Histone H2AK119 Mono-Ubiquitination Is Essential for Polycomb-Mediated Transcriptional Repression.

Polycomb group proteins (PcGs) maintain transcriptional repression to preserve cellular identity in two distinct repressive complexes, PRC1 and PRC2, that modify histones by depositing H2AK119ub1 and H3K27me3, respectively. PRC1 and PRC2 exist in different variants and show a complex regulatory cross-talk. However, the contribution that H2AK119ub1 plays in mediating PcG repressive functions remains largely controversial. Using a fully catalytic inactive RING1B mutant, we demonstrated that H2AK119ub1 deposition is essential to maintain PcG-target gene repression in embryonic stem cells (ESCs). Loss of H2AK119ub1 induced a rapid displacement of PRC2 activity and a loss of H3K27me3 deposition. This preferentially affected PRC2.2 variant with respect to PRC2.1, destabilizing canonical PRC1 activity. Finally, we found that variant PRC1 forms can sense H2AK119ub1 deposition, which contributes to their stabilization specifically at sites where this modification is highly enriched. Overall, our data place H2AK119ub1 deposition as a central hub that mounts PcG repressive machineries to preserve cell transcriptional identity.

R-Loops Enhance Polycomb Repression at a Subset of Developmental Regulator Genes.

R-loops are three-stranded nucleic acid structures that form during transcription, especially over unmethylated CpG-rich promoters of active genes. In mouse embryonic stem cells (mESCs), CpG-rich developmental regulator genes are repressed by the Polycomb complexes PRC1 and PRC2. Here, we show that R-loops form at a subset of Polycomb target genes, and we investigate their contribution to Polycomb repression. At R-loop-positive genes, R-loop removal leads to decreased PRC1 and PRC2 recruitment and Pol II activation into a productive elongation state, accompanied by gene derepression at nascent and processed transcript levels. Stable removal of PRC2 derepresses R-loop-negative genes, as expected, but does not affect R-loops, PRC1 recruitment, or transcriptional repression of R-loop-positive genes. Our results highlight that Polycomb repression does not occur via one mechanism but consists of different layers of repression, some of which are gene specific. We uncover that one such mechanism is mediated by an interplay between R-loops and RING1B recruitment.

Functional Landscape of PCGF Proteins Reveals Both RING1A/B-Dependent-and RING1A/B-Independent-Specific Activities.

Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) control cell identity by establishing facultative heterochromatin repressive domains at common sets of target genes. PRC1, which deposits H2Aub1 through the E3 ligases RING1A/B, forms six biochemically distinct subcomplexes depending on the assembled PCGF protein (PCGF1-PCGF6); however, it is yet unclear whether these subcomplexes have also specific activities. Here we show that PCGF1 and PCGF2 largely compensate for each other, while other PCGF proteins have high levels of specificity for distinct target genes. PCGF2 associates with transcription repression, whereas PCGF3 and PCGF6 associate with actively transcribed genes. Notably, PCGF3 and PCGF6 complexes can assemble and be recruited to several active sites independently of RING1A/B activity (therefore, of PRC1). For chromatin recruitment, the PCGF6 complex requires the combinatorial activities of its MGA-MAX and E2F6-DP1 subunits, while PCGF3 requires an interaction with the USF1 DNA binding transcription factor.

PRC1 catalytic unit RING1B regulates early neural differentiation of human pluripotent stem cells.

BACKGROUND: Polycomb group (PcG) proteins are histone modifiers which control gene expression by assembling into large repressive complexes termed - Polycomb repressive complex (PRC); RING1B, core catalytic subunit of PRC1 that performs H2AK119 monoubiquitination leading to gene repression. The role of PRC1 complex during early neural specification in humans is unclear; we have tried to uncover the role of PRC1 in neuronal differentiation using human pluripotent stem cells as an in vitro model. RESULTS: We differentiated both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) towards neural progenitor stage evident from the expression of NESTIN, TUJ1, NCAD, and PAX6. When we checked the total expression of RING1B and BMI1, we saw that they were significantly upregulated in differentiated neural progenitors compared to undifferentiated cells. Further, we used Chromatin Immunoprecipitation coupled with qPCR to determine the localization of RING1B, and the repressive histone modification H2AK119ub1 at the promoters of neuronal specific genes. We observed that RING1B localized to and catalyzed H2AK119ub1 modification at promoters of TUJ1, NCAM, and NESTIN during early differentiation and later RING1B was lost from its promoter leading their expression; while functional RING1B persisted significantly on mature neuronal genes such as IRX3, GSX2, SOX1, NEUROD1 and FOXG1 in neural progenitors. CONCLUSION: The results of our study show that PRC1 catalytic component RING1B occupies neuronal gene promoters in human pluripotent stem cells and may prevent their precocious expression. However, when neuronal inductive signals are given, RING1B is not only removed from neuronal gene promoters, but the inhibitory H2AK119ub1 modification is also lost.

Polycomb repressive complex 1: Regulators of neurogenesis from embryonic to adult stage.

Development of vertebrate nervous system is a complex process which involves differential gene expression and disruptions in this process or in the mature brain, may lead to neurological disorders and diseases. Extensive work that spanned several decades using rodent models and recent work on stem cells have helped uncover the intricate process of neuronal differentiation and maturation. There are various morphological changes, genetic and epigenetic modifications which occur during normal mammalian neural development, one of the chromatin modifications that controls vital gene expression are the posttranslational modifications on histone proteins, that controls accessibility of translational machinery. Among the histone modifiers, polycomb group proteins (PcGs), such as Ezh2, Eed and Suz12 form large protein complexes-polycomb repressive complex 2 (PRC2); while Ring1b and Bmi1 proteins form core of PRC1 along with accessory proteins such as Cbx, Hph, Rybp and Pcgfs catalyse histone modifications such as H3K27me3 and H2AK119ub1. PRC1 proteins are known to play critical role in X chromosome inactivation in females but they also repress the expression of key developmental genes and tightly regulate the mammalian neuronal development. In this review we have discussed the signalling pathways, morphogens and nuclear factors that initiate, regulate and maintain cells of the nervous system. Further, we have extensively reviewed the recent literature on the role of Ring1b and Bmi1 in mammalian neuronal development and differentiation; as well as highlighted questions that are still unanswered.

Inhibition of RING1B alters lineage specificity in human embryonic stem cells.

Polycomb group (PcG) proteins are histone modifiers which are known to perform transcriptional repression and have been shown to be critical during murine embryonic development. PcGs are broadly characterized into polycomb repressive complex 1 (PRC1) and 2 and (PRC2). RING1B, core catalytic unit of PRC1 performs H2AK119 monoubiquitination leading to transcriptional repression. We used human embryonic stem cell (hESC) line to study the fate of pluripotent stem cells (PSCs) under inhibition of RING1B, as its role in human development is still to be completely explored. Embryoid bodies (EBs) were generated to differentiate hESCs using hanging drop method. PRT4165 (synthetic RING1B catalytic activity inhibitor) was added to undifferentiated and differentiated cells for 24 h. When we inhibited RING1B in undifferentiated cells, OCT4 levels remained unchanged indicating RING1B does not regulate pluripotency. The drug when added to differentiated cells led to decrease in the levels of RING1B, BMI1, and H2AK119ub1. Interestingly, we also report that the differentiated cells show an increased expression of neuroectodermal markers: SOX1 and PAX6 as well as expression of other neuroectodermal markers such as TUJ1, FOXG1, and NCAM. However, there was reduction in expression of endodermal (SOX17 and FOXA2) mesodermal marker BRACHYURY and TBX5 in treated EBs compared with control EBs. In summary, alteration of RING1B catalytic activity in hESCs during differentiation promotes neuroectodermal differentiation thus, we demonstrate critical role of RING1B in regulating neural differentiation. The strategy of inhibiting RING1B could be used to direct PSCs towards early neuronal fate.

Intrinsically disordered chromatin protein NUPR1 binds to the C-terminal region of Polycomb RING1B.

Intrinsically disordered proteins (IDPs) are ubiquitous in eukaryotes, and they are often associated with diseases in humans. The protein NUPR1 is a multifunctional IDP involved in chromatin remodeling and in the development and progression of pancreatic cancer; however, the details of such functions are unknown. Polycomb proteins are involved in specific transcriptional cascades and gene silencing. One of the proteins of the Polycomb complex is the Ring finger protein 1 (RING1). RING1 is related to aggressive tumor features in multiple cancer types. In this work we characterized the interaction between NUPR1 and the paralogue RING1B in vitro, in silico, and in cellulo. The interaction occurred through the C-terminal region of RING1B (C-RING1B), with an affinity in the low micromolar range (∼10 µM). The binding region of NUPR1, mapped by NMR, was a hydrophobic polypeptide patch at the 30s region of its sequence, as pinpointed by computational results and site-directed mutagenesis at Ala33. The association between C-RING1B and wild-type NUPR1 also occurred in cellulo as tested by protein ligation assays; this interaction is inhibited by trifluoperazine, a drug known to hamper binding of wild-type NUPR1 with other proteins. Furthermore, the Thr68Gln and Ala33Gln/Thr68Gln mutants had a reduction in the binding toward C-RING1B as shown by in vitro, in silico, and in cellulo studies. This is an example of a well-folded partner of NUPR1, because its other interacting proteins are also unfolded. We hypothesize that NUPR1 plays an active role in chromatin remodeling and carcinogenesis, together with Polycomb proteins.

Ring1b-dependent epigenetic remodelling is an essential prerequisite for pancreatic carcinogenesis.

BACKGROUND AND AIMS: Besides well-defined genetic alterations, the dedifferentiation of mature acinar cells is an important prerequisite for pancreatic carcinogenesis. Acinar-specific genes controlling cell homeostasis are extensively downregulated during cancer development; however, the underlying mechanisms are poorly understood. Now, we devised a novel in vitro strategy to determine genome-wide dynamics in the epigenetic landscape in pancreatic carcinogenesis. DESIGN: With our in vitro carcinogenic sequence, we performed global gene expression analysis and ChIP sequencing for the histone modifications H3K4me3, H3K27me3 and H2AK119ub. Followed by a comprehensive bioinformatic approach, we captured gene clusters with extensive epigenetic and transcriptional remodelling. Relevance of Ring1b-catalysed H2AK119ub in acinar cell reprogramming was studied in an inducible Ring1b knockout mouse model. CRISPR/Cas9-mediated Ring1b ablation as well as drug-induced Ring1b inhibition were functionally characterised in pancreatic cancer cells. RESULTS: The epigenome is vigorously modified during pancreatic carcinogenesis, defining cellular identity. Particularly, regulatory acinar cell transcription factors are epigenetically silenced by the Ring1b-catalysed histone modification H2AK119ub in acinar-to-ductal metaplasia and pancreatic cancer cells. Ring1b knockout mice showed greatly impaired acinar cell dedifferentiation and pancreatic tumour formation due to a retained expression of acinar differentiation genes. Depletion or drug-induced inhibition of Ring1b promoted tumour cell reprogramming towards a less aggressive phenotype. CONCLUSIONS: Our data provide substantial evidence that the epigenetic silencing of acinar cell fate genes is a mandatory event in the development and progression of pancreatic cancer. Targeting the epigenetic repressor Ring1b could offer new therapeutic options.

Polycomb RING1A- and RING1B-dependent histone H2A monoubiquitylation at pericentromeric regions promotes S-phase progression.

The functions of polycomb products extend beyond their well-known activity as transcriptional regulators to include genome duplication processes. Polycomb activities during DNA replication and DNA damage repair are unclear, particularly without induced replicative stress. We have used a cellular model of conditionally inactive polycomb E3 ligases (RING1A and RING1B), which monoubiquitylate lysine 119 of histone H2A (H2AK119Ub), to examine DNA replication in unperturbed cells. We identify slow elongation and fork stalling during DNA replication that is associated with the accumulation of mid and late S-phase cells. Signs of replicative stress and colocalisation of double-strand breaks with chromocenters, the sites of coalesced pericentromeric heterocromatic (PCH) domains, were enriched in cells at mid S-phase, the stage at which PCH is replicated. Altered replication was rescued by targeted monoubiquitylation of PCH through methyl-CpG binding domain protein 1. The acute senescence associated with the depletion of RING1 proteins, which is mediated by p21 (also known as CDKN1A) upregulation, could be uncoupled from a response to DNA damage. These findings link cell proliferation and the polycomb proteins RING1A and RING1B to S-phase progression through a specific function in PCH replication.

The FBXL10/KDM2B scaffolding protein associates with novel polycomb repressive complex-1 to regulate adipogenesis.

Polycomb repressive complex 1 (PRC1) plays an essential role in the epigenetic repression of gene expression during development and cellular differentiation via multiple effector mechanisms, including ubiquitination of H2A and chromatin compaction. However, whether it regulates the stepwise progression of adipogenesis is unknown. Here, we show that FBXL10/KDM2B is an anti-adipogenic factor that is up-regulated during the early phase of 3T3-L1 preadipocyte differentiation and in adipose tissue in a diet-induced model of obesity. Interestingly, inhibition of adipogenesis does not require the JmjC demethylase domain of FBXL10, but it does require the F-box and leucine-rich repeat domains, which we show recruit a noncanonical polycomb repressive complex 1 (PRC1) containing RING1B, SKP1, PCGF1, and BCOR. Knockdown of either RING1B or SKP1 prevented FBXL10-mediated repression of 3T3-L1 preadipocyte differentiation indicating that PRC1 formation mediates the inhibitory effect of FBXL10 on adipogenesis. Using ChIP-seq, we show that FBXL10 recruits RING1B to key specific genomic loci surrounding the key cell cycle and the adipogenic genes Cdk1, Uhrf1, Pparg1, and Pparg2 to repress adipogenesis. These results suggest that FBXL10 represses adipogenesis by targeting a noncanonical PRC1 complex to repress key genes (e.g. Pparg) that control conversion of pluripotent cells into the adipogenic lineage.

Analysis of Polycomb Epigenetic Marks in HeLa Spheroids.

Biochemical composition of the nucleus affects both its physical properties and its morphology. In recent years, several studies demonstrated the formation of f-actin in the nuclei. These filaments intermingle with the chromatin fibers underlying the crucial role of the mechanical force in chromatin remodeling, being thus involved in transcription, differentiation, replication, and DNA repair. Given the suggested role of Ezh2 in the cross-talk between f-actin and chromatin, we describe here how to obtain HeLa cell spheroids and a method to perform immunofluorescence analysis of nuclear epigenetic marks in a 3D cell culture system.

Targeted Degradation of PRC1 Components, BMI1 and RING1B, via a Novel Protein Complex Degrader Strategy.

Polycomb repressive complex 1 (PRC1) is an essential epigenetic regulator that mainly controls histone H2A Lys119 mono-ubiquitination (H2AK119ub). B cell-specific Moloney murine leukemia virus Integration site 1 (BMI1) and really interesting new gene 1B (RING1B) are PRC1 core components and play critical roles in the development of various cancers. However, therapeutic agents targeting PRC1 are very limited. In this study, MS147, the first degrader of PRC1 core components, BMI1 and RING1B, is discovered via a novel protein complex degradation strategy that utilizes the target protein's interacting partner protein (embryonic ectoderm development (EED)). MS147, which comprises an EED small-molecule binder linked to a ligand of the E3 ligase von Hippel-Lindau (VHL), degrades BMI1/RING1B in an EED-, VHL-, ubiquitination-, and time-dependent manner. MS147 preferentially degrades BMI1/RING1B over polycomb repressive complex 2 (PRC2) core components. Consequently, MS147 effectively reduces H2AK119ub, but not histone H3 Lys27 tri-methylation (H3K27me3), which is catalyzed by PRC2. Furthermore, MS147 effectively inhibits the proliferation of cancer cell lines that are insensitive to PRC2 inhibitors/degraders. Overall, this study provides a novel BMI1/RING1B degrader, which is a useful chemical tool to further investigate the roles of PRC1 in cancer, and a novel protein complex degradation strategy, which can potentially expand the degradable human proteome.

ChIP-qPCR for Polycomb Group Proteins During Neuronal Differentiation of Human Pluripotent Stem Cells.

Neuronal differentiation is an intricate and a complex process which involves crosstalk among various signaling pathways, growth factors, transcription factors, and epigenetic modifiers. During different stages of neuronal development, there are various histone modifiers which drive the expression of lineage-specific genes. Polycomb group proteins are one of the histone modifiers that control transcriptional repression of specific genes in development, differentiation, and functionality of various tissues. Chromatin immunoprecipitation (ChIP) is a widely used technique to investigate the interaction of proteins and DNA; ChIP combined with quantitative real-time PCR (qPCR) gives a quantitative data about the occupancy of specific protein on a particular stretch of DNA, and this can help us investigate how a protein regulates expression of a specific gene. In this chapter, we describe a protocol for ChIP coupled to qPCR during early neuronal differentiation to identify the specific genomic targets regulated by components of Polycomb repressive complex 1.

Bmi-1-RING1B prevents GATA4-dependent senescence-associated pathological cardiac hypertrophy by promoting autophagic degradation of GATA4.

AIMS: Senescence-associated pathological cardiac hypertrophy (SA-PCH) is associated with upregulation of foetal genes, fibrosis, senescence-associated secretory phenotype (SASP), cardiac dysfunction and increased morbidity and mortality. Therefore, we conducted experiments to investigate whether GATA4 accumulation induces SA-PCH, and whether Bmi-1-RING1B promotes GATA4 ubiquitination and its selective autophagic degradation to prevent SA-PCH. METHODS AND RESULTS: Bmi-1-deficient (Bmi-1-/- ), transgenic Bmi-1 overexpressing (Bmi-1Tg ) and wild-type (WT) mice were infused with angiotensin II (Ang II) to stimulate the development of SA-PCH. Through bioinformatics analysis with RNA sequencing data from cardiac tissues, we found that Bmi-1-RING1B and autophagy are negatively related to SA-PCH. Bmi-1 deficiency promoted GATA4-dependent SA-PCH by increasing GATA4 protein and hypertrophy-related molecules transcribed by GATA4 such as ANP and BNP. Bmi-1 deficiency stimulated NF-κB-p65-dependent SASP, leading to cardiac dysfunction, cardiomyocyte hypertrophy and senescence. Bmi-1 overexpression repressed GATA4-dependent SA-PCH. GATA4 degraded by Bmi-1 was mainly dependent on autophagy rather than proteasome. In human myocardium, p16 positively correlated with ANP and GATA4 and negatively correlated with LC3B, Bmi-1 and RING1B; GATA4 positively correlated with p62 and negatively correlated with Bmi-1 and LC3B. With increased p16 protein levels, ANP-, BNP- and GATA4-positive cells or areas increased; however, LC3B-positive cells or areas decreased in human myocardium. GATA4 is ubiquitinated after combining with Bmi-1-RING1B, which is then recognised by p62, is translocated to autophagosomes to form autophagolysosomes and degraded. Downregulated GATA4 ameliorated SA-PCH and cardiac dysfunction by reducing GATA4-dependent hypertrophy and SASP-related molecules. Bmi-1 combined with RING1B (residues 1-179) and C-terminus of GATA4 (residues 206-443 including zinc finger domains) through residues 1-95, including a RING-HC-finger. RING1B combined with C-terminus of GATA4 through the C-terminus (residues 180-336). Adeno-associated viral vector serotype 9 (AAV9)-cytomegalovirus (CMV)-Bmi-1-RING1B treatment significantly attenuated GATA4-dependent SA-PCH through promoting GATA4 autophagic degradation. CONCLUSIONS: Bmi-1-RING1B maintained cardiac function and prevented SA-PCH by promoting selective autophagy for degrading GATA4. TRANSLATIONAL PERSPECTIVE: AAV9-CMV-Bmi-1-RING1B could be used for translational gene therapy to ubiquitinate GATA4 and prevent GATA4-dependent SA-PCH. Also, the combined domains between Bmi-1-RING1B and GATA4 in aging cardiomyocytes could be therapeutic targets for identifying stapled peptides in clinical applications to promote the combination of Bmi-1-RING1B with GATA4 and the ubiquitination of GATA4 to prevent SA-PCH and heart failure. We found that degradation of cardiac GATA4 by Bmi-1 was mainly dependent on autophagy rather than proteasome, and autophagy agonists metformin and rapamycin could ameliorate the SA-PCH, suggesting that activation of autophagy with metformin or rapamycin could also be a promising method to prevent SA-PCH.

Functions of Polycomb Proteins on Active Targets.

Chromatin regulators of the Polycomb group of genes are well-known by their activities as transcriptional repressors. Characteristically, their presence at genomic sites occurs with specific histone modifications and sometimes high-order chromatin structures correlated with silencing of genes involved in cell differentiation. However, evidence gathered in recent years, on flies and mammals, shows that in addition to these sites, Polycomb products bind to a large number of active regulatory regions. Occupied sites include promoters and also intergenic regions, containing enhancers and super-enhancers. Contrasting with occupancies at repressed targets, characteristic histone modifications are low or undetectable. Functions on active targets are dual, restraining gene expression at some targets while promoting activity at others. Our aim here is to summarize the evidence available and discuss the convenience of broadening the scope of research to include Polycomb functions on active targets.

Pancreas development and the Polycomb group protein complexes.

The dual nature of pancreatic tissue permits both endocrine and exocrine functions. Enzymatic secretions by the exocrine pancreas help digestive processes while the pancreatic hormones regulate glucose homeostasis and energy metabolism. Pancreas organogenesis is defined by a conserved array of signaling pathways that act on common gut progenitors to bring about the generation of diverse cell types. Multiple cellular processes characterize development of the mature organ. These processes are mediated by signaling pathways that regulate lineage-specific transcription factors and chromatin modifications guiding long-term gene expression programs. The chromatin landscape is altered chiefly by DNA or histone modifications, chromatin remodelers, and non-coding RNAs. Amongst histone modifiers, several studies have identified Polycomb group (PcG) proteins as crucial determinants mediating transcriptional repression of genes involved in developmental processes. Although PcG-mediated chromatin modifications define cellular transitions and influence cell identity of multipotent progenitors, much remains to be understood regarding coordination between extracellular signals and their impact on Polycomb functions during the pancreas lineage progression. In this review, we discuss interactions between sequence-specific DNA binding proteins and chromatin regulators underlying pancreas development and insulin producing ß-cells, with particular focus on Polycomb group proteins. Understanding such basic molecular mechanisms would improve current strategies for stem cell-based differentiation while also help elucidate the pathogenesis of several pancreas-related maladies, including diabetes and pancreatic cancer.

Writing Histone Monoubiquitination in Human Malignancy-The Role of RING Finger E3 Ubiquitin Ligases.

There is growing evidence highlighting the importance of monoubiquitination as part of the histone code. Monoubiquitination, the covalent attachment of a single ubiquitin molecule at specific lysines of histone tails, has been associated with transcriptional elongation and the DNA damage response. Sites function as scaffolds or docking platforms for proteins involved in transcription or DNA repair; however, not all sites are equal, with some sites resulting in actively transcribed chromatin and others associated with gene silencing. All events are written by E3 ubiquitin ligases, predominantly of the RING (really interesting new gene) finger type. One of the most well-studied events is monoubiquitination of histone H2B at lysine 120 (H2Bub1), written predominantly by the RING finger complex RNF20-RNF40 and generally associated with active transcription. Monoubiquitination of histone H2A at lysine 119 (H2AK119ub1) is also well-studied, its E3 ubiquitin ligase constituting part of thePolycomb Repressor Complex 1 (PRC1), RING1B-BMI1, associated with transcriptional silencing. Both modifications are activated as part of the DNA damage response. Histone monoubiquitination is a key epigenomic event shaping the chromatin landscape of malignancy and influencing how cells respond to DNA damage. This review discusses a number of these sites and the E3 RING finger ubiquitin ligases that write them.

4-Nonylphenol-enhanced EZH2 and RNF2 expression, H3K27me3 and H2AK119ub1 marks resulting in silencing of p21CDKN1A in vitro.

Aim: To examine the impact of 4-nonylphenol (4-NP), on the expression of polycomb repressive complexes and cellular proliferation. Materials & methods: Cell proliferation assays, quantitative PCR, Western blotting, luciferase reporter assay, chromatin immunoprecipitation-quantitative PCR were used for the study. Results: The 4-NP at 100 nM concentration significantly increased proliferation of MCF-7 cells. It enhanced the expression of RNF2-BMI1 and EZH2-SUZ12 and concomitantly increased H2AK119ub1 and H3K27me3 repressive marks at p21 proximal promoter resulting in its reduced expression. Selective inhibition of RNF2 or EZH2 reverted the 4-NP action. The phospho-CREB, SP1 and E2F-1 are enriched at proximal promoter of RNF2 and EZH2 and cyclin D1, but not p21. Conclusion: The 4-NP-mediated upregulation of RNF2 and EZH2 resulted in epigenetic silencing of p21.

Regulation of Mesenchymal Stem to Transit-Amplifying Cell Transition in the Continuously Growing Mouse Incisor.

In adult tissues and organs with high turnover rates, the generation of transit-amplifying cell (TAC) populations from self-renewing stem cells drives cell replacement. The role of stem cells is to provide a renewable source of cells that give rise to TACs to provide the cell numbers that are necessary for cell differentiation. Regulation of the formation of TACs is thus fundamental to controlling cell replacement. Here, we analyze the properties of a population of mesenchymal TACs in the continuously growing mouse incisor to identify key components of the molecular regulation that drives proliferation. We show that the polycomb repressive complex 1 acts as a global regulator of the TAC phenotype by its direct action on the expression of key cell-cycle regulatory genes and by regulating Wnt/ß-catenin-signaling activity. We also identify an essential requirement for TACs in maintaining mesenchymal stem cells, which is indicative of a positive feedback mechanism.

Smchd1 Targeting to the Inactive X Is Dependent on the Xist-HnrnpK-PRC1 Pathway.

We and others have recently reported that the SMC protein Smchd1 is a regulator of chromosome conformation. Smchd1 is critical for the structure of the inactive X chromosome and at autosomal targets such as the Hox genes. However, it is unknown how Smchd1 is recruited to these sites. Here, we report that Smchd1 localizes to the inactive X via the Xist-HnrnpK-PRC1 (polycomb repressive complex 1) pathway. Contrary to previous reports, Smchd1 does not bind Xist or other RNA molecules with any specificity. Rather, the localization of Smchd1 to the inactive X is H2AK119ub dependent. Following perturbation of this interaction, Smchd1 is destabilized, which has consequences for gene silencing genome-wide. Our work adds Smchd1 to the PRC1 silencing pathway for X chromosome inactivation.