query: Smc5/6 engagment drives the activation of SUMO E3 ligase Mms21 by ATP-dependent remolding.

positive: ATPase-Dependent Control of the Mms21 SUMO Ligase during DNA Repair Modification of proteins by SUMO is essential for the maintenance of genome integrity. During DNA replication, the Mms21-branch of the SUMO pathway counteracts recombination intermediates at damaged replication forks, thus facilitating sister chromatid disjunction. The Mms21 SUMO ligase docks to the arm region of the Smc5 protein in the Smc5/6 complex; together, they cooperate during recombinational DNA repair. Yet how the activity of the SUMO ligase is controlled remains unknown. Here we show that the SUMO ligase and the chromosome disjunction functions of Mms21 depend on its docking to an intact and active Smc5/6 complex, indicating that the Smc5/6-Mms21 complex operates as a large SUMO ligase in vivo. In spite of the physical distance separating the E3 and the nucleotide-binding domains in Smc5/6, Mms21-dependent sumoylation requires binding of ATP to Smc5, a step that is part of the ligase mechanism that assists Ubc9 function. The communication is enabled by the presence of a conserved disruption in the coiled coil domain of Smc5, pointing to potential conformational changes for SUMO ligase activation. In accordance, scanning force microscopy of the Smc5-Mms21 heterodimer shows that the molecule is physically remodeled in an ATP-dependent manner. Our results demonstrate that the ATP-binding activity of the Smc5/6 complex is coordinated with its SUMO ligase, through the coiled coil domain of Smc5 and the physical remodeling of the molecule, to promote sumoylation and chromosome disjunction during DNA repair.

negative: The SMC5/6 complex maintains telomere length in ALT cancer cells through SUMOylation of telomere-binding proteins Most cancer cells activate telomerase to elongate telomeres and achieve unlimited replicative potential. Some cancer cells cannot activate telomerase and use telomere homologous recombination (HR) to elongate telomeres, a mechanism termed alternative lengthening of telomeres (ALT). A hallmark of ALT cells is the recruitment of telomeres to PML bodies (termed APBs). Here, we show that the SMC5/6 complex localizes to APBs in ALT cells and is required for targeting telomeres to APBs. The MMS21 SUMO ligase of the SMC5/6 complex SUMOylates multiple telomere-binding proteins, including TRF1 and TRF2. Inhibition of TRF1 or TRF2 SUMOylation prevents APB formation. Depletion of SMC5/6 subunits by RNA interference inhibits telomere HR, causing telomere shortening and senescence in ALT cells. Thus, the SMC5/6 complex facilitates telomere HR and elongation in ALT cells by promoting APB formation through SUMOylation of telomere-binding proteins.

negative: A SUMO-Dependent Protein Network Regulates Chromosome Congression during Oocyte Meiosis During Caenorhabditis elegans oocyte meiosis, a multi-protein ring complex (RC) localized between homologous chromosomes, promotes chromosome congression through the action of the chromokinesin KLP-19. While some RC components are known, the mechanism of RC assembly has remained obscure. We show that SUMO E3 ligase GEI-17/PIAS is required for KLP-19 recruitment to the RC, and proteomic analysis identified KLP-19 as a SUMO substrate in vivo. In vitro analysis revealed that KLP-19 is efficiently sumoylated in a GEI-17-dependent manner, while GEI-17 undergoes extensive auto-sumoylation. GEI-17 and another RC component, the kinase BUB-1, contain functional SUMO interaction motifs (SIMs), allowing them to recruit SUMO modified proteins, including KLP-19, into the RC. Thus, dynamic SUMO modification and the presence of SIMs in RC components generate a SUMO-SIM network that facilitates assembly of the RC. Our results highlight the importance of SUMO-SIM networks in regulating the assembly of dynamic protein complexes.

negative: Function and regulation of SUMO proteases Covalent attachment of small ubiquitin-like modifier (SUMO) to proteins is highly dynamic, and both SUMO–protein conjugation and cleavage can be regulated. Protein desumoylation is carried out by SUMO proteases, which control cellular mechanisms ranging from transcription and cell division to ribosome biogenesis. Recent advances include the discovery of two novel classes of SUMO proteases, insights regarding SUMO protease specificity, and revelations of previously unappreciated SUMO protease functions in several key cellular pathways. These developments, together with new connections between SUMO proteases and the recently discovered SUMO-targeted ubiquitin ligases (STUbLs), make this an exciting period to study these enzymes.

negative: De novo assembly of a PML nuclear subcompartment occurs through multiple pathways and induces telomere elongation. Telomerase-negative tumor cells use an alternative lengthening of telomeres (ALT) pathway that involves DNA recombination and repair to maintain their proliferative potential. The cytological hallmark of this process is the accumulation of promyelocytic leukemia (PML) nuclear protein at telomeric DNA to form ALT-associated PML bodies (APBs). Here, the de novo formation of a telomeric PML nuclear subcompartment was investigated by recruiting APB protein components. We show that functionally distinct proteins were able to initiate the formation of bona fide APBs with high efficiency in a self-organizing and self-propagating manner. These included: (1) PML and Sp100 as the constituting components of PML nuclear bodies, (2) telomere repeat binding factors 1 and 2 (TRF1 and TRF2, respectively), (3) the DNA repair protein NBS1 and (4) the SUMO E3 ligase MMS21, as well as the isolated SUMO1 domain, through an interacting domain of another protein factor. By contrast, the repair factors Rad9, Rad17 and Rad51 were less efficient in APB nucleation but were recruited to preassembled APBs. The artificially created APBs induced telomeric extension through a DNA repair mechanism, as inferred from their colocalization with sites of non-replicative DNA synthesis and histone H2A.X phosphorylation, and an increase of the telomere repeat length. These activities were absent after recruitment of the APB factors to a pericentric locus and establish APBs as functional intermediates of the ALT pathway.

negative: Identifying and Characterizing Interplay between Hepatitis B Virus X Protein and Smc5/6 Hepatitis B X protein (HBx) plays an essential role in the hepatitis B virus (HBV) replication cycle, but the function of HBx has been elusive until recently. It was recently shown that transcription from the HBV genome (covalently-closed circular DNA, cccDNA) is inhibited by the structural maintenance of chromosome 5/6 complex (Smc5/6), and that a key function of HBx is to redirect the DNA-damage binding protein 1 (DDB1) E3 ubiquitin ligase to target this complex for degradation. By doing so, HBx alleviates transcriptional repression by Smc5/6 and stimulates HBV gene expression. In this review, we discuss in detail how the interplay between HBx and Smc5/6 was identified and characterized. We also discuss what is known regarding the repression of cccDNA transcription by Smc5/6, the timing of HBx expression, and the potential role of HBx in promoting hepatocellular carcinoma (HCC).

negative: Noxa controls Mule-dependent Mcl-1 ubiquitination through the regulation of the Mcl-1/USP9X interaction. The level of the Mcl-1 pro-survival protein is highly regulated, and the down-regulation of Mcl-1 expression favors the apoptotic process. Mcl-1 physically interacts with different BH3-only proteins; particularly, Noxa is involved in the modulation of Mcl-1 expression. In this study, we demonstrated that Noxa triggers the degradation of Mcl-1 at the mitochondria according to the exclusive location of Noxa at this compartment. The Noxa-induced degradation of Mcl-1 required the E3 ligase Mule, which is responsible for the polyubiquitination of Mcl-1. Because the USP9X deubiquitinase was recently demonstrated to be involved in Mcl-1 protein turnover by preventing its degradation through the removal of conjugated ubiquitin, we investigated whether Noxa affected the deubiquitination process. Interestingly, Noxa over-expression caused a decrease in the USP9X/Mcl-1 interaction associated with an increase in the Mcl-1 polyubiquitinated forms. Additionally, Noxa over-expression triggered an increase in the Mule/Mcl-1 interaction in parallel with the decrease in Mule/USP9X complex formation. Taken together, these modifications result in the degradation of Mcl-1 by the proteasome machinery. The implication of Noxa in the regulation of Mcl-1 proteasomal degradation adds complexity to this process, which is governed by multiple interactions.

negative: Roles of histone acetylation and chromatin remodeling factor in a meiotic recombination hotspot. Histone acetyltransferases (HATs) and ATP-dependent chromatin remodeling factors (ADCRs) are involved in selective gene regulation via modulation of local chromatin configuration. Activation of the recombination hotspot ade6-M26 of Schizosaccharomyces pombe is mediated by a cAMP responsive element (CRE)-like sequence, M26, and a heterodimeric ATF/CREB transcription factor, Atf1.Pcr1. Chromatin remodeling occurs meiotically around M26. We examined the roles of HATs and ADCRs in chromatin remodeling around M26. Histones H3 and H4 around M26 were hyperacetylated in an M26- and Atf1-dependent manner early in meiosis. SpGcn5, the S. pombe homolog of Gcn5p, was required for the majority of histone H3 acetylation around M26 in vivo. Deletion of gcn5+ caused a significant delay in chromatin remodeling but only partial reduction of M26 meiotic recombination frequency. The snf22+ (a Swi2/Snf2-ADCR homologue) deletion and snf22+ gcn5+ double deletion abolished chromatin remodeling and significant reduction of meiotic recombination around M26. These results suggest that HATs and ADCRs cooperatively alter local chromatin structure, as in selective transcription activation, to activate meiotic recombination at M26 in a site-specific manner.

negative: Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL Acute lymphoblastic leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors. Despite intensive chemotherapy, 20% of pediatric patients and over 50% of adult patients with ALL do not achieve a complete remission or relapse after intensified chemotherapy, making disease relapse and resistance to therapy the most substantial challenge in the treatment of this disease. Using whole-exome sequencing, we identify mutations in the cytosolic 5'-nucleotidase II gene (NT5C2), which encodes a 5'-nucleotidase enzyme that is responsible for the inactivation of nucleoside-analog chemotherapy drugs, in 20/103 (19%) relapse T cell ALLs and 1/35 (3%) relapse B-precursor ALLs. NT5C2 mutant proteins show increased nucleotidase activity in vitro and conferred resistance to chemotherapy with 6-mercaptopurine and 6-thioguanine when expressed in ALL lymphoblasts. These results support a prominent role for activating mutations in NT5C2 and increased nucleoside-analog metabolism in disease progression and chemotherapy resistance in ALL.

negative: A gp130–Src–YAP module links inflammation to epithelial regeneration Inflammation promotes regeneration of injured tissues through poorly understood mechanisms, some of which involve interleukin (IL)-6 family members, the expression of which is elevated in many diseases including inflammatory bowel diseases and colorectal cancer. Here we show in mice and human cells that gp130, a co-receptor for IL-6 cytokines, triggers activation of YAP and Notch, transcriptional regulators that control tissue growth and regeneration, independently of the gp130 effector STAT3. Through YAP and Notch, intestinal gp130 signalling stimulates epithelial cell proliferation, causes aberrant differentiation and confers resistance to mucosal erosion. gp130 associates with the related tyrosine kinases Src and Yes, which are activated on receptor engagement to phosphorylate YAP and induce its stabilization and nuclear translocation. This signalling module is strongly activated upon mucosal injury to promote healing and maintain barrier function.

negative: The INO80 ATP-dependent chromatin remodeling complex is a nucleosome spacing factor. The mobilization of nucleosomes by the ATP-dependent remodeler INO80 is quite different from another remodeler (SWI/SNF) that is also involved in gene activation. Unlike that recently shown for SWI/SNF, INO80 is unable to disassemble nucleosomes when remodeling short nucleosomal arrays. Instead, INO80 more closely resembles, although with notable exceptions, the nucleosome spacing activity of ISW2 and ISW1a, which are generally involved in transcription repression. INO80 required a minimum of 33 to 43 bp of extranucleosomal DNA for mobilizing nucleosomes, with 70 bp being optimal. INO80 prefers to move mononucleosomes to the center of DNA, like ISW2 and ISW1a, but does so with higher precision. Unlike ISW2/1a, INO80 does not require the H4 tail for nucleosome mobilization; instead, the H2A histone tail negatively regulates nucleosome movement by INO80. INO80 moved arrays of two or three nucleosomes with 50 or 79 bp of linker DNA closer together, with a final length of ∼30 bp of linker DNA or a repeat length of ∼177 bp. A minimum length of >30 bp of linker DNA was required for nucleosome movement and spacing by INO80 in arrays.

negative: The ubiquitin ligase Drosophila Mind bomb promotes Notch signaling by regulating the localization and activity of Serrate and Delta. The receptor Notch and its ligands of the Delta/Serrate/LAG2 (DSL) family are the central components in the Notch pathway, a fundamental cell signaling system that regulates pattern formation during animal development. Delta is directly ubiquitinated by Drosophila and Xenopus Neuralized, and by zebrafish Mind bomb, two unrelated RING-type E3 ubiquitin ligases with common abilities to promote Delta endocytosis and signaling activity. Although orthologs of both Neuralized and Mind bomb are found in most metazoan organisms, their relative contributions to Notch signaling in any single organism have not yet been assessed. We show here that a Drosophila ortholog of Mind bomb (D-mib) is a positive component of Notch signaling that is required for multiple Neuralized-independent, Notch-dependent developmental processes. Furthermore, we show that D-mib associates physically and functionally with both Serrate and Delta. We find that D-mib uses its ubiquitin ligase activity to promote DSL ligand activity, an activity that is correlated with its ability to induce the endocytosis and degradation of both Delta and Serrate (see also Le Borgne et al., 2005). We further demonstrate that D-mib can functionally replace Neuralized in multiple cell fate decisions that absolutely require endogenous Neuralized, a testament to the highly similar activities of these two unrelated ubiquitin ligases in regulating Notch signaling. We conclude that ubiquitination of Delta and Serrate by Neuralized and D-mib is an obligate feature of DSL ligand activation throughout Drosophila development.

negative: Ubiquitination on nonlysine residues by a viral E3 ubiquitin ligase. Ubiquitination controls a broad range of cellular functions. The last step of the ubiquitination pathway is regulated by enzyme type 3 (E3) ubiquitin ligases. E3 enzymes are responsible for substrate specificity and catalyze the formation of an isopeptide bond between a lysine residue of the substrate (or the N terminus of the substrate) and ubiquitin. MIR1 and MIR2 are two E3 ubiquitin ligases encoded by Kaposi's sarcoma-associated herpesvirus that mediate the ubiquitination of major histocompatibility complex class I (MHC I) molecules and subsequent internalization. Here, we found that MIR1, but not MIR2, promoted down-regulation of MHC I molecules lacking lysine residues in their intracytoplasmic domain. In the presence of MIR1, these MHC I molecules were ubiquitinated, and their association with ubiquitin was sensitive to beta2-mercaptoethanol, unlike lysine-ubiquitin bonds. This form of ubiquitination required a cysteine residue in the intracytoplasmic tail of MHC I molecules. An MHC I molecule containing a single cysteine residue in an artificial glycine and alanine intracytoplasmic domain was endocytosed and degraded in the presence of MIR1. Thus, ubiquitination can occur on proteins lacking accessible lysines or an accessible N terminus.

negative: High Km soluble 5'-nucleotidase from human placenta. Properties and allosteric regulation by IMP and ATP. A human placental soluble "high Km" 5'-nucleotidase has been separated from "low Km" 5'-nucleotidase and nonspecific phosphatase by AMP-Sepharose affinity chromatography. The enzyme was purified 8000-fold to a specific activity of 25.6 mumol/min/mg. The subunit molecular mass is 53 kDa, and the native molecular mass is 210 kDa, suggesting a tetrameric structure. Soluble high Km 5'-nucleotidase is most active with IMP and GMP and their deoxy derivatives. IMP is hydrolyzed 15 times faster than AMP. The enzyme has a virtually absolute requirement for magnesium ions and is regulated by them. Purine nucleoside 5'-triphosphates strongly activate the enzyme with the potency order dATP greater than ATP greater than GTP. 2,3-Diphosphoglycerate activates the enzyme as potently as ATP. Three millimolar ATP decreased the Km for IMP from 0.33 to 0.09 mM and increased the Vmax 12-fold. ATP activation was modified by the IMP concentration. At 20 microM IMP the ATP-dependent activation curve was sigmoidal, while at 2 mM IMP it was hyperbolic. The A0.5 values for ATP were 2.26 and 0.70 mM, and the relative maximal velocities were 32.9 and 126.0 nmol/min, respectively. Inorganic phosphate shifts the hyperbolic substrate velocity relationship for IMP to a sigmoidal one. With physiological concentrations of cofactors (3 mM ATP, 1-4 mM Pi, 150 mM KCl) at pH 7.4, the enzyme is 25-35 times more active toward 100 microM IMP than 100 microM AMP. These data show that: (a) soluble human placental high Km 5'-nucleotidase coexists in human placenta with the low Km enzyme; (b) under physiological conditions the enzyme favors the hydrolysis of IMP and is critically regulated by IMP, ATP, and Pi levels; and (c) kinetic properties of ATP and IMP are each modified by the other compound suggesting complex interaction of the associated binding sites.

negative: The Ulp1 SUMO isopeptidase distinct domains required for viability, nuclear envelope localization, and substrate specificity Protein modification by the ubiquitin-like SUMO protein contributes to many cellular regulatory mechanisms. In Saccharomyces cerevisiae, both sumoylating and desumoylating activities are essential for viability. Of its two known desumoylating enzymes, Ubl-specific protease (Ulp)1 and Ulp2/Smt4, Ulp1 is specifically required for cell cycle progression. A ∼200-residue segment, the Ulp domain (UD), is conserved among Ulps and includes a core cysteine protease domain that is even more widespread. Here we demonstrate that the Ulp1 UD by itself can support wild-type growth rates and in vitro can cleave SUMO from substrates. However, in cells expressing only the UD of Ulp1, many SUMO conjugates accumulate to high levels, indicating that the nonessential Ulp1 NH2-terminal domain is important for activity against a substantial fraction of sumoylated targets. The NH2-terminal domain also includes sequences necessary and sufficient to concentrate Ulp1 at nuclear envelope sites. Remarkably, NH2-terminally deleted Ulp1 variants are able, unlike full-length Ulp1, to suppress defects of cells lacking the divergent Ulp2 isopeptidase. Thus, the NH2-terminal regulatory domain of Ulp1 restricts Ulp1 activity toward certain sumoylated proteins while enabling the cleavage of others. These data define key functional elements of Ulp1 and strongly suggest that subcellular localization is a physiologically significant constraint on SUMO isopeptidase specificity.

negative: MG53 nucleates assembly of cell membrane repair machinery Dynamic membrane repair and remodelling is an elemental process that maintains cell integrity and mediates efficient cellular function. Here we report that MG53, a muscle-specific tripartite motif family protein (TRIM72), is a component of the sarcolemmal membrane-repair machinery. MG53 interacts with phosphatidylserine to associate with intracellular vesicles that traffic to and fuse with sarcolemmal membranes. Mice null for MG53 show progressive myopathy and reduced exercise capability, associated with defective membrane-repair capacity. Injury of the sarcolemmal membrane leads to entry of the extracellular oxidative environment and MG53 oligomerization, resulting in recruitment of MG53-containing vesicles to the injury site. After vesicle translocation, entry of extracellular Ca2+ facilitates vesicle fusion to reseal the membrane. Our data indicate that intracellular vesicle translocation and Ca2+-dependent membrane fusion are distinct steps involved in the repair of membrane damage and that MG53 may initiate the assembly of the membrane repair machinery in an oxidation-dependent manner.

negative: ATP binding to PAN or the 26S ATPases causes association with the 20S proteasome, gate opening, and translocation of unfolded proteins. The archaeal ATPase complex PAN, the homolog of the eukaryotic 26S proteasome-regulatory ATPases, was shown to associate transiently with the 20S proteasome upon binding of ATP or ATPgammaS, but not ADP. By electron microscopy (EM), PAN appears as a two-ring structure, capping the 20S, and resembles two densities in the 19S complex. The N termini of the archaeal 20S alpha subunits were found to function as a gate that prevents entry of seven-residue peptides but allows entry of tetrapeptides. Upon association with the 20S particle, PAN stimulates gate opening. Although degradation of globular proteins requires ATP hydrolysis, the PAN-20S complex with ATPgammaS translocates and degrades unfolded and denatured proteins. Rabbit 26S proteasomes also degrade these unfolded proteins upon ATP binding, without hydrolysis. Thus, although unfolding requires energy from ATP hydrolysis, ATP binding alone supports ATPase-20S association, gate opening, and translocation of unfolded substrates into the proteasome, which can occur by facilitated diffusion through the ATPase.

negative: The Translation Initiation Factor 3f (eIF3f) Exhibits a Deubiquitinase Activity Regulating Notch Activation Activation of the mammalian Notch receptor after ligand binding relies on a succession of events including metalloprotease-cleavage, endocytosis, monoubiquitination, and eventually processing by the gamma-secretase, giving rise to a soluble, transcriptionally active molecule. The Notch1 receptor was proposed to be monoubiquitinated before its gamma-secretase cleavage; the targeted lysine has been localized to its submembrane domain. Investigating how this step might be regulated by a deubiquitinase (DUB) activity will provide new insight for understanding Notch receptor activation and downstream signaling. An immunofluorescence-based screening of an shRNA library allowed us to identify eIF3f, previously known as one of the subunits of the translation initiation factor eIF3, as a DUB targeting the activated Notch receptor. We show that eIF3f has an intrinsic DUB activity. Knocking down eIF3f leads to an accumulation of monoubiquitinated forms of activated Notch, an effect counteracted by murine WT eIF3f but not by a catalytically inactive mutant. We also show that eIF3f is recruited to activated Notch on endocytic vesicles by the putative E3 ubiquitin ligase Deltex1, which serves as a bridging factor. Finally, catalytically inactive forms of eIF3f as well as shRNAs targeting eIF3f repress Notch activation in a coculture assay, showing that eIF3f is a new positive regulator of the Notch pathway. Our results support two new and provocative conclusions: (1) The activated form of Notch needs to be deubiquitinated before being processed by the gamma-secretase activity and entering the nucleus, where it fulfills its transcriptional function. (2) The enzyme accounting for this deubiquitinase activity is eIF3f, known so far as a translation initiation factor. These data improve our knowledge of Notch signaling but also open new avenues of research on the Zomes family and the translation initiation factors.

negative: The 1.9 A structure of a proteasome-11S activator complex and implications for proteasome-PAN/PA700 interactions. Proteasomes are cylindrical structures that function in multiple cellular processes by degrading a wide variety of cytosolic and nuclear proteins. Substrate access and product release from the enclosed catalytic chamber occurs through axial pores that are opened by activator complexes. Here, we report high-resolution structures of wild-type and mutant archaeal proteasomes bound to the activator PA26. These structures support the proposal that an ordered open conformation is required for proteolysis and that its formation can be triggered by outward displacement of surrounding residues. The structures and associated biochemical assays reveal the mechanism of binding, which involves an interaction between the PA26 C terminus and a conserved lysine. Surprisingly, biochemical observations implicate an equivalent interaction for the unrelated ATP-dependent activators PAN and PA700.

negative: Regulation of autophagy by cytoplasmic p53 Multiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that deletion, depletion or inhibition of p53 can induce autophagy in human, mouse and nematode cells subjected to knockout, knockdown or pharmacological inhibition of p53. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53−/− cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53.

negative: Mesenchymal Inflammation Drives Genotoxic Stress in Hematopoietic Stem Cells and Predicts Disease Evolution in Human Pre-leukemia. Mesenchymal niche cells may drive tissue failure and malignant transformation in the hematopoietic system, but the underlying molecular mechanisms and relevance to human disease remain poorly defined. Here, we show that perturbation of mesenchymal cells in a mouse model of the pre-leukemic disorder Shwachman-Diamond syndrome (SDS) induces mitochondrial dysfunction, oxidative stress, and activation of DNA damage responses in hematopoietic stem and progenitor cells. Massive parallel RNA sequencing of highly purified mesenchymal cells in the SDS mouse model and a range of human pre-leukemic syndromes identified p53-S100A8/9-TLR inflammatory signaling as a common driving mechanism of genotoxic stress. Transcriptional activation of this signaling axis in the mesenchymal niche predicted leukemic evolution and progression-free survival in myelodysplastic syndrome (MDS), the principal leukemia predisposition syndrome. Collectively, our findings identify mesenchymal niche-induced genotoxic stress in heterotypic stem and progenitor cells through inflammatory signaling as a targetable determinant of disease outcome in human pre-leukemia.

negative: Multiple degradation pathways regulate versatile CIP/KIP CDK inhibitors. The mammalian CIP/KIP family of cyclin-dependent kinase (CDK) inhibitors (CKIs) comprises three proteins--p21(Cip1/WAF1), p27(Kip1), and p57(Kip2)--that bind and inhibit cyclin-CDK complexes, which are key regulators of the cell cycle. CIP/KIP CKIs have additional independent functions in regulating transcription, apoptosis and actin cytoskeletal dynamics. These divergent functions are performed in distinct cellular compartments and contribute to the seemingly contradictory observation that the CKIs can both suppress and promote cancer. Multiple ubiquitin ligases (E3s) direct the proteasome-mediated degradation of p21, p27 and p57. This review analyzes recent data highlighting our current understanding of how distinct E3 pathways regulate subpopulations of the CKIs to control their diverse functions.

negative: KDM5B regulates embryonic stem cell self-renewal and represses cryptic intragenic transcription. Although regulation of histone methylation is believed to contribute to embryonic stem cell (ESC) self-renewal, the mechanisms remain obscure. We show here that the histone H3 trimethyl lysine 4 (H3K4me3) demethylase, KDM5B, is a downstream Nanog target and critical for ESC self-renewal. Although KDM5B is believed to function as a promoter-bound repressor, we find that it paradoxically functions as an activator of a gene network associated with self-renewal. ChIP-Seq reveals that KDM5B is predominantly targeted to intragenic regions and that it is recruited to H3K36me3 via an interaction with the chromodomain protein MRG15. Depletion of KDM5B or MRG15 increases intragenic H3K4me3, increases cryptic intragenic transcription, and inhibits transcriptional elongation of KDM5B target genes. We propose that KDM5B activates self-renewal-associated gene expression by repressing cryptic initiation and maintaining an H3K4me3 gradient important for productive transcriptional elongation.

negative: Alternative Lengthening of Telomeres Mediated by Mitotic DNA Synthesis Engages Break-Induced Replication Processes. Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. By analyzing telomerase-positive cells and their human TERC knockout-derived ALT human cell lines, we show that ALT cells harbor more fragile telomeres representing telomere replication problems. ALT-associated replication defects trigger mitotic DNA synthesis (MiDAS) at telomeres in a RAD52-dependent, but RAD51-independent, manner. Telomeric MiDAS is a conservative DNA synthesis process, potentially mediated by break-induced replication, similar to type II ALT survivors in Saccharomyces cerevisiae Replication stresses induced by ectopic oncogenic expression of cyclin E, G-quadruplexes, or R-loop formation facilitate the ALT pathway and lead to telomere clustering, a hallmark of ALT cancers. The TIMELESS/TIPIN complex suppresses telomere clustering and telomeric MiDAS, whereas the SMC5/6 complex promotes them. In summary, ALT cells exhibit more telomere replication defects that result in persistent DNA damage responses at telomeres, leading to the engagement of telomeric MiDAS (spontaneous mitotic telomere synthesis) that is triggered by DNA replication stress, a potential driver of genomic duplications in cancer.

negative: Key role of Ubc5 and lysine-63 polyubiquitination in viral activation of IRF3. The mitochondrial antiviral signaling protein (MAVS; also known as IPS-1, VISA, and CARDIF) is essential for innate immune response against RNA viruses. MAVS transduces signals from the cytosolic RIG-I-like receptors, which bind to viral RNAs. But how MAVS activates downstream transcription factors such as IRF3 to induce type-I interferons is not well understood. We have established a cell-free system in which mitochondria derived from virus-infected cells activate IRF3 in the cytosol. Fractionation of the cytosol led to the identification of Ubc5 as a ubiquitin-conjugating enzyme (E2) required for IRF3 activation. Using an inducible RNAi strategy, we demonstrate that catalytically active Ubc5 is required for IRF3 activation by viral infection. The activation of IRF3 also requires two ubiquitin-binding domains of NEMO. Furthermore, we show that replacement of endogenous ubiquitin with its K63R mutant abolishes viral activation of IRF3, demonstrating that K63 polyubiquitination plays a key role in IRF3 activation.

negative: The INO80 chromatin remodeling complex in transcription, replication and repair. The Ino80 ATPase is a member of the SNF2 family of ATPases and functions as an integral component of a multisubunit ATP-dependent chromatin remodeling complex. Although INO80 complexes from yeast and higher eukaryotes share a common core of conserved subunits, the complexes have diverged substantially during evolution and have acquired new subunits with apparently species-specific functions. Recent studies have shown that the INO80 complex contributes to a wide variety of chromatin-dependent nuclear transactions, including transcription, DNA repair and DNA replication.

negative: PCNA functions as a molecular platform to trigger Cdt1 destruction and prevent re-replication Ubiquitin-mediated proteolysis of the replication licensing factor Cdt1 (Cdc10-dependent transcript 1) in S phase is a key mechanism that limits DNA replication to a single round per cell cycle in metazoans. In Xenopus egg extracts, Cdt1 is destroyed on chromatin during DNA replication. Here, we report that replication-dependent proteolysis of Cdt1 requires its interaction with proliferating cell nuclear antigen (PCNA), a homotrimeric processivity factor for DNA polymerases. Cdt1 binds to PCNA through a consensus PCNA-interaction motif that is conserved in Cdt1 of all metazoans, and removal of PCNA from egg extracts inhibits replication-dependent Cdt1 destruction. Mutation of the PCNA-interaction motif yields a stabilized Cdt1 protein that induces re-replication. DDB1, a component of the Cul4 E3 ubiquitin ligase that mediates human Cdt1 proteolysis in response to DNA damage, is also required for replication-dependent Cdt1 destruction. Cdt1 and DDB1 interact in extracts, and DDB1 chromatin loading is dependent on the binding of Cdt1 to PCNA, which indicates that PCNA docking activates the pre-formed Cdt1–Cul4DDB1 ligase complex. Thus, PCNA functions as a platform for Cdt1 destruction, ensuring efficient and temporally restricted inactivation of a key cell-cycle regulator.

negative: Endocytic Trafficking of Rac Is Required for the Spatial Restriction of Signaling in Cell Migration The small GTPases, Rab5 and Rac, are essential for endocytosis and actin remodeling, respectively. Coordination of these processes is critical to achieve spatial restriction of intracellular signaling, which is essential for a variety of polarized functions. Here, we show that clathrin- and Rab5-mediated endocytosis are required for the activation of Rac induced by motogenic stimuli. Rac activation occurs on early endosomes, where the RacGEF Tiam1 is also recruited. Subsequent recycling of Rac to the plasma membrane ensures localized signaling, leading to the formation of actin-based migratory protrusions. Thus, membrane trafficking of Rac is required for the spatial resolution of Rac-dependent motogenic signals. We further demonstrate that a Rab5-to-Rac circuitry controls the morphology of motile mammalian tumor cells and primordial germinal cells during zebrafish development, suggesting that this circuitry is relevant for the regulation of migratory programs in various cells, in both in vitro settings and whole organisms.

negative: The fidelity of the ligation step determines how ends are resolved during Nonhomologous end joining Nonhomologous end joining (NHEJ) can effectively resolve chromosome breaks despite diverse end structures; however, it is unclear how the steps employed for resolution are determined. We sought to address this question by analysing cellular NHEJ of ends with systematically mispaired and damaged termini. We show NHEJ is uniquely proficient at bypassing subtle terminal mispairs and radiomimetic damage by direct ligation. Nevertheless, bypass ability varies widely, with increases in mispair severity gradually reducing bypass products from 85 to 6%. End-processing by nucleases and polymerases is increased to compensate, although paths with the fewest number of steps to generate a substrate suitable for ligation are favoured. Thus, both the frequency and nature of end processing are tailored to meet the needs of the ligation step. We propose a model where the ligase organizes all steps during NHEJ within the stable paired-end complex to limit end processing and associated errors.

negative: Phosphorylation of histone H3(T118) alters nucleosome dynamics and remodeling Nucleosomes, the fundamental units of chromatin structure, are regulators and barriers to transcription, replication and repair. Post-translational modifications (PTMs) of the histone proteins within nucleosomes regulate these DNA processes. Histone H3(T118) is a site of phosphorylation [H3(T118ph)] and is implicated in regulation of transcription and DNA repair. We prepared H3(T118ph) by expressed protein ligation and determined its influence on nucleosome dynamics. We find H3(T118ph) reduces DNA-histone binding by 2  kcal/mol, increases nucleosome mobility by 28-fold and increases DNA accessibility near the dyad region by 6-fold. Moreover, H3(T118ph) increases the rate of hMSH2-hMSH6 nucleosome disassembly and enables nucleosome disassembly by the SWI/SNF chromatin remodeler. These studies suggest that H3(T118ph) directly enhances and may reprogram chromatin remodeling reactions.

negative: The anaphase promoting complex/cyclosome: a machine designed to destroy The anaphase promoting complex/cyclosome (APC/C) is a ubiquitin ligase that has essential functions in and outside the eukaryotic cell cycle. It is the most complex molecular machine that is known to catalyse ubiquitylation reactions, and it contains more than a dozen subunits that assemble into a large 1.5-MDa complex. Recent discoveries have revealed an unexpected multitude of mechanisms that control APC/C activity, and have provided a first insight into how this unusual ubiquitin ligase recognizes its substrates.

negative: TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Human embryonic stem cells (hESCs) self-renew indefinitely and give rise to derivatives of all three primary germ layers, yet little is known about the signaling cascades that govern their pluripotent character. Because it plays a prominent role in the early cell fate decisions of embryonic development, we have examined the role of TGFbeta superfamily signaling in hESCs. We found that, in undifferentiated cells, the TGFbeta/activin/nodal branch is activated (through the signal transducer SMAD2/3) while the BMP/GDF branch (SMAD1/5) is only active in isolated mitotic cells. Upon early differentiation, SMAD2/3 signaling is decreased while SMAD1/5 signaling is activated. We next tested the functional role of TGFbeta/activin/nodal signaling in hESCs and found that it is required for the maintenance of markers of the undifferentiated state. We extend these findings to show that SMAD2/3 activation is required downstream of WNT signaling, which we have previously shown to be sufficient to maintain the undifferentiated state of hESCs. Strikingly, we show that in ex vivo mouse blastocyst cultures, SMAD2/3 signaling is also required to maintain the inner cell mass (from which stem cells are derived). These data reveal a crucial role for TGFbeta signaling in the earliest stages of cell fate determination and demonstrate an interconnection between TGFbeta and WNT signaling in these contexts.

negative: Histone levels are regulated by phosphorylation and ubiquitylation dependent proteolysis Histone levels are tightly regulated to prevent harmful effects such as genomic instability and hypersensitivity to DNA-damaging agents due to the accumulation of these highly basic proteins when DNA replication slows down or stops. Although chromosomal histones are stable, excess (non-chromatin bound) histones are rapidly degraded in a Rad53 (radiation sensitive 53) kinase-dependent manner in Saccharomyces cerevisiae. Here we demonstrate that excess histones associate with Rad53 in vivo and seem to undergo modifications such as tyrosine phosphorylation and polyubiquitylation, before their proteolysis by the proteasome. We have identified the Tyr 99 residue of histone H3 as being critical for the efficient ubiquitylation and degradation of this histone. We have also identified the ubiquitin conjugating enzymes (E2) Ubc4 and Ubc5, as well as the ubiquitin ligase (E3) Tom1 (temperature dependent organization in mitotic nucleus 1), as enzymes involved in the ubiquitylation of excess histones. Regulated histone proteolysis has major implications for the maintenance of epigenetic marks on chromatin, genomic stability and the packaging of sperm DNA.