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DNMT3L Modulates Significant and Distinct Flanking Sequence Preference for DNA Methylation by DNMT3A and DNMT3B In Vivo
Wienholz, Bethany L.
Kareta, Michael S.
Moarefi, Amir H.
Gordon, Catherine A.
Ginno, Paul A.
Chédin, Frédéric
PLoS Genetics
The DNTM3A and DNMT3B de novo DNA methyltransferases (DNMTs) are responsible for setting genomic DNA methylation patterns, a key layer of epigenetic information. Here, using an in vivo episomal methylation assay and extensive bisulfite methylation sequencing, we show that human DNMT3A and DNMT3B possess significant and distinct flanking sequence preferences for target CpG sites. Selection for high or low efficiency sites is mediated by the base composition at the −2 and +2 positions flanking the CpG site for DNMT3A, and at the −1 and +1 positions for DNMT3B. This intrinsic preference reproducibly leads to the formation of specific de novo methylation patterns characterized by up to 34-fold variations in the efficiency of DNA methylation at individual sites. Furthermore, analysis of the distribution of signature methylation hotspot and coldspot motifs suggests that DNMT flanking sequence preference has contributed to shaping the composition of CpG islands in the human genome. Our results also show that the DNMT3L stimulatory factor modulates the formation of de novo methylation patterns in two ways. First, DNMT3L selectively focuses the DNA methylation machinery on properly chromatinized DNA templates. Second, DNMT3L attenuates the impact of the intrinsic DNMT flanking sequence preference by providing a much greater boost to the methylation of poorly methylated sites, thus promoting the formation of broader and more uniform methylation patterns. This study offers insights into the manner by which DNA methylation patterns are deposited and reveals a new level of interplay between members of the de novo DNMT family.
Author Summary
The methylation of cytosine bases in DNA represents an extra layer of heritable biological information necessary for regulating gene expression and ensuring genomic stability in mammals. In this paper, we examine the function of the proteins responsible for laying down the initial DNA methylation patterns in the human genome. These proteins, called de novo DNA methyltransferases (DNMTs), comprise two active enzymes, DNMT3A and DNMT3B, and one stimulatory factor, DNMT3L. Our study clearly establishes that DNMT3A and DNMT3B do not methylate DNA at random but rather that they show strong and distinct preferences for their target sites in vivo. These preferences lead to the deposition of unique and reproducible patterns of methylation and may have contributed to shaping segments of the human genome. In contrast, we show that DNMT3L stimulates DNA methylation mostly at sites that are poorly methylated on their own, thus leading to patterns that are more uniform. This modulation is proposed to result from DNMT3L anchoring the DNA methylation machinery onto chromatin, the physiological form under which DNA exists in our cells. This study furthers our understanding of how genomic DNA methylation patterns are established in vivo.
Related Articles
Dnmt3a Protects Active Chromosome Domains against Cancer-Associated Hypomethylation
Raddatz, Günter
Gao, Qing
Bender, Sebastian
Jaenisch, Rudolf
Lyko, Frank
PLoS Genetics
Changes in genomic DNA methylation patterns are generally assumed to play an important role in the etiology of human cancers. The Dnmt3a enzyme is required for the establishment of normal methylation patterns, and mutations in Dnmt3a have been described in leukemias. Deletion of Dnmt3a in a K-ras–dependent mouse lung cancer model has been shown to promote tumor progression, which suggested that the enzyme might suppress tumor development by stabilizing DNA methylation patterns. We have used whole-genome bisulfite sequencing to comprehensively characterize the methylomes from Dnmt3a wildtype and Dnmt3a-deficient mouse lung tumors. Our results show that profound global methylation changes can occur in K-ras–induced lung cancer. Dnmt3a wild-type tumors were characterized by large hypomethylated domains that correspond to nuclear lamina-associated domains. In contrast, Dnmt3a-deficient tumors showed a uniformly hypomethylated genome. Further data analysis revealed that Dnmt3a is required for efficient maintenance methylation of active chromosome domains and that Dnmt3a-deficient tumors show moderate levels of gene deregulation in these domains. In summary, our results uncover conserved features of cancer methylomes and define the role of Dnmt3a in maintaining DNA methylation patterns in cancer.
Author Summary
Dnmt3a is generally assumed to be a de novo DNA methyltransferase that plays an important role in establishing DNA methylation patterns during embryogenesis. However, mutations in the human DNMT3A gene have been detected in various cancers, suggesting that the enzyme might also be relevant for DNA methylation in adult tissues and in tumors. We have established genome-wide methylation profiles at single base pair resolution to define Dnmt3a-dependent methylation changes in a mouse tumor model. Our results show that mouse tumors with a functional Dnmt3a enzyme are characterized by regional hypomethylation, while Dnmt3a-deficient tumors showed a uniformly hypomethylated genome. Further data analysis revealed that Dnmt3a is required for maintaining normal DNA methylation patterns specifically in gene bodies and in active chromosome domains. Our study thus defines the role of Dnmt3a in maintaining DNA methylation patterns and provides a paradigm for understanding the effects of DNMT3A mutations on human cancer methylomes.
Related Articles
Regulation of Lineage Specific DNA Hypomethylation in Mouse Trophectoderm
Oda, Masaaki
Oxley, David
Dean, Wendy
Reik, Wolf
PLoS ONE
DNA methylation is reprogrammed during early embryogenesis by active and passive mechanisms in advance of the first differentiation event producing the embryonic and extraembryonic lineage cells which contribute to the future embryo proper and to the placenta respectively. Embryonic lineage cells re-acquire a highly methylated genome dependent on the DNA methyltransferases (DNMTs) Dnmt3a and Dnmt3b that are required for de novo methylation. By contrast, extraembryonic lineage cells remain globally hypomethylated but the mechanisms that underlie this hypomethylation remain unknown.
Methodology/Principal Findings
We have employed an inducible system that supports differentiation between these two lineages and recapitulates the DNA methylation asymmetry generated in vivo. We find that in vitro down-regulation of Oct3/4 in ES cells recapitulates the decline in global DNA methylation associated with trophoblast. The de novo DNMTs Dnmt3a2 and Dnmt3b are down-regulated during trophoblast differentiation. Dnmt1, which is responsible for maintenance methylation, is expressed comparably in embryonic and trophoblast lineages, however importantly in trophoblast giant cells Dnmt1fails to be attracted to replication foci, thus allowing loss of DNA methylation while implicating a passive demethylation mechanism. Interestingly, Dnmt1 localization was restored by exogenous Np95/Uhrf1, a Dnmt1 chaperone required for Dnmt1-targeting to replication foci, yet DNA methylation levels remained low. Over-expression of de novo DNMTs also failed to increase DNA methylation in target sequences.
We propose that induced trophoblast cells may have a mechanism to resist genome-wide increases of DNA methylation, thus reinforcing the genome-wide epigenetic distinctions between the embryonic and extraembryonic lineages in the mouse. This resistance may be based on transcription factors or on global differences in chromatin structure.
Related Articles
5-Aza-Deoxycytidine Induces Selective Degradation of DNA Methyltransferase 1 by a Proteasomal Pathway That Requires the KEN Box, Bromo-Adjacent Homology Domain, and Nuclear Localization Signal
Ghoshal, Kalpana
Datta, Jharna
Majumder, Sarmila
Bai, Shoumei
Kutay, Huban
Motiwala, Tasneem
Jacob, Samson T.
Molecular and Cellular Biology
5-Azacytidine- and 5-aza-deoxycytidine (5-aza-CdR)-mediated reactivation of tumor suppressor genes silenced by promoter methylation has provided an alternate approach in cancer therapy. Despite the importance of epigenetic therapy, the mechanism of action of DNA-hypomethylating agents in vivo has not been completely elucidated. Here we report that among three functional DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), the maintenance methyltransferase, DNMT1, was rapidly degraded by the proteasomal pathway upon treatment of cells with these drugs. The 5-aza-CdR-induced degradation, which occurs in the nucleus, could be blocked by proteasomal inhibitors and required a functional ubiquitin-activating enzyme. The drug-induced degradation occurred even in the absence of DNA replication. Treatment of cells with other nucleoside analogs modified at C-5, 5-fluorodeoxyuridine and 5-fluorocytidine, did not induce the degradation of DNMT1. Mutation of cysteine at the catalytic site of Dnmt1 (involved in the formation of a covalent intermediate with cytidine in DNA) to serine (CS) did not impede 5-aza-CdR-induced degradation. Neither the wild type nor the catalytic site mutant of Dnmt3a or Dnmt3b was sensitive to 5-aza-CdR-mediated degradation. These results indicate that covalent bond formation between the enzyme and 5-aza-CdR-incorporated DNA is not essential for enzyme degradation. Mutation of the conserved KEN box, a targeting signal for proteasomal degradation, to AAA increased the basal level of Dnmt1 and blocked its degradation by 5-aza-CdR. Deletion of the catalytic domain increased the expression of Dnmt1 but did not confer resistance to 5-aza-CdR-induced degradation. Both the nuclear localization signal and the bromo-adjacent homology domain were essential for nuclear localization and for the 5-aza-CdR-mediated degradation of Dnmt1. Polyubiquitination of Dnmt1 in vivo and its stabilization upon treatment of cells with a proteasomal inhibitor indicate that the level of Dnmt1 is controlled by ubiquitin-dependent proteasomal degradation. Overexpression of the substrate recognition component, Cdh1 but not Cdc20, of APC (anaphase-promoting complex)/cyclosome ubiquitin ligase reduced the level of Dnmt1 in both untreated and 5-aza-CdR-treated cells. In contrast, the depletion of Cdh1 with small interfering RNA increased the basal level of DNMT1 that blocked 5-aza-CdR-induced degradation. Dnmt1 interacted with Cdh1 and colocalized in the nucleus at discrete foci. Both Dnmt1 and Cdh1 were phosphorylated in vivo, but only Cdh1 was significantly dephosphorylated upon 5-aza-CdR treatment, suggesting its involvement in initiating the proteasomal degradation of DNMT1. These results demonstrate a unique mechanism for the selective degradation of DNMT1, the maintenance DNA methyltransferase, by well-known DNA-hypomethylating agents.
Related Articles
Loss of DNMT1o Disrupts Imprinted X Chromosome Inactivation and Accentuates Placental Defects in Females
McGraw, Serge
Oakes, Christopher C.
Martel, Josée
Cirio, M. Cecilia
de Zeeuw, Pauline
Mak, Winifred
Plass, Christoph
Bartolomei, Marisa S.
Chaillet, J. Richard
Trasler, Jacquetta M.
PLoS Genetics
The maintenance of key germline derived DNA methylation patterns during preimplantation development depends on stores of DNA cytosine methyltransferase-1o (DNMT1o) provided by the oocyte. Dnmt1omat−/− mouse embryos born to Dnmt1Δ1o/Δ1o female mice lack DNMT1o protein and have disrupted genomic imprinting and associated phenotypic abnormalities. Here, we describe additional female-specific morphological abnormalities and DNA hypomethylation defects outside imprinted loci, restricted to extraembryonic tissue. Compared to male offspring, the placentae of female offspring of Dnmt1Δ1o/Δ1o mothers displayed a higher incidence of genic and intergenic hypomethylation and more frequent and extreme placental dysmorphology. The majority of the affected loci were concentrated on the X chromosome and associated with aberrant biallelic expression, indicating that imprinted X-inactivation was perturbed. Hypomethylation of a key regulatory region of Xite within the X-inactivation center was present in female blastocysts shortly after the absence of methylation maintenance by DNMT1o at the 8-cell stage. The female preponderance of placental DNA hypomethylation associated with maternal DNMT1o deficiency provides evidence of additional roles beyond the maintenance of genomic imprints for DNA methylation events in the preimplantation embryo, including a role in imprinted X chromosome inactivation.
Author Summary
During oocyte growth and maturation, vital proteins and enzymes are produced to ensure that, when fertilized, a healthy embryo will arise. When this natural process is interrupted, one or more of these essential elements can fail to be produced thus compromising the health of the future embryo. We are using a mouse model, lacking an enzyme (DNMT1o) produced in the oocyte and only required post-fertilization in the early embryo for the maintenance of inherited DNA methylation marks. Here, we reveal that oocytes lacking DNMT1o, when fertilized, generated conceptuses with a wide variety of placental abnormalities. These placental abnormalities were more frequent and severe in females, and showed specific genomic regions constantly deprived of their normal methylation marks. The affected genomic regions were concentrated on the X chromosome. Interestingly, we also found that a region important for the regulation of the X chromosome inactivation process was hypomethylated in female blastocysts and was associated with sex-specific abnormalities in the placenta, relaxation of imprinted X chromosome inactivation, and disruption of DNA methylation later in development. Our findings provide a novel unanticipated role for DNA methylation events taking place within the first few days of life specifically in female preimplantation embryos.
Related Articles
Role of DNA Methyltransferases in Regulation of Human Ribosomal RNA Gene Transcription*
Majumder, Sarmila
Ghoshal, Kalpana
Datta, Jharna
Smith, David Spencer
Bai, Shoumei
Jacob, Samson T.
The Journal of biological chemistry
We have previously demonstrated that the expression of human ribosomal RNA genes (rDNA) in normal and cancer cells is differentially regulated by methylation of the promoter CpG islands. Furthermore, we showed that the methyl CpG-binding protein MBD2 plays a selective role in the methylation-mediated block in rDNA expression. Here, we analyzed the role of three functional mammalian DNA methyltransferases (DNMTs) in regulating the rDNA promoters activity. Immunofluorescence analysis and biochemical fractionation showed that all three DNMTs (DNMT1, DNMT3A, and DNMT3B) are associated with the inactive rDNA in the nucleolus. Although DNMTs associate with both methylated and unmethylated rDNA promoters, DNMT1 preferentially associates with the methylated genes. The rDNA primary transcript level was significantly elevated in DNMT1−/− or DNMT3B−/− human colon carcinoma (HCT116) cells. Southern blot analysis demonstrated a moderate level of rDNA promoter hypomethylation in DNMT1−/− cells and a dramatic loss of rDNA promoter methylation in double knockout cells. Transient overexpression of DNMT1 or DNMT3B suppressed the luciferase expression from both methylated and unmethylated pHrD-IRES-Luc, a reporter plasmid where the rDNA promoter drives luciferase expression. DNMT1-mediated suppression of the unmethylated promoter involves de novo methylation of the promoter, whereas histone deacetylase 2 cooperates with DNMT1 to inhibit the methylated rDNA promoter. Unlike DNMT1, both the wild type and catalytically inactive DNMT3B mutant can suppress rDNA promoter irrespective of its methylation status. DNMT3B-mediated suppression of the rDNA promoter also involves histone deacetylation. Treatment of HCT116 cells with Decitabine (a DNMT inhibitor) or trichostatin A (a histone deacetylase inhibitor) up-regulated endogenous rDNA expression. These inhibitors synergistically activated methylated pHrD-IRES-Luc, whereas they exhibited additive effects on the unmethylated promoter. These results demonstrate localization of DNMTs with the inactive rDNA in the nucleolus, the specific role of DNMT1 and DNMT3B in rDNA expression and the differential regulation of rDNA expression from the methylated and unmethylated rDNA promoters.
Related Articles
DNMT1 genetic polymorphisms affect breast cancer risk in the central European Caucasian population
Kullmann, Kathrin
Deryal, Mustafa
Ong, Mei Fang
Schmidt, Werner
Mahlknecht, Ulrich
Clinical Epigenetics
DNA methylation of CpG islands within the promoter region of genes is an epigenetic modification with an important role in the development of cancer and it is typically mediated by DNA methyltransferases (DNMTs). In cancer cells, global hypomethylation of the genome as a whole and regional hypermethylation of CpG islands have been reported. Four groups of DNMTs have been identified: DNMT1, DNMT2 (TRDMT1), DNMT3A and DNMT3B. DNMT2 uses the catalytic mechanism of DNMTs, but does in fact methylate RNA. Little is known about the significance of these genes in human breast cancer. In the study presented herein, we analyzed five distinct DNMT single SNPs with regard to potential associations with breast cancer risk.
Case description
In this study, we genotyped 221 female Caucasian breast cancer patients and 221 female Caucasian healthy controls, and we used five allele-specific real-time polymerase chain reaction (qPCR) assays. We selected one locus within the DNMT1 gene and two loci within the DNMT3A and DNMT3B genes, respectively. Statistics were calculated using the chi-squared and Fisher's exact tests, and correlated with clinical parameters such as age, diagnosis, histology, TNM stage, hormonal receptor status, human epidermal growth factor receptor 2 (HER2) status, response to treatment and survival. Statistically significant results were obtained for correlations with the DNMT1 gene.
Discussion and Evaluation
Five genomic loci within the DNMT1, DNMT3A and DNMT3B genes were assessed. Statistical significance (P = 0.030) was identified for DNMT1 SNP (A201G, rs2228612): six women within the control group were GG homozygous (variant), while this mutation was absent in the breast cancer group.
We conclude that women with the DNMT1 SNP (A201G, rs2228612) GG homozygous genotype (variant) have a lower risk of developing breast cancer compared to heterozygous or wildtype genotypes. To date, alterations within the DNMT1 gene have not been reported to be associated with cancer in the Caucasian population.
DNMT; SNP; Breast cancer
Related Articles
Mouse Oocyte Methylomes at Base Resolution Reveal Genome-Wide Accumulation of Non-CpG Methylation and Role of DNA Methyltransferases
Shirane, Kenjiro
Toh, Hidehiro
Kobayashi, Hisato
Miura, Fumihito
Chiba, Hatsune
Ito, Takashi
Kono, Tomohiro
Sasaki, Hiroyuki
PLoS Genetics
DNA methylation is an epigenetic modification that plays a crucial role in normal mammalian development, retrotransposon silencing, and cellular reprogramming. Although methylation mainly occurs on the cytosine in a CG site, non-CG methylation is prevalent in pluripotent stem cells, brain, and oocytes. We previously identified non-CG methylation in several CG-rich regions in mouse germinal vesicle oocytes (GVOs), but the overall distribution of non-CG methylation and the enzymes responsible for this modification are unknown. Using amplification-free whole-genome bisulfite sequencing, which can be used with minute amounts of DNA, we constructed the base-resolution methylome maps of GVOs, non-growing oocytes (NGOs), and mutant GVOs lacking the DNA methyltransferase Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3L. We found that nearly two-thirds of all methylcytosines occur in a non-CG context in GVOs. The distribution of non-CG methylation closely resembled that of CG methylation throughout the genome and showed clear enrichment in gene bodies. Compared to NGOs, GVOs were over four times more methylated at non-CG sites, indicating that non-CG methylation accumulates during oocyte growth. Lack of Dnmt3a or Dnmt3L resulted in a global reduction in both CG and non-CG methylation, showing that non-CG methylation depends on the Dnmt3a-Dnmt3L complex. Dnmt3b was dispensable. Of note, lack of Dnmt1 resulted in a slight decrease in CG methylation, suggesting that this maintenance enzyme plays a role in non-dividing oocytes. Dnmt1 may act on CG sites that remain hemimethylated in the de novo methylation process. Our results provide a basis for understanding the mechanisms and significance of non-CG methylation in mammalian oocytes.
Author Summary
Methylation of cytosine bases in DNA is an epigenetic modification crucial for normal development, retrotransposon silencing, and cellular reprogramming. In mammals, the vast majority of 5-methylcytosine occurs at CG dinucleotides, and thus most studies to date have focused on this dinucleotide. However, recent studies have shown that 5-methylcytosine is abundant at non-CG (CA, CT, and CC) sites in certain tissues and certain cell types in human and mouse. We previously identified non-CG methylation in CG-rich sequences, including the imprint control regions in mouse germinal vesicle oocytes, but its global distribution and the enzymes responsible are unknown. Using advanced high-throughput sequencing technology applicable to minute amounts of DNA, we obtained high-resolution methylation maps of newborn non-growing oocytes, adult germinal vesicle oocytes, and mutant germinal vesicle oocytes lacking any of the four DNA methyltransferase family proteins. Our results revealed that non-CG methylation accumulates genome-wide in close proximity to highly methylated CG sites during the oocyte growth stage. We also found that the de novo DNA methyltransferase proteins Dnmt3a and Dnmt3L are responsible for non-CG methylation in oocytes. Unexpectedly, we found that the maintenance methyltransferase Dnmt1 has a role in de novo CG methylation. Our study provides a basis for understanding the mechanisms and significance of non-CG methylation in mammalian oocytes.
Related Articles
Investigating the Potential Role of Genetic and Epigenetic Variation of DNA Methyltransferase Genes in Hyperplastic Polyposis Syndrome
Drini, Musa
Wong, Nicholas C.
Scott, Hamish S.
Craig, Jeffrey M.
Dobrovic, Alexander
Hewitt, Chelsee A.
Dow, Christofer
Young, Joanne P.
Jenkins, Mark A.
Saffery, Richard
Macrae, Finlay A.
PLoS ONE
Hyperplastic Polyposis Syndrome (HPS) is a condition associated with multiple serrated polyps, and an increased risk of colorectal cancer (CRC). At least half of CRCs arising in HPS show a CpG island methylator phenotype (CIMP), potentially linked to aberrant DNA methyltransferase (DNMT) activity. CIMP is associated with methylation of tumor suppressor genes including regulators of DNA mismatch repair (such as MLH1, MGMT), and negative regulators of Wnt signaling (such as WIF1). In this study, we investigated the potential for interaction of genetic and epigenetic variation in DNMT genes, in the aetiology of HPS.
We utilized high resolution melting (HRM) analysis to screen 45 cases with HPS for novel sequence variants in DNMT1, DNMT3A, DNMT3B, and DNMT3L. 21 polyps from 13 patients were screened for BRAF and KRAS mutations, with assessment of promoter methylation in the DNMT1, DNMT3A, DNMT3B, DNMT3L MLH1, MGMT, and WIF1 gene promoters.
No pathologic germline mutations were observed in any DNA-methyltransferase gene. However, the T allele of rs62106244 (intron 10 of DNMT1 gene) was over-represented in cases with HPS (p<0.01) compared with population controls. The DNMT1, DNMT3A and DNMT3B promoters were unmethylated in all instances. Interestingly, the DNMT3L promoter showed low levels of methylation in polyps and normal colonic mucosa relative to matched disease free cells with methylation level negatively correlated to expression level in normal colonic tissue. DNMT3L promoter hypomethylation was more often found in polyps harbouring KRAS mutations (p = 0.0053). BRAF mutations were common (11 out of 21 polyps), whilst KRAS mutations were identified in 4 of 21 polyps.
Genetic or epigenetic alterations in DNMT genes do not appear to be associated with HPS, but further investigation of genetic variation at rs62106244 is justified given the high frequency of the minor allele in this case series.
Related Articles
Tumor Suppressor Gene Inactivation during Cadmium-Induced Malignant Transformation of Human Prostate Cells Correlates with Overexpression of de Novo DNA Methyltransferase
Benbrahim-Tallaa, Lamia
Waterland, Robert A.
Dill, Anna L.
Webber, Mukta M.
Waalkes, Michael P.
Environmental Health Perspectives
Aberrant DNA methylation is common in carcinogenesis. The typical pattern appears to involve reduced expression of maintenance DNA methyltransferase, DNMT1, inducing genomic hypomethylation, whereas increased expression of de novo DNMT3a or 3b causes gene-specific hypermethylation.
During cadmium-induced malignant transformation, an unusual pattern of genomic hypermethylation occurred that we studied to provide insight into the roles of specific DNMTs in oncogenesis.
Gene expression and DNA methylation were assessed in control and chronic cadmium-transformed prostate epithelial cells (CTPE) using reverse transcription–polymerase chain reaction (RT-PCR), Western blot analysis, methylation-specific PCR, and methyl acceptance assay.
During the 10-weeks of cadmium exposure that induced malignant transformation, progressive increases in generalized DNMT enzymatic activity occurred that were associated with over-expression of DNMT3b without changes in DNMT1 expression. Increased DNMT3b expression preceded increased DNMT enzymatic activity. Procainamide, a specific DNMT1 inhibitor, reversed cadmium-induced genomic DNA hypermethylation. Reduced expression of the tumor suppressor genes, RASSF1A and p16, began about the time DNMT3b overexpression first occurred and progressively decreased thereafter. RASSF1A and p16 promoter regions were heavily methylated in CTPE cells, indicating silencing by hypermethylation, while the DNA demethylating agent, 5-aza-2′-deoxycytidine, reversed this silencing. DNMT1 inhibition only modestly increased RASSF1A and p16 expression in CTPE cells and did not completely reverse silencing.
These data indicate that DNMT3b overexpression can result in generalized DNA hypermethylation and gene silencing but that DNMT1 is required to maintain these effects. The pattern of genomic DNA hypermethylation together with up-regulation of DNMT3b may provide a unique set of biomarkers to specifically identify cadmium-induced human prostate cancers.
cadmium; carcinogenesis; DNA methylation; DNMT3b; p16; prostate; RASSF1A
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Clinicopathological Significance and Prognostic Value of DNA Methyltransferase 1, 3a, and 3b Expressions in Sporadic Epithelial Ovarian Cancer
Bai, Xuefeng
Song, Zhiguo
Fu, Yingzi
Yu, Zhaojin
Zhao, Lin
Zhao, Haishan
Yao, Weifan
Huang, Desheng
Mi, Xiaoyi
Wang, Enhua
Zheng, Zhihong
Wei, Minjie
PLoS ONE
Altered DNA methylation of tumor suppressor gene promoters plays a role in human carcinogenesis and DNA methyltransferases (DNMTs) are responsible for it. This study aimed to determine aberrant expression of DNMT1, DNMT3a, and DNMT3b in benign and malignant ovarian tumor tissues for their association with clinicopathological significance and prognostic value. A total of 142 ovarian cancers and 44 benign ovarian tumors were recruited for immunohistochemical analysis of their expression. The data showed that expression of DNMT1, DNMT3a, and DNMT3b was observed in 76 (53.5%), 92 (64.8%) and 79 (55.6%) of 142 cases of ovarian cancer tissues, respectively. Of the serious tumors, DNMT3a protein expression was significantly higher than that in benign tumor samples (P = 0.001); DNMT3b was marginally significant down regulated in ovarian cancers compared to that of the benign tumors (P = 0.054); DNMT1 expression has no statistical difference between ovarian cancers and benign tumor tissues (P = 0.837). Of the mucious tumors, the expression of DNMT3a, DNMT3b, and DNMT1 was not different between malignant and benign tumors. Moreover, DNMT1 expression was associated with DNMT3b expression (P = 0.020, r = 0.195). DNMT1 expression was associated with age of the patients, menopause status, and tumor localization, while DNMT3a expression was associated with histological types and serum CA125 levels and DNMT3b expression was associated with lymph node metastasis. In addition, patients with DNMT1 or DNMT3b expression had a trend of better survival than those with negative expression. Co-expression of DNMT1 and DNMT3b was significantly associated with better overall survival (P = 0.014). The data from this study provided the first evidence for differential expression of DNMTs proteins in ovarian cancer tissues and their associations with clinicopathological and survival data in sporadic ovarian cancer patients.
Related Articles
Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation*
Estève, Pierre-Olivier
Terragni, Jolyon
Deepti, Kanneganti
Chin, Hang Gyeong
Dai, Nan
Espejo, Alexsandra
Corrêa, Ivan R.
Bedford, Mark T.
Pradhan, Sriharsa
The Journal of Biological Chemistry
Background: SET7 monomethylates DNMT1, promoting its proteasomal degradation, yet methylated DNMT1 still remains throughout the cell cycle.
Results: The methyllysine reader PHF20L1 stabilizes methylated DNMT1. Disruption of PHF20L1 induces DNMT1 degradation and genome hypomethylation.
Conclusion: PHF20L1, an epigenetic reader, cooperates with writer and eraser to regulate epigenetic inheritance.
Significance: PHF20L1 can be targeted as a means of regulating DNMT1 activity and DNA methylation in cells.
Inheritance of DNA cytosine methylation pattern during successive cell division is mediated by maintenance DNA (cytosine-5) methyltransferase 1 (DNMT1). Lysine 142 of DNMT1 is methylated by the SET domain containing lysine methyltransferase 7 (SET7), leading to its degradation by proteasome. Here we show that PHD finger protein 20-like 1 (PHF20L1) regulates DNMT1 turnover in mammalian cells. Malignant brain tumor (MBT) domain of PHF20L1 binds to monomethylated lysine 142 on DNMT1 (DNMT1K142me1) and colocalizes at the perinucleolar space in a SET7-dependent manner. PHF20L1 knockdown by siRNA resulted in decreased amounts of DNMT1 on chromatin. Ubiquitination of DNMT1K142me1 was abolished by overexpression of PHF20L1, suggesting that its binding may block proteasomal degradation of DNMT1K142me1. Conversely, siRNA-mediated knockdown of PHF20L1 or incubation of a small molecule MBT domain binding inhibitor in cultured cells accelerated the proteasomal degradation of DNMT1. These results demonstrate that the MBT domain of PHF20L1 reads and controls enzyme levels of methylated DNMT1 in cells, thus representing a novel antagonist of DNMT1 degradation.
DNA-binding Protein; DNA Methylation; DNA Methyltransferase; Protein Degradation; Protein Methylation; MBT Domain; PHF20L1; SET7; UNC1215
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Nucleosomes Containing Methylated DNA Stabilize DNA Methyltransferases 3A/3B and Ensure Faithful Epigenetic Inheritance
Sharma, Shikhar
De Carvalho, Daniel D.
Jeong, Shinwu
Jones, Peter A.
Liang, Gangning
PLoS Genetics
How epigenetic information is propagated during somatic cell divisions is still unclear but is absolutely critical for preserving gene expression patterns and cellular identity. Here we show an unanticipated mechanism for inheritance of DNA methylation patterns where the epigenetic mark not only recruits the catalyzing enzyme but also regulates the protein level, i.e. the enzymatic product (5-methylcytosine) determines the level of the methylase, thus forming a novel homeostatic inheritance system. Nucleosomes containing methylated DNA stabilize de novo DNA methyltransferases, DNMT3A/3B, allowing little free DNMT3A/3B enzymes to exist in the nucleus. Stabilization of DNMT3A/3B on nucleosomes in methylated regions further promotes propagation of DNA methylation. However, reduction of cellular DNA methylation levels creating more potential CpG substrates counter-intuitively results in a dramatic decrease of DNMT3A/3B proteins due to diminished nucleosome binding and subsequent degradation of the unstable free proteins. These data show an unexpected self-regulatory inheritance mechanism that not only ensures somatic propagation of methylated states by DNMT1 and DNMT3A/3B enzymes but also prevents aberrant de novo methylation by causing degradation of free DNMT3A/3B enzymes.
Author Summary
Proper inheritance of DNA methylation patterns is essential for preserving cellular identity and preventing malignant cellular transformation. In mammals, DNMT3A/3B, the de novo methyltransferases, establish the DNA methylation patterns during development and then maintain them in co-operation with the maintenance methyltransferase, DNMT1, through cell divisions. However, the mechanisms by which DNMT3A/3B assist DNMT1 in faithful inheritance of methylation patterns in somatic cells while guarding against aberrant de novo DNA methylation are still unclear. In this study, we present a novel principle of enzyme regulation where the levels of the catalyzing enzymes, DNMT3A/3B, are determined by the level of their own enzymatic product, i.e. 5-methylcytosine itself. Through biochemical analyses, we have shown that binding of DNMT3A/3B to nucleosomes with methylated DNA stabilizes these proteins, enabling faithful propagation of methylation patterns through cell divisions. However, reduction in DNA methylation results in diminished nucleosome binding of DNMT3A/3B and subsequent degradation of the free DNMT3A/3B proteins. This novel self-regulatory inheritance mechanism not only ensures faithful somatic propagation of methylated states but also prevents aberrant de novo methylation by causing degradation of free DNMT3A/3B enzymes.
Related Articles
A novel DNMT3B splice variant expressed in tumor and pluripotent cells modulates genomic DNA methylation patterns and displays altered DNA binding
Gopalakrishnan, Suhasni
Van Emburgh, Beth O.
Shan, Jixiu
Su, Zhen
Fields, C. Robert
Vieweg, Johannes
Hamazaki, Takashi
Schwartz, Philip H.
Terada, Naohiro
Robertson, Keith D.
Molecular cancer research : MCR
DNA methylation is an epigenetic mark essential for mammalian development, genomic stability, and imprinting. DNA methylation patterns are established and maintained by three DNA methyltransferases: DNMT1, DNMT3A, and DNMT3B. Interestingly, all three DNMTs make use of alternative splicing. DNMT3B has nearly 40 known splice variants expressed in a tissue- and disease-specific manner, but very little is known about the role of these splice variants in modulating DNMT3B function. We describe here the identification and characterization of a novel alternatively spliced form of DNMT3B lacking exon 5 within the N-terminal regulatory domain. This variant, which we term DNMT3B3Δ5 because it is closely related in structure to the ubiquitously expressed DNMT3B3 isoform, is highly expressed in pluripotent cells and brain tissue, is downregulated during differentiation, and is conserved in the mouse. Creation of pluripotent iPS cells from fibroblasts results in marked induction of DNMT3B3Δ5. DNMT3B3Δ5 expression is also altered in human disease, with tumor cell lines displaying elevated or reduced expression depending on their tissue of origin. We then compared the DNA binding and subcellular localization of DNMT3B3Δ5 versus DNMT3B3, revealing that DNMT3B3Δ5 possessed significantly enhanced DNA binding affinity and displayed an altered nuclear distribution. Finally, ectopic overexpression of DNMT3B3Δ5 resulted in repetitive element hypomethylation and enhanced cell growth in a colony formation assay. Taken together, these results demonstrate that DNMT3B3Δ5 may play an important role in stem cell maintenance or differentiation and suggest that sequences encoded by exon 5 influence the functional properties of DNMT3B.
Related Articles
Essential Role for Dnmt1 in the Prevention and Maintenance of MYC-Induced T-Cell Lymphomas
Peters, Staci L.
Hlady, Ryan A.
Opavska, Jana
Klinkebiel, David
Novakova, Slavomira
Smith, Lynette M.
Lewis, Robert E.
Karpf, Adam R.
Simpson, Melanie A.
Wu, Lizhao
Opavsky, Rene
Molecular and Cellular Biology
DNA cytosine methylation is an epigenetic modification involved in the transcriptional repression of genes controlling a variety of physiological processes, including hematopoiesis. DNA methyltransferase 1 (Dnmt1) is a key enzyme involved in the somatic inheritance of DNA methylation and thus plays a critical role in epigenomic stability. Aberrant methylation contributes to the pathogenesis of human cancer and of hematologic malignancies in particular. To gain deeper insight into the function of Dnmt1 in lymphoid malignancies, we genetically inactivated Dnmt1 in a mouse model of MYC-induced T-cell lymphomagenesis. We show that loss of Dnmt1 delays lymphomagenesis by suppressing normal hematopoiesis and impairing tumor cell proliferation. Acute inactivation of Dnmt1 in primary lymphoma cells rapidly induced apoptosis, indicating that Dnmt1 is required to sustain T-cell lymphomas. Using high-resolution genome-wide profiling, we identified differentially methylated regions between control and Dnmt1-deficient lymphomas, demonstrating a locus-specific function for Dnmt1 in both maintenance and de novo promoter methylation. Dnmt1 activity is independent of the presence of Dnmt3a or Dnmt3b in de novo promoter methylation of the H2-Ab1 gene. Collectively, these data show for the first time that Dnmt1 is critical for the prevention and maintenance of T-cell lymphomas and contributes to aberrant methylation by both de novo and maintenance methylation.
Related Articles
FOXM1 Induces a Global Methylation Signature That Mimics the Cancer Epigenome in Head and Neck Squamous Cell Carcinoma
Teh, Muy-Teck
Gemenetzidis, Emilios
Patel, Deeviyaben
Tariq, Rameez
Nadir, Ayesha
Bahta, Adiam W.
Waseem, Ahmad
Hutchison, Iain L.
PLoS ONE
The oncogene FOXM1 has been implicated in all major types of human cancer. We recently showed that aberrant FOXM1 expression causes stem cell compartment expansion resulting in the initiation of hyperplasia. We have previously shown that FOXM1 regulates HELLS, a SNF2/helicase involved in DNA methylation, implicating FOXM1 in epigenetic regulation. Here, we have demonstrated using primary normal human oral keratinocytes (NOK) that upregulation of FOXM1 suppressed the tumour suppressor gene p16INK4A (CDKN2A) through promoter hypermethylation. Knockdown of HELLS using siRNA re-activated the mRNA expression of p16INK4A and concomitant downregulation of two DNA methyltransferases DNMT1 and DNMT3B. The dose-dependent upregulation of endogenous FOXM1 (isoform B) expression during tumour progression across a panel of normal primary NOK strains (n = 8), dysplasias (n = 5) and head and neck squamous cell carcinoma (HNSCC) cell lines (n = 11) correlated positively with endogenous expressions of HELLS, BMI1, DNMT1 and DNMT3B and negatively with p16INK4A and involucrin. Bisulfite modification and methylation-specific promoter analysis using absolute quantitative PCR (MS-qPCR) showed that upregulation of FOXM1 significantly induced p16INK4A promoter hypermethylation (10-fold, P<0.05) in primary NOK cells. Using a non-bias genome-wide promoter methylation microarray profiling method, we revealed that aberrant FOXM1 expression in primary NOK induced a global hypomethylation pattern similar to that found in an HNSCC (SCC15) cell line. Following validation experiments using absolute qPCR, we have identified a set of differentially methylated genes, found to be inversely correlated with in vivo mRNA expression levels of clinical HNSCC tumour biopsy samples. This study provided the first evidence, using primary normal human cells and tumour tissues, that aberrant upregulation of FOXM1 orchestrated a DNA methylation signature that mimics the cancer methylome landscape, from which we have identified a unique FOXM1-induced epigenetic signature which may have clinical translational potentials as biomarkers for early cancer screening, diagnostic and/or therapeutic interventions.
Related Articles
Haploinsufficiency of the paternal-effect gene Dnmt3L results in transient DNA hypomethylation in progenitor cells of the male germline
Niles, K.M.
Yeh, J.R.
Chan, D.
Landry, M.
Nagano, M.C.
Trasler, J.M.
Human Reproduction (Oxford, England)
How does haploinsufficiency of the paternal-effect gene Dnmt3L affect DNA methylation establishment and stability in the male germline?
Reduced expression of DNMT3L in male germ cells, associated with haploinsufficiency of the paternal-effect gene Dnmt3L, results in abnormal hypomethylation of prenatal germline progenitor cells.
The DNA methyltransferase regulator Dnmt3-Like (Dnmt3L) is a paternal-effect gene required for DNA methylation acquisition in male germline stem cells and their precursors. In males, DNMT3L deficiency causes meiotic abnormalities and infertility. While Dnmt3L heterozygous males are fertile, they have abnormalities in X chromosome compaction and postmeiotic gene expression and sire offspring with sex chromosome aneuploidy. It has been proposed that the paternal effects of Dnmt3L haploinsufficiency are due to epigenetic defects in early male germ cells. DNA methylation is an essential epigenetic modification essential for normal germ cell development. Since patterns of DNA methylation across the genome are initially acquired in prenatal male germ cells, perturbations in methylation could contribute to the epigenetic basis of the paternal effects in Dnmt3L+/− males.
This is a cross-sectional study of DNA methylation in Dnmt3L+/+ versus Dnmt3L+/− male germ cells collected from mice at 16.5 days post-coitum (dpc), Day 6 and Day 70 (n = 3 per genotype, each n represents a pool of 2–20 animals). Additionally, DNA methylation was compared in enriched populations of spermatogonial stem cells (SSC)/progenitor cells from Dnmt3L+/+ and Dnmt3L+/− males following ∼2 months in culture.
DNA methylation at intergenic loci along chromosomes 9 and X was examined by quantitative analysis of DNA methylation by real-time polymerase chain reaction at the time of initial acquisition of epigenetic patterns in the prenatal male germline (16.5 dpc) and compared with patterns in early post-natal spermatogonia (Day 6) and in spermatozoa in mice. DNA methylation status at CpG-rich sites across the genome was assessed in spermatogonial precursors from Day 4 male mice using restriction landmark genomic scanning.
At 16.5 dpc, 42% of intergenic loci examined along chromosome 9 and 10% of those along chromosome X were hypomethylated in Dnmt3L heterozygotes. By Day 6 and in spermatozoa, germ cell DNA methylation was similar in heterozygous and wild-type mice. DNA methylation stability of acquired patterns in wild-type and Dnmt3L+/− SSC/progenitor cell culture was analyzed at numerous loci across the genome in cells cultured in vitro and collected at passages 6–28. While the methylation of most loci was stable in culture over time, differences at ∼1% of sites were found between Dnmt3L+/− and Dnmt3L+/+ cultures.
Evaluation of DNA methylation in SSCs can only be performed after a period of culture limiting the investigation to changes observed during culture when compared with DNA methylation differences between genotypes that could be present at the beginning of culture establishment.
The DNA methylation defects described here in prenatal male germline progenitor cells and SSC culture are the earliest epigenetic perturbations yet identified for a mammalian paternal-effect gene and may influence downstream epigenetic events in germ cells at later stages of development. Together, the results provide evidence of a 'window' of susceptibility in prenatal male germ cell precursors for the induction of epimutations due to genetic perturbations and, potentially, in utero environmental exposures.
Canadian Institutes of Health Research (CIHR) provided funding for J.M.T. (MOP229913) and M.C.N. (MOP86532). The authors have no conflicts of interest to declare.
epigenetics; DNA methylation; Dnmt3L; spermatogonial stem cells; paternal effect
Related Articles
The De Novo Methyltransferases DNMT3a and DNMT3b Target the Murine Gammaherpesvirus Immediate-Early Gene 50 Promoter during Establishment of Latency▿
Gray, Kathleen S.
Forrest, J. Craig
Speck, Samuel H.
Journal of Virology
The role of epigenetic modifications in the regulation of gammaherpesvirus latency has been a subject of active study for more than 20 years. DNA methylation, associated with transcriptional silencing in mammalian genomes, has been shown to be an important mechanism in the transcriptional control of several key gammaherpesvirus genes. In particular, DNA methylation of the functionally conserved immediate-early replication and transcription activator (RTA) has been shown to regulate Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus Rta expression. Here we demonstrate that the murine gammaherpesvirus (MHV68) homolog, encoded by gene 50, is also subject to direct repression by DNA methylation, both in vitro and in vivo. We observed that the treatment of latently MHV68-infected B-cell lines with a methyltransferase inhibitor induced virus reactivation. In addition, we show that the methylation of the recently characterized distal gene 50 promoter represses activity in a murine macrophage cell line. To evaluate the role of de novo methyltransferases (DNMTs) in the establishment of these methylation marks, we infected mice in which conditional DNMT3a and DNMT3b alleles were selectively deleted in B lymphocytes. DNMT3a/DNMT3b-deficient B cells were phenotypically normal, displaying no obvious compromise in cell surface marker expression or antibody production either in naïve mice or in the context of nonviral and viral immunogens. However, mice lacking functional DNMT3a and DNMT3b in B cells exhibited hallmarks of deregulated MHV68 lytic replication, including increased splenomegaly and the presence of infectious virus in the spleen at day 18 following infection. In addition, total gene 50 transcript levels were elevated in the spleens of these mice at day 18, which correlated with the hypomethylation of the distal gene 50 promoter. However, by day 42 postinfection, aberrant virus replication was resolved, and we observed wild-type frequencies of viral genome-positive splenocytes in mice lacking functional DNMT3a and DNMT3b in B lymphocytes. The latter correlated with increased CpG methylation in the distal gene 50 promoter, which was restored to levels similar to those of littermate controls harboring functional DNMT3a and DNMT3b alleles in B lymphocytes, suggesting the existence of an alternative mechanism for the de novo methylation of the MHV68 genome. Importantly, this DNMT3a/DNMT3b-independent methylation appeared to be targeted specifically to the gene 50 promoter, as we observed that the promoters for MHV68 gene 72 (v-cyclin) and M11 (v-bcl2) remained hypomethylated at day 42 postinfection. Taken together, these data provide the first evidence of the importance of DNA methylation in regulating gammaherpesvirus RTA/gene 50 transcription during virus infection in vivo and provide insight into the hierarchy of host machinery required to establish this modification.
Related Articles
Low-Level Environmental Cadmium Exposure Is Associated with DNA Hypomethylation in Argentinean Women
Hossain, Mohammad Bakhtiar
Vahter, Marie
Concha, Gabriela
Broberg, Karin
Environmental Health Perspectives
Background: Cadmium, a common food pollutant, alters DNA methylation in vitro. Epigenetic effects might therefore partly explain cadmium's toxicity, including its carcinogenicity; however, human data on epigenetic effects are lacking.
Objective: We evaluated the effects of dietary cadmium exposure on DNA methylation, considering other environmental exposures, genetic predisposition, and gene expression.
Methods: Concentrations of cadmium, arsenic, selenium, and zinc in blood and urine of nonsmoking women (n = 202) from the northern Argentinean Andes were measured by inductively coupled mass spectrometry. Methylation in CpG islands of LINE-1 (long interspersed nuclear element-1; a proxy for global DNA methylation) and promoter regions of p16 [cyclin-dependent kinase inhibitor 2A (CDKN2A)] and MLH1 (mutL homolog 1) in peripheral blood were measured by bisulfite polymerase chain reaction pyrosequencing. Genotyping (n = 172) for the DNA (cytosine-5-)-methyltransferase 1 gene (DNMT1 rs10854076 and rs2228611) and DNA (cytosine-5-)-methyltransferase 3 beta gene (DNMT3B rs2424913 and rs2424932) was performed with Sequenom iPLEX GOLD SNP genotyping; and gene expression (n = 90), with DirectHyb HumanHT-12 (version 3.0).
Results: Cadmium exposure was low: median concentrations in blood and urine were 0.36 and 0.23 µg/L, respectively. Urinary cadmium (natural log transformed) was inversely associated with LINE-1 methylation (β = –0.50, p = 0.0070; β = –0.44, p = 0.026, adjusted for age and coca chewing) but not with p16 or MLH1 methylation. Both DNMT1 rs10854076 and DNMT1 rs2228611 polymorphisms modified associations between urinary cadmium and LINE-1 (p-values for interaction in adjusted models were 0.045 and 0.064, respectively). The rare genotypes demonstrated stronger hypomethylation with increasing urinary cadmium concentrations. Cadmium was inversely associated with DNMT3B (rS = –0.28, p = 0.0086) but not with DNMT1 expression (rS = –0.075, p = 0.48).
Conclusion: Environmental cadmium exposure was associated with DNA hypomethylation in peripheral blood, and DNMT1 genotypes modified this association. The role of epigenetic modifications in cadmium-associated diseases needs clarification.
cadmium; DNMT1; DNMT3B; epigenetic; genotype; LINE-1; MLH1; p16; pyrosequencing
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Deregulation between miR-29b/c and DNMT3A Is Associated with Epigenetic Silencing of the CDH1 Gene, Affecting Cell Migration and Invasion in Gastric Cancer
Cui, He
Wang, Ling
Gong, Pihai
Zhao, Chengcheng
Zhang, Shaodan
Zhang, Kun
Zhou, Rongping
Zhao, Zhujiang
Fan, Hong
PLoS ONE
The de-regulation of the miR-29 family and DNA methyltransferase 3A (DNMT3A) is associated with gastric cancer (GC). While increasing evidence indicates miR-29b/c could regulate DNA methylation by targeting DNMT3A, it is currently unknown if epigenetic silencing of miR-29b/c via promoter hypermethylation in GC is caused by abnormal expression of DNMT3A. Thus, we aimed to evaluate whether cross-talk regulation exists between miR-29b/c and DNMT3A and whether it is associated with a malignant phenotype in GC. First, wound healing and Transwell assays revealed that miR-29b/c suppresses tumor metastasis in GC. A luciferase reporter assay demonstrated that DNMT3A is a direct target of miR-29b/c. We used bisulfite genomic sequencing to analyze the DNA methylation status of miR-29b/c. The percentage of methylated CpGs was significantly decreased in DNMT3A-depleted cells compared to the controls. Furthermore, the involvement of DNMT3A in promoting GC cell migration was associated with the promoter methylation-mediated repression of CDH1. In 50 paired clinical GC tissue specimens, decreased miR-29b/c was significantly correlated with the degree of differentiation and invasion of the cells and was negatively correlated with DNMT3A expression. Together, our preliminary results suggest that the following process may be involved in GC tumorigenesis. miR-29b/c suppresses the downstream gene DNMT3A, and in turn, miR-29b/c is suppressed by DNMT3A in a DNA methylation-dependent manner. The de-regulation of both of miR-29b/c and DNMT3A leads to the epigenetic silencing of CDH1 and contributes to the metastasis phenotype in GC. This finding reveals that DNA methylation-associated silencing of miR-29b/c is critical for GC development and thus may be a therapeutic target.
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Transition of LINE-1 DNA Methylation Status and Altered Expression in First and Third Trimester Placentas
He, Zhi-ming
Li, Jinping
Hwa, Yi Lisa
Brost, Brian
Fang, Qun
Jiang, Shi-Wen
PLoS ONE
DNA methylation plays a critical role in the regulation of gene expression, genomic DNA stability, cell proliferation, and malignant transformation. Common cellular features including fast tissue expansion, invasive growth, and active angiogenesis, have been noticed between placental development and tumorigenesis by many investigators. While the DNA hypomethylation and transcriptional activation of LINE-1 has been found to be a feature of tumorigenesis, it is not clear if similar changes could be involved in placental development. In this study, we assessed LINE-1 methylation in human placentas from different gestational ages and observed a significant decrease of LINE-1 methylation levels in third trimester placentas compared to first trimester placentas. Accompanying with this change is the significantly increased LINE-1 mRNA levels in third trimester placentas. Since no global DNA methylation change was detected between first and third trimesters, LINE-1 methylation changes appeared to be a specific epigenetic entity contributing to placental development. Indeed, further analyses showed that LINE-1 upregulation was correlated with higher levels of PCNA, suggesting a link between LINE-1 activation and fast proliferation of certain cellular components in third trimester placentas. Measurement of the DNMT1, DNMT3A, and DNMT3B expression found a significant reduction of DNMT3B between third and first trimesters, pointing to the possible involvement of this enzyme in the regulation of LINE-1 methylation. Taken together these results provided evidence for a dynamic temporal regulation of LINE-1 methylation and activation during placental development. These studies have laid a foundation for future investigation on the function of LINE-1 expression in human placenta under different patho-physiological conditions.
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Distinct roles of DNMT1-dependent and DNMT1-independent methylation patterns in the genome of mouse embryonic stem cells
Li, Zhiguang
Dai, Hongzheng
Martos, Suzanne N.
Xu, Beisi
Gao, Yang
Li, Teng
Zhu, Guangjing
Schones, Dustin E.
Wang, Zhibin
Genome Biology
DNA methylation patterns are initiated by de novo DNA methyltransferases DNMT3a/3b adding methyl groups to CG dinucleotides in the hypomethylated genome of early embryos. These patterns are faithfully maintained by DNMT1 during DNA replication to ensure epigenetic inheritance across generations. However, this two-step model is based on limited data.
We generated base-resolution DNA methylomes for a series of DNMT knockout embryonic stem cells, with deep coverage at highly repetitive elements. We show that DNMT1 and DNMT3a/3b activities work complementarily and simultaneously to establish symmetric CG methylation and CHH (H = A, T or C) methylation. DNMT3a/3b can add methyl groups to daughter strands after each cycle of DNA replication. We also observe an unexpected division of labor between DNMT1 and DNMT3a/3b in suppressing retrotransposon long terminal repeats and long interspersed elements, respectively. Our data suggest that mammalian cells use a specific CG density threshold to predetermine methylation levels in wild-type cells and the magnitude of methylation reduction in DNMT knockout cells. Only genes with low CG density can be induced or, surprisingly, suppressed in the hypomethylated genome. Lastly, we do not find any association between gene body methylation and transcriptional activity.
We show the concerted actions of DNMT enzymes in the establishment and maintenance of methylation patterns. The finding of distinct roles of DNMT1-dependent and -independent methylation patterns in genome stability and regulation of transcription provides new insights for understanding germ cell development, neuronal diversity, and transgenerational epigenetic inheritance and will help to develop next-generation DNMT inhibitors.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-015-0685-2) contains supplementary material, which is available to authorized users.
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Mouse ES cells overexpressing DNMT1 produce abnormal neurons with upregulated NR1
D'Aiuto, Leonardo
Di Maio, Roberto
Mohan, K. Naga
Minervini, Crescenzio
Saporiti, Federica
Soreca, Isabella
Greenamyre, J. Timothy
Chaillet, J. Richard
Differentiation; research in biological diversity
High levels of DNA methyltransferase 1 (DNMT1), hypermethylation, and downregulation of GAD67 and reelin have been described in GABAergic interneurons of patients with schizophrenia (SZ) and bipolar (BP) disorders. However, overexpression of DNMT1 is lethal, making it difficult to assess the direct effect of high levels of DNMT1 on neuronal development in vivo. We therefore used Dnmt1tet/tet mouse ES cells that overexpress DNMT1 as an in vitro model to investigate the impact of high levels of DNMT1 on neuronal differentiation. Although there is down-regulation of DNMT1 during early stages of differentiation in wild type and Dnmt1tet/tet ES cell lines, neurons derived from Dnmt1tet/tet cells showed abnormal dendritic arborization and branching. The Dnmt1tet/tet neuronal cells also showed elevated levels of functional N-methyl D-aspartate receptor (NMDAR), a feature also reported in some neurological and neurodegenerative disorders. Considering the roles of reelin and GAD67 in neuronal networking and excitatory/inhibitory balance respectively, we studied methylation of these genes' promoters in Dnmt1tet/tet ES cells and neurons. Both reelin and GAD67 promoters were not hypermethylated in the Dnmt1tet/tet ES cells and neurons, suggesting that overexpression of DNMT1 may not directly result in methylation-mediated repression of these two genes. Taken together, our results suggest that overexpression of DNMT1 in ES cells results in an epigenetic change prior to the onset of differentiation. This epigenetic change in turn results in abnormal neuronal differentiation and upregulation of functional NMDA receptor.
DNA methylation; Dnmt1; neuronal differentiation; NMDA receptor; epigenetic
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Expression of DNMT1 and DNMT3a Are Regulated by GLI1 in Human Pancreatic Cancer
He, ShanShan
Wang, Feng
Yang, LiJuan
Guo, ChuanYong
Wan, Rong
Ke, AiWu
Xu, Ling
Hu, GuoYong
Xu, XuanFu
Shen, Jie
Wang, XingPeng
PLoS ONE
Background and Aims
GLI1, as an indispensable transcriptional factor of Hedgehog signaling pathway, plays an important role in the development of pancreatic cancer (PC). DNA methyltransferases (DNMTs) mediate the methylation of quantity of tumor-related genes. Our study aimed to explore the relationship between GLI1 and DNMTs.
Expressions of GLI1 and DNMTs were detected in tumor and adjacent normal tissues of PC patients by immunohistochemistry (IHC). PANC-1 cells were treated by cyclopamine and GLI1-siRNA, while BxPC-3 cells were transfected with overexpression-GLI1 lentiviral vector. Then GLI1 and DNMTs expression were analyzed by qRT-PCR and western blot (WB). Then we took chromatin immunoprecipitation (ChIP) to demonstrate GLI1 bind to DNMT1. Finally, nested MSP was taken to valuate the methylation levels of APC and hMLH1, when GLI1 expression altered.
IHC result suggested the expressions of GLI1, DNMT1 and DNMT3a in PC tissues were all higher than those in adjacent normal tissues (p<0.05). After GLI1 expression repressed by cyclopamine in mRNA and protein level (down-regulation 88.1±2.2%, 86.4±2.2%, respectively), DNMT1 and DNMT3a mRNA and protein level decreased by 91.6%±2.2% and 83.8±4.8%, 87.4±2.7% and 84.4±1.3%, respectively. When further knocked down the expression of GLI1 by siRNA (mRNA decreased by 88.6±2.1%, protein decreased by 63.5±4.5%), DNMT1 and DNMT3a mRNA decreased by 80.9±2.3% and 78.6±3.8% and protein decreased by 64.8±2.8% and 67.5±5.6%, respectively. Over-expression of GLI1 by GLI1 gene transfection (mRNA increased by 655.5±85.9%, and protein increased by 272.3±14.4%.), DNMT1 and DNMT3a mRNA and protein increased by 293.0±14.8% and 578.3±58.5%, 143.5±17.4% and 214.0±18.9%, respectively. ChIP assays showed GLI1 protein bound to DNMT1 but not to DNMT3a. Results of nested MSP demonstrated GLI1 expression affected the DNA methylation level of APC but not hMLH1 in PC.
DNMT1 and DNMT3a are regulated by GLI1 in PC, and DNMT1 is its direct target gene.
Related Articles
DNMT1 but not its interaction with the replication machinery is required for maintenance of DNA methylation in human cells
Spada, Fabio
Haemmer, Andrea
Kuch, David
Rothbauer, Ulrich
Schermelleh, Lothar
Kremmer, Elisabeth
Carell, Thomas
Längst, Gernot
Leonhardt, Heinrich
The Journal of Cell Biology
DNA methylation plays a central role in the epigenetic regulation of gene expression in vertebrates. Genetic and biochemical data indicated that DNA methyltransferase 1 (Dnmt1) is indispensable for the maintenance of DNA methylation patterns in mice, but targeting of the DNMT1 locus in human HCT116 tumor cells had only minor effects on genomic methylation and cell viability. In this study, we identified an alternative splicing in these cells that bypasses the disrupting selective marker and results in a catalytically active DNMT1 protein lacking the proliferating cell nuclear antigen–binding domain required for association with the replication machinery. Using a mechanism-based trapping assay, we show that this truncated DNMT1 protein displays only twofold reduced postreplicative DNA methylation maintenance activity in vivo. RNA interference–mediated knockdown of this truncated DNMT1 results in global genomic hypomethylation and cell death. These results indicate that DNMT1 is essential in mouse and human cells, but direct coupling of the replication of genetic and epigenetic information is not strictly required.
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text:
Elegant Pins!
These Silver Queen Pins can be used with ribbon sashes or just as pins. Each 4 inch pin says Queen and features a metal clasp. Present your new Prom or Homecoming Queen with an elegant Silver Queen Pin!
Please allow ample time for delivery. Our ship date for this product is noted in your shopping cart. Many items are shipped separately, so please allow extra time for these items to arrive. Shipping charges are based on the value of the merchandise and not the number of shipments. For additional shipping information, contact our Customer Service Department at (firstname.lastname@example.com or 1-800-227-2789).
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subset: commoncrawl


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text:
when you sit in a car and smoke weed
yo, lets have a calm bake
dodane przez ryan luty 06, 2004
A group of women with shaved beavers sunbathing in the buff. The aim is to leave no tan lines. Very common on European nude beaches.
A lot of voyeur sites just feature clambake beach pics.
dodane przez wolfbait51 styczeń 28, 2011
See Dutch Oven and Queef. Similar to the Dutch oven however it can only be done by a woman. The woman queefs under the covers and pulls them over her bedmate's head to keep them trapped in the stink.
Did you see my youtube video of me doing a crazy Clam Bake my boyfriend? For some reason he is no longer returning my phone calls....
dodane przez Mr. Jinchao wrzesień 20, 2010
A setting or event where the number of woman is overly concentrated resulting in general catty or bitchy behavior. The exact opposite of a Sausage Festival.
They need to hire some dudes on the sales team; it's a clam bake over there with all that gossiping.
dodane przez fair_claire kwiecień 27, 2010
A party at which females greatly outnumber males.
You go to a house party but it's filled with women who just went to a baby shower together, you are one of a handful of men so it's a "clambake".
dodane przez Oliver L. the wordsmith lipiec 16, 2013
When a woman queefs in bed, then pulls the blanket over her man's face.
female version of a Dutch oven. Guys, don't give your woman a Dutch Oven. She might give you a clam bake.
dodane przez Leatherneck65 październik 08, 2012
A Clam Bake is when you are at a party where there are more girls then there are boys. It is a "sausage fest," but with girls.
"Dude, this party is a clam bake."
dodane przez MillyB sierpień 19, 2011


text: Andrea 587pages on this wiki Edit History Talk0 Andrea Andrea is a character in Cars. Cars Andrea is a great reporter! Hailing from Germany, Andrea studied journalism at one of the most prestigious schools in the country and speaks over three different languages. She is known for her signature phrase, "We have the right to know what's going on around us, 85 miles per hour, 7 days a week!" And she can say it in all three languages. Retrieved from "http://worldofcars.wikia.com/wiki/Andrea?oldid=16898" Categories: Cars Characters Characters Movie Characters


text:
Sharing the hurt
SO CLOSE: Shiloh McVay consoles daughter, Destiny McVay, after Destiny barely missed a chance to compete at the state cross country meet in Kearney on Friday. Destiny crossed the line in 16th place during the C-4 district competition at Ogallala on Thursday. The top 15 individuals in each class, in addition to the top three teams, qualify for state. Shiloh competed on two Swede teams that made it to state in 1994 and 1995. See Sports for the details and more photos.
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TV4 Sport träffade "The Mauler" efter att det blivit klart att han ska gå en titelmatch mot Daniel Cormier. Hör vad Gustafsson ska göra för att prestera toppklass i buren. Swedish MMA gives you the watch video >>
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Sean O'Connell explains weigh-ins prank, slow start in victory over Matt Van Buren watch video >>
Matt "Gutter" Van Buren and Chris Fields call each other out during some down time in the house. watch video >>
The Ultimate Fighter 19 elimination fight between Daniel Vizcaya and Matt Van Buren, uncut and uninterrupted. watch video >>
Matt Van Buren and Team Edgar coaches go over strategy before his prelim bout against Chris Fields. watch video >>
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The Northwest Texas Healthcare Auxiliary will sponsor the Texas Association Hospital Auxiliaries District Meeting 1-A and 1-B on Friday at the Ambassador Hotel.
Nancy Hays, state president of the hospital auxiliaries association, will talk at the meeting, which will begin at 8:30 a.m. and conclude about 2:30 p.m.
Hays received a physical education degree and taught school for several years. She has been an active volunteer in church, school, community and hospital organizations.
Hays became a hospital auxiliary member while attending high school. In 1991, she became a member of the Texas Association of Hospital Auxiliaries Board of Directors.
TAHA was organized for charitable and educational purposes. The association sponsors scholarships for students in the field of health care. The TAHA District meeting 1-A and 1-B includes 19 hospitals in the Panhandle area.
For more information, call Marilyn Riecken, volunteer service coordinator for NWTH auxiliary, at 354-1722.
© 2016. All Rights Reserved. | Contact Us


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2012 BMW 5 Series Sedan 550i 4dr Sedan (4.4L 8cyl Turbo 6M)
If your looking for good gas mileage the automatic 550i 2012 is not your car! But if you want a mustang/ vette killer this is your car! The torque and speed is hair-raising! I timed my 0-60 @ 4.4 . The 550i paddle shifters launches you out the box so fast that no crappy rice burner or Mustang 5.0 will ever recover. I havent loss a street race yet. ( lol) The fit and finish is first class and the seat position feels custom fitted. You will never find a a better combo of performance and luxury for under 35k.
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I have a confession to make: I found Bleacher Report due to the CBSSports.com correspondent application process.
So there, I've said it.
According to the "rules", journalism experience was preferred, not required.
What I did have was a wealth of experience in and around professional sports and the entertainment industry.
My Saints have opened so many doors during my life, how could I not jump at the chance to cover them for CBSSports via Bleacher report?
Sure, I'll have to come up with five articles, but there is a list of compulsory topics. That I can do, I have been around football all my life. Truth be told, the New Orleans Saints, in the '70s were my second favorite team.
My dad's semi-pro team, the Blue Knights, was my first.
Yes, I knew I was going into this with a bit of a handicap, but I would NEVER forgive myself if I didn't try.
Now the contest is over, and I am ecstatic to say, I was one of the finalist. So I didn't get the "job," but the experience sure outweighed the outcome. Here's a brief look at how it went, with the help of 'Ole Blue Eyes, the Chairman of the board, Mr. Frank Sinatra.
And now, the end is near,
And so I face the final curtain.
My friends, I'll say it clear;
I'll state my case of which I'm certain.


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In fallout new vegas where's camp mcCarrans missing supplies
kgb answers » Arts & Entertainment » Video Games » In fallout new vegas where's camp mcCarrans missing supplies
Sunday, February 05 2012
Source: http://cheats.gamespy.com/xbox-360-cheats/fallout-new-vegas/
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I hate to sound like a broken record, because every football piece written in the last two months says something about free agency with the condition that it will only happen if the league and the players' union come to terms on a new collective bargaining agreement.
That is the reality of the situation, however, and the teams have until March 4 to work out a deal that will allow free agency to operate as normal, because that is the date when the new league year (and free agency) would officially begin.
After a year with no CBA and a pretty shallow pool of free agents thanks to the accrued seasons requirement for unrestricted free agency increasing from four years to six, we could potentially go back to the old system of just four years if a new CBA is reached.
In that event, a very large and talented class of players will hit the open market, which is sure to excite fans of all teams at the prospects of landing a game-changing talent.
Of course, some of these players might be retained by their old clubs with the franchise tag (which itself is another matter of contention regarding the new CBA).
That being said, here are the top 20 players in the NFL with expiring contracts in 2011—all of which could go a long way in turning a franchise's fortunes...
Chris J. Nelson majored in journalism at Georgia State University and is currently a programming coordinator for Turner Sports in Atlanta. He operates his own Miami Dolphins website, The Miami Dolphins Spotlight, and he can be followed on Twitter here.


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2 hurt in Elk County crash involving ATV, minivan
RIDGWAY -- A 38-year-old man and a 12-year-old girl were injured when the all-terrain vehicle they were riding collided with a minivan in Elk County.
The girl from Kersey, whose name wasn't released, suffered a minor injury while Jason P. Swanson, also of Kersey, received a major injury, state police at Ridgway said. Swanson was taken to Elk Regional Health Center, police said.
The driver of the minivan, Elaine M. Smith, 40, of Brockport, and three passengers in her vehicle -- Althea L. Donahue, 68; a 14-year-old girl; and an 8-year-old boy, all of Brockport -- weren't hurt, police said.
The crash happened Friday at 5:25 p.m. in the 800 block of Brandy Camp Road in Fox Township, police said. The minivan was southbound when the ATV attempted to cross the road from a driveway, police said. Smith tried to swerve to avoid the collision, police said.


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Danny Garcia Discusses Cotto-Margarito II, Ortiz-Berto II
There was a sense of jubilation on Tuesday when it was revealed that the New York State Athletic Commission had approved the license of former welterweight champion Antonio Margarito, paving the way for his December 3rd rematch with Miguel Cotto to land inside of Madison Square Garden. Margarito had suffered a broken orbital bone in his face and developed a cataract in his right eye in a brutal points loss to Manny Pacquiao last year, leaving the commission with some hesitation towards approving him.
It seems fitting that the fight will take place in the Garden, as there will be a heavy contingent of Puerto Ricans in the building rooting on Cotto, who hails from the island's town of Caguas. Miguel suffered his first loss as a professional to Margarito in July of 2008 when he was stopped in the 11th round at the MGM Grand in Las Vegas and will surely be drawing support from his rabid followers when the bell rings.
While several people breathed a sigh of relief upon Margarito's approval, not everyone was taken back by the news. Trainer Danny Garcia, known best for his work with former champion Victor Ortiz, feels that staging the fight in New York won't affect the outcome of the fight.
"Well, I don't know about that," Garcia said during our conversation. "That's where Cotto wanted it but it's not going to help anybody. A fight is a fight and the best guy is going to win. The better fighter is going to take it."
The first Cotto-Margarito bout showcased a classic comeback by Margarito, as he overcame an early lead before eventually breaking Cotto down and dropping him twice late in the fight before registering the stoppage. Garcia was asked if that mental burden would be hard for Cotto to overcome but he quickly turned the tables.
"No. Not in this fight, because he knows and we all know that Margarito was using something in his hand wraps," Garcia continued. "I think Cotto wants the fight real bad. He wants to beat Margarito and he's going to be ready to do anything to beat him."
True enough, one fight after his victory over Cotto, Margarito was found to have the tampered hand wraps that Garcia is referring to prior to his loss to Shane Mosley. A one-year suspension would hit the Tijuana fighter after the discovery and speculation ran wild as people fathomed if he also had illegal wraps during his victory over Cotto.
If so, Garcia feels we won't be seeing the same fighter on the 3rd.
"Margarito is going to do the same thing but if he used something in his hand wraps the first time, and this second time he's not going to have it again, he's going to have problems on his mind. He's going to feel weaker," Garcia stated.
Garcia was reached for this interview for a particular reason, as his younger brother and fellow trainer Robert has been working with Margarito for the past three camps now. The relationship between the two men is often-strained these days, part of which is due to the fashion in which Ortiz, who used to work with Robert, elected to join forces with Danny a few years back.
Danny was asked if he still finds it in him to root for his sibling and offered up a response that wasn't particularly convincing at first.
"Yeah, why not?" stated Garcia. "He's my brother and it's for his family and his kids, like me. We all work for our family and I wish him the best. He's my brother and I hope he does good."
Garcia's star pupil Ortiz looks to be in line for January 28th rematch with Andre Berto, who he upset dramatically earlier this year to capture the WBC welterweight championship before losing it to Floyd Mayweather in his following bout. Garcia noted that he has been working with Ortiz sparingly but expects him to fully dive into camp in a few days' time.
There definitely is a little bad blood left over from the first fight and Berto and his trainer Tony Morgan have expressed a different level of confidence heading into the second fight.
But it simply won't matter, says Garcia.
"I just think Victor is the better fighter and we're going to beat him again. Because we already did and it wasn't even close."
Looks like the fight will get ugly-with elbows, head butts and low blows if neccessary. I don't see any sportsman-like conduct happening here. Worse case senario is a riot braking up if Margarito does something stupid- for example, punching after…Comment by flint on 11-24-2011
[QUOTE=.::|ULTIMATE|::.;11467831]Danny Garcia is an ******* he is always throwing jabs at his brother's fighters.[/QUOTE]that goes both ways. robert does the same.they're good trainers,but more importantly they're brothers and need to fix this thing quick before their parents pass on to…Comment by sd southpaw on 11-24-2011
berto didnt respect ortiz before that fight and ortiz got in that a**. this time around i think berto will do better and i think ortiz lost some confidence after that mayweather 2 pieceComment by .::|ULTIMATE|::. on 11-24-2011
Danny Garcia is an ******* he is always throwing jabs at his brother's fighters.Comment by HattonFaithful on 11-24-2011
[QUOTE=hougigo;11467694]True... but I was trying to make a slight at Victor Conte being on his team now. I know they're going to have random testing... but it's more for Berto than Ortiz because with Conte in his side, people automatically…Post a Comment - View More User Comments (11)


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Information Morning Cape Breton
Information Morning Cape Breton | Weekdays 6 a.m. to 8:37 a.m. | CBC Radio One
Join Information Morning's host Steve Sutherland and news editor Hal Higgins starting at 6 a.m. on weekdays.
Archbishop retires from Sydney 'labour of joy' to Halifax
The patriarch of the African Orthodox Church is saying goodbye to the congregation he has served for 33 years in Sydney, N.S.
North Sydney group lobbies for Archibald's Wharf replacement
A new Cape Breton group has plans to turn a vacant area of the North Sydney waterfront into a mixed-use recreational space.
Surf's up: New competition coming to Cape Breton this October
A new surf competition, the Schooner Cliffs Surf Classic, will be held in early October at Schooner Pond Cove. Prize money totaling $2,000 will be up for grabs.
Information Morning Cape Breton Clips
Thursday September 29, 2016
Audio Medal of Bravery - Nicole Foran
When a plane in Jamaica was hijacked in 2009, a Nova Scotia flight attendant played a pivotal role in bringing the stressful situation to a safe ending. Nicole Foran of Halifax has been recognized for that role with a Medal of Bravery.
Thursday September 29, 2016
Audio Breakfast Biographies - Tuma Young
Today, a profile featuring Tuma Young, who is originally from Malagawatch, Inverness County. He was the first Mi'kmaq-speaking lawyer in NS. And, now, he's an assistant professor of Indigenous Studies at Cape Breton University.
Thursday September 29, 2016
Audio Nurse Practitioners
The Department of Health announced it will hire 7 nurse practitioners and family practice nurses to work in various locations across Cape Breton. Kathy Bell is the director of Primary Health Care, Eastern Zone, Nova Scotia Health Authority.
Wednesday September 28, 2016
Audio Jim St. Clair - Kidston Island
Treasure Island? There may not be gold buried on Kidston Island, but there's a wealth of fascinating stories to be unearthed on the island near Baddeck. Information Morning host Steve Sutherland and community historian Jim St. Clair take a trip.
Wednesday September 28, 2016
Audio Richmond Candidates District 2
Today, we speak with three of the five candidates looking to represent District 2 in Richmond County - Larry Keating, Alvin Martell and Gina Stanley. Candidates Victor David and Marcel Boudreau were unable to attend our discussion.
Tuesday September 27, 2016
Audio CBRM Candidates District 10
Our roundtable discussions with candidates in various Cape Breton districts continues. Today, District 10, where Darren Bruckschwaiger, Dave Wilson, and Brian Shaw are vying for your vote in Dominion and parts of Glace Bay.
Monday September 26, 2016
Audio Breakfast Biographies - Joan Jessome
Most people in Nova Scotia know the name Joan Jessome because of her work with the NSGEU. Learn about her fascinating life as labour leader, fighter, grandmother, and "the most hated woman in Nova Scotia" in "The Breakfast Biographies."
Friday September 23, 2016
Audio Issue Panel 0923
Now, a brand new season of the Information Morning Issue Panel with panelists Kim Desveaux, Jenn Power, and Chris Milburn. Every second week, a rotating roster of talkative and opinionated Cape Bretoners offer their take on the stories of the week.
Severe weather warnings or watches in effect for:
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Annapolis Royal
- Shimon Peres remembered as 'man of vision' at funeral
- Trump broke U.S. law with dealings in Cuba, Newsweek report says
- Police officer who fatally shot Tulsa man had 'auditory exclusion,' lawyer says
- Scientists bid farewell to Rosetta spacecraft ahead of Friday's crash landing
- At least 6 dead in typhoon-linked landslides in China
- 'Never seen anything that intense': Storms lead to state of emergency in Windsor, Tecumseh, Ont.
- What's next for Pacific NorthWest LNG project? 4 questions answered
- Canada takes World Cup title after furious comeback in final minutes
- Her husband's sex assault trial ended with a bizarre twist, so this woman went public
- 'Top of my bucket list': Royal hugs thrill Yukon moms
- Foot doctor used experimental implants unapproved for surgery on unsuspecting patients
- Senator reports herself to ethics czar over tax-haven revelations
- 3 Canadian banks served 2,000 offshore corporations, Bahamas leak shows
- Canadian ties exposed in leaked Bahamas tax-haven data
- Canadian immigration DNA test would force kids through dangerous area under travel warning
- Shimon Peres remembered as 'man of vision' at funeral
- PM's top adviser compares Maryam Monsef criticism to U.S. 'birther' movement
- Man in Trudeau selfie linked to RCMP terror investigation
- Dominic LeBlanc under fire for agreeing to attend private event hosted by law, communications firms
- Jane Philpott says 3% a 'reasonable escalator' for health-care spending
- Canada's population tops 36 million as immigrants, refugees swell numbers
- OPEC deal optimism leads oil, TSX higher
- Japanese retailer Uniqlo dips its toes in Canada's cutthroat retail sector
- 'Fraud is fraud. Theft is theft': U.S. lawmakers grill Wells Fargo CEO
- First phones, now washing machines: Samsung washers pose risk, CPSC warns
- Why Canada recommends nasal-mist flu vaccine against U.S. advice
- Canada 'incredibly average' on reducing premature mortality compared with peer countries: report
- Zika outbreak: travel consideration issued by CDC for 11 Southeast Asian countries
- Common painkillers linked to increased risk of heart failure: study
- Zika outbreak: Utah man may have contracted virus from dying father's tears
- NFL fans go Gaga: Super Bowl halftime performer named
- Queen of Katwe a refreshingly positive African story
- Brad Pitt skips movie premiere to focus on 'family situation'
- MGM TV acquires rights for Margaret Atwood's The Heart Goes Last
- Singer Cyndi Lauper calls pit bull ban a 'sad day for Montreal' as thousands threaten to boycott city
- Sugar puts bumblebees in a good mood, says research showing emotion-like behaviour
- Scientists bid farewell to Rosetta spacecraft ahead of Friday's crash landing
- 'Pepe is not hate symbol,' says internet-famous frog's creator
- 63% of Albertans oppose NDP carbon tax, poll suggests
- Moncton Wildcats put 'human hockey puck hovercraft' on ice
- Make America White Again: Why Donald Trump plays the race card
- Clinton vs. Trump: Follow the race for the White House with CBC's presidential poll tracker
- Looking for a place to happen: Canadian stories behind Tragically Hip's lyrics
- Battling the Beast: The untold story of the fight to save Fort McMurray
- Nothing 'more beautiful than to lend a hand': Meet 7 inspiring Aboriginal community builders
- Not just the face of climate change: Inuit want a say in Canada's climate strategy
- Thunder Bay police investigate Facebook comments deemed 'not acceptable'
- Semiahmoo First Nation talking to Surrey about water supply to native land
- Wilbur Dedam found guilty on 6 sex charges
- UNESCO hears fears that Wood Buffalo National Park 'is going to dry out'
- Canada's core, led by Sidney Crosby, continues world hockey domination
- Late collapse ends Team Europe's unexpected run to final
- Canada takes World Cup title after furious comeback in final minutes
- 5 things we'd like to see at the next World Cup of Hockey
- Orioles shut out Jays to pull even for wild card


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Spectrum Research: Can Nicotinamide Riboside Improve Endurance and Strength
Nicotinamide Riboside, which is marketed by the brand name of Niagen by an American pharmaceutical company, ChromaDex is a naturally occurring vitamin B3 metabolite that is known for its anti-aging properties. The compound majorly acts on the powerhouses of the cell, Mitochondria, and help improve energy generation.
Because of this action, Niagen is often promoted as a sport supplement that can improve endurance and strength.
In this article, we are providing evidence regarding whether or not Niagen improves performance of athletes. The article also summarizes the mechanism of action through which Niagen promotes energy production by mitochondria.
Mechanism of Action of Niagen
Niagen produces its effects by acting as a precursor of Nicotinamide adenine dinucleotide (NAD+), which is an important component of many cellular pathways – which can be seen in athletes who're on the paleo diet or do intense strength training. NAD+ plays significant role in energy production and metabolism and, therefore, increasing its level can help a person feel more energetic. In addition to this, NAD+ also acts a rate-limiting co-substrate for sirtuins, which are also known as longevity genes.
Niagen in Sport Nutrition — What Evidence Do We Have?
While mechanism of action of Niagen is quite clearly understood, its role as a sport supplement has not been investigated thoroughly. However, based on the scientific studies that suggest that increased levels of NAD+ can improve endurance by reducing oxidative stress induced suppression of aerobic respiration, we can assume that Niagen has a potential role in sports nutrition.
The findings of a study conducted in 2010 were published in a journal named 'Nutrition', which suggest that strenuous exercises result in reduced NAD+ levels, which ultimately result in impeding muscle performance due to fatigue. In order to evaluate the effect of an anti-oxidant supplement on NAD+ levels and muscle performance, the same study inducted 13 participants. The participants performed continuous progressive exercises and then received an anti-oxidant cocktail which improved NAD+ levels and muscle endurance.
In addition to this, the first ever clinical trial on Niagen suggests that the supplement effectively increases the level of NAD+ without producing any side effects. The trial was conducted by ChromaDex — the company that manufactures and markets Niagen. During the clinical trial, 12 healthy adult subjects received 100mg, 300mg, and 1 gm doses of Niagen. After supplementation, NAD+ levels were found to increase by 2.7 folds.
Experts believe that the findings of the first-ever human clinical trial of Niagen are quite impressive and another trial to investigate the effects of Niagen has already been initiated. No side effects were reported by the subjects who participated in the clinical trial. Therefore, one can suggest that Niagen is a relatively safer sports supplement compared to the rest.
While Nicotinamide Riboside is known to be an effective precursor of NAD+, its safety in human subjects us yet to be established. Therefore, athletes should use the supplement in suggested therapeutic doses and report any side effects immediately to their healthcare provider.


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Visit Shipping & Returns and Terms of Use pages to learn about our policies
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Hundreds of styles starting at $6
When building an EyeWear collection fit and style are often the two most important factors in making frame decisions. That's why at EyeBuyDirect, we offer a Fit and Style guarantee. If for any reason, you are unhappy with your glasses, return them within 14 days for a free exchange or refund.
Frames and lenses are fully warranted against defects in materials and workmanship for one full year from time of purchase. If your glasses fail due to a manufacturing defect, we will replace them for a new pair at equal value, free.
Free shipping applies for orders above $99 or to new customers with orders above $15. It applies to US shipments via USPS only.
Product warranty does not cover damage caused by accidents, negligence, or improper care.


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Actress to star in CTV special
Shirley MacLaine will star as matriarch Amelia Thomas in Canuck telepic "Anne of Green Gables: A New Beginning," which will air on commercial web CTV next year.
The movie is a prequel to the original miniseries based on Lucy Maud Montgomery's novels, with a script by helmer-exec producer Kevin Sullivan, for his Sullivan Entertainment.
Hannah Endicott-Douglas stars as the young Anne Shirley, Barbara Hershey as the grown-up Anne Shirley.


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Video: Lollapalooza 2013 Day One Highlights, Plus Watch Saturday and Sunday Webcasts Here
Even if you're lucky enough to have snagged a ticket to the sold out Lollapalooza, you can't see everything. So the fest is doing what it can to get people access to a variety of shows. »Read Full Blog Post
Comedian John Pinette, 50, found dead
Comedian John Pinette died Saturday in a Pittsburgh hotel room, according to a report published by the Pittsburgh Post-Gazette.
Pinette, 50, had suffered from liver and heart disease, according to the Allegheny County Medical Examiner's office. Authorities did not suspect foul play, the Post-Gazette reported. »Read Full Article(4)
Soulstice Theatre paints a wrenching 'Still Life'
It doesn't take a grand scale to tell a stirring story.
Soulstice Theatre opened Alexander Dinelaris' "Still Life" this weekend, a wrenching story about some of life's weightiest topics: death, dignity, love, loss and the emotional wounds we all carry to the grave with us. »Read Full Article
Renaissance Theaterworks brings back older, wiser 'Skin Tight'
Director Laura Gordon admits in her program notes for "Skin Tight" that she'd initially had "very mixed feelings" about Renaissance Theaterworks' decision to bring it back — not quite 10 years after she made her professional directorial debut with the same play.
I felt the same way. I cherish my memories of Renaissance's 2004 production of Gary Henderson's gorgeous, hourlong memory play, in which actors Braden Moran and Leah Dutchin gave us the moving story of long-married New Zealanders Tom and Elizabeth. I didn't want a new production messing with those images — or, worse, cheapening them. »Read Full Article
Florentine Opera Company party, MAM After Dark, Tomato Dodgeball
Hit a high note
The Florentine Opera Company knows how to enthrall its audience, so its announcement of next season's productions deserves to be made with the proper dose of drama. It will announce its season with a party Tuesday at the Florentine Opera Center, 926 E. Burleigh St. The celebration starts at 6 p.m. with light hors d'oeuvres and beverages. Then, the company's studio artists will entertain, followed by the announcement. It is a free event. Let them know if they can expect you by emailing email@example.com. »Read Full Article


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Monte Pittman
To hear 'Inverted Grasp of Balance' (due out this Friday, September 23rd) in full before its release, please visit the Wall Street Journal - where an exclusive interview with Monte Pittman can be read, alongside the album stream!Read More
Charred Walls of the Damned
Today, Charred Walls Of The Damned's album Creatures Watching Over The Dead is available as an exclusive stream in advance of the release on HowardStern.com. Check out the new record in its entirety.Read More
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