights into the effects of linker histone phosphorylation in chromatin condensation. PMID:25870416
Prothymosin alpha interacts with free core histones in the nucleus of dividing cells.
Covelo, Guillermo; Sarandeses, Concepción S; Díaz-Jullien, Cristina; Freire, Manuel
The acidic protein prothymosin alpha (ProTalpha), with a broad presence in mammalian cells, has been widely considered to have a role in cell division, through an unrevealed mechanism in which histones may be involved in view of their ability to interact with ProTalpha in vitro. Results of co-immunoprecipitation experiments presented here demonstrate that ProTalpha interacts in vivo with core histones in proliferating B-lymphocytes (NC-37 cells). This interaction occurs with histones H3, H2A, H2B and H4 located free in the nucleoplasm, whereas no interaction was detected with histone H1, mono-nucleosome particles or chromatin. Moreover, the core histones form part of a nuclear multiprotein complex of about 700 kDa separated by ProTalpha-Sepharose affinity, with components including H3 and H4 acetyltranferases, H3 methyltransferases, hnRNP isotypes A3, A2/B1 and R, ATP-dependent and independent DNA helicases II, beta-actin and vimentin, all co-purifying by gel filtration. This indicates that the interaction of ProTalpha with core histones in the nucleus may be related to the structural modification of histones H3 and H4, and hence to chromatin activity, raising the possibility that the other proteins in the nuclear complex may play a role in this process.
Small-molecule-based inhibition of histone demethylation in cells assessed by quantitative mass spectrometry.
Mackeen, Mukram M; Kramer, Holger B; Chang, Kai-Hsuan; Coleman, Matthew L; Hopkinson, Richard J; Schofield, Christopher J; Kessler, Benedikt M
Post-translational modifications on histones are an important mechanism for the regulation of gene expression and are involved in all aspects of cell growth and differentiation, as well as pathological processes including neurodegeneration, autoimmunity, and cancer. A major challenge within the chromatin field is to develop methods for the quantitative analysis of histone modifications. Here we report a mass spectrometry (MS) approach based on ultraperformance liquid chromatography high/low collision switching (UPLC-MS(E)) to monitor histone modifications in cells. This approach is exemplified by the analysis of trimethylated lysine-9 levels in histone H3, following a simple chemical derivatization procedure with d(6)-acetic anhydride. This method was used to study the inhibition of histone demethylases with pyridine-2,4-dicarboxylic acid (PDCA) derivatives in cells. Our results show that the PDCA-dimethyl ester inhibits JMJD2A catalyzed demethylation of lysine-9 on histone H3 in human HEK 293T cells. Demethylase inhibition, as observed by MS analyses, was supported by immunoblotting with modification-specific antibodies. The results demonstrate that PDCA derived small molecules are cell permeable demethylase inhibitors and reveal that quantitative MS is a useful tool for measuring post-translational histone modifications in cells.
Synergistic effects between natural histone mixtures and polyethylenimine in non-viral gene delivery in vitro.
Schneeweiss, Anne; Buyens, Kevin; Giese, Matthias; Sanders, Niek; Ulbert, Sebastian
Nanoparticles made of plasmid DNA (pDNA) and cationic polymers are promising strategies for non-viral gene delivery. However, many cationic polymers are toxic to cells when used in higher concentrations. Positively charged proteins, such as histones, are biodegradable and a good alternative, especially for potential in vivo applications. It has previously been shown that histones are able to complex DNA and mediate transfection of cells. To investigate possible synergistic effects between the different histone types and to avoid the use of recombinant proteins, we analysed whether natural histone mixtures would be functional as gene carriers. Core and linker histones from calf thymus and from chicken erythrocytes were used to transfect different cell lines. The protein mixtures efficiently complexed the pDNA, and the resulting particles entered the cells. However, only marginal expression of the gene encoded by the pDNA was observed. Transfection rates increased drastically when minimal amounts of the basic polymer polyethylenimine (PEI) were added to the particles. Neither PEI nor histones alone mediated any transfection under the conditions where a combination of both worked efficiently, and the combined particles were well tolerated by the cells. These results demonstrate that histone mixtures from natural sources in combination with minimal amounts of PEI can be used as gene carriers. This might have consequences for the development of novel gene delivery strategies, such as DNA vaccines, with minimal side-effects. Copyright © 2010 Elsevier B.V. All rights reserved.
Structure of the human histone chaperone FACT Spt16 N-terminal domain
International Nuclear Information System (INIS)
Marcianò, G.; Huang, D. T.
The Spt16–SSRP1 heterodimer is a histone chaperone that plays an important role in regulating chromatin assembly. Here, a crystal structure of the N-terminal domain of human Spt16 is presented and it is shown that this domain may contribute to histone binding. The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding
Histones from Dying Renal Cells Aggravate Kidney Injury via TLR2 and TLR4
Allam, Ramanjaneyulu; Scherbaum, Christina Rebecca; Darisipudi, Murthy Narayana; Mulay, Shrikant R.; Hägele, Holger; Lichtnekert, Julia; Hagemann, Jan Henrik; Rupanagudi, Khader Valli; Ryu, Mi; Schwarzenberger, Claudia; Hohenstein, Bernd; Hugo, Christian; Uhl, Bernd; Reichel, Christoph A.; Krombach, Fritz; Monestier, Marc; Liapis, Helen; Moreth, Kristin; Schaefer, Liliana
In AKI, dying renal cells release intracellular molecules that stimulate immune cells to secrete proinflammatory cytokines, which trigger leukocyte recruitment and renal inflammation. Whether the release of histones, specifically, from dying cells contributes to the inflammation of AKI is unknown. In this study, we found that dying tubular epithelial cells released histones into the extracellular space, which directly interacted with Toll-like receptor (TLR)-2 (TLR2) and TLR4 to induce MyD88, NF-κB, and mitogen activated protein kinase signaling. Extracellular histones also had directly toxic effects on renal endothelial cells and tubular epithelial cells in vitro. In addition, direct injection of histones into the renal arteries of mice demonstrated that histones induce leukocyte recruitment, microvascular vascular leakage, renal inflammation, and structural features of AKI in a TLR2/TLR4-dependent manner. Antihistone IgG, which neutralizes the immunostimulatory effects of histones, suppressed intrarenal inflammation, neutrophil infiltration, and tubular cell necrosis and improved excretory renal function. In summary, the release of histones from dying cells aggravates AKI via both its direct toxicity to renal cells and its proinflammatory effects. Because the induction of proinflammatory cytokines in dendritic cells requires TLR2 and TLR4, these results support the concept that renal damage triggers an innate immune response, which contributes to the pathogenesis of AKI. PMID:22677551
Real-Time Tracking of Parental Histones Reveals Their Contribution to Chromatin Integrity Following DNA Damage.
Adam, Salomé; Dabin, Juliette; Chevallier, Odile; Leroy, Olivier; Baldeyron, Céline; Corpet, Armelle; Lomonte, Patrick; Renaud, Olivier; Almouzni, Geneviève; Polo, Sophie E
Chromatin integrity is critical for cell function and identity but is challenged by DNA damage. To understand how chromatin architecture and the information that it conveys are preserved or altered following genotoxic stress, we established a system for real-time tracking of parental histones, which characterize the pre-damage chromatin state. Focusing on histone H3 dynamics after local UVC irradiation in human cells, we demonstrate that parental histones rapidly redistribute around damaged regions by a dual mechanism combining chromatin opening and histone mobilization on chromatin. Importantly, parental histones almost entirely recover and mix with new histones in repairing chromatin. Our data further define a close coordination of parental histone dynamics with DNA repair progression through the damage sensor DDB2 (DNA damage-binding protein 2). We speculate that this mechanism may contribute to maintaining a memory of the original chromatin landscape and may help preserve epigenome stability in response to DNA damage. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.
Structure of the human histone chaperone FACT Spt16 N-terminal domain
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Marcianò, G.; Huang, D. T., E-mail: firstname.lastname@example.com [Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow G61 1BD, Scotland (United Kingdom)
The Spt16–SSRP1 heterodimer is a histone chaperone that plays an important role in regulating chromatin assembly. Here, a crystal structure of the N-terminal domain of human Spt16 is presented and it is shown that this domain may contribute to histone binding. The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding.
Extracting histones for the specific purpose of label-free MS.
Govaert, Elisabeth; Van Steendam, Katleen; Scheerlinck, Ellen; Vossaert, Liesbeth; Meert, Paulien; Stella, Martina; Willems, Sander; De Clerck, Laura; Dhaenens, Maarten; Deforce, Dieter
Extracting histones from cells is the first step in studies that aim to characterize histones and their post-translational modifications (hPTMs) with MS. In the last decade, label-free quantification is more frequently being used for MS-based histone characterization. However, many histone extraction protocols were not specifically designed for label-free MS. While label-free quantification has its advantages, it is also very susceptible to technical variation. Here, we adjust an established histone extraction protocol according to general label-free MS guidelines with a specific focus on minimizing sample handling. These protocols are first evaluated using SDS-PAGE. Hereafter, a selection of extraction protocols was used in a complete histone workflow for label-free MS. All protocols display nearly identical relative quantification of hPTMs. We thus show that, depending on the cell type under investigation and at the cost of some additional contaminating proteins, minimizing sample handling can be done during histone isolation. This allows analyzing bigger sample batches, leads to reduced technical variation and minimizes the chance of in vitro alterations to the hPTM snapshot. Overall, these results allow researchers to determine the best protocol depending on the resources and goal of their specific study. Data are available via ProteomeXchange with identifier PXD002885. © 2016 The Authors. Proteomics Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Extracellular histones, cell-free DNA, or nucleosomes: differences in immunostimulation.
Marsman, Gerben; Zeerleder, Sacha; Luken, Brenda M
In inflammation, extensive cell death may occur, which results in the release of chromatin components into the extracellular environment. Individually, the purified chromatin components double stranded (ds)DNA and histones have been demonstrated, both in vitro and in vivo, to display various immunostimulatory effects, for example, histones induce cytotoxicity and proinflammatory signaling through toll-like receptor (TLR)2 and 4, while DNA induces signaling through TLR9 and intracellular nucleic acid sensing mechanisms. However, DNA and histones are organized in nucleosomes in the nucleus, and evidence suggests that nucleosomes are released as such in inflammation. The cytotoxicity and proinflammatory signaling induced by nucleosomes have not been studied as extensively as the separate effects brought abo