Polycomb-mediated Gene Repression Research Articles

Loss of Ezh2 function remodels the DNA replication initiation landscape

In metazoan cells, DNA replication initiates from thousands of genomic loci scattered throughout the genome called DNA replication origins. Origins are strongly associated with euchromatin, particularly open genomic regions such as promoters and enhancers. However, over a third of transcriptionally silent genes are associated with DNA replication initiation. Most of these genes are bound and repressed by the Polycomb repressive complex-2 (PRC2) through the repressive H3K27me3 mark. This is the strongest overlap observed for a chromatin regulator with replication origin activity. Here, we asked whether Polycomb-mediated gene repression is functionally involved in recruiting DNA replication origins to transcriptionally silent genes. We show that the absence of EZH2, the catalytic subunit of PRC2, results in increased DNA replication initiation, specifically in the vicinity of EZH2 binding sites. The increase in DNA replication initiation does not correlate with transcriptional de-repression or the acquisition of activating histone marks but does correlate with loss of H3K27me3 from bivalent promoters.

RedChIP identifies noncoding RNAs associated with genomic sites occupied by Polycomb and CTCF proteins

Nuclear noncoding RNAs (ncRNAs) are key regulators of gene expression and chromatin organization. The progress in studying nuclear ncRNAs depends on the ability to identify the genome-wide spectrum of contacts of ncRNAs with chromatin. To address this question, a panel of RNA–DNA proximity ligation techniques has been developed. However, neither of these techniques examines proteins involved in RNA–chromatin interactions. Here, we introduce RedChIP, a technique combining RNA–DNA proximity ligation and chromatin immunoprecipitation for identifying RNA–chromatin interactions mediated by a particular protein. Using antibodies against architectural protein CTCF and the EZH2 subunit of the Polycomb repressive complex 2, we identify a spectrum of cis- and trans-acting ncRNAs enriched at Polycomb- and CTCF-binding sites in human cells, which may be involved in Polycomb-mediated gene repression and CTCF-dependent chromatin looping. By providing a protein-centric view of RNA–DNA interactions, RedChIP represents an important tool for studies of nuclear ncRNAs.

PRC1 drives Polycomb-mediated gene repression by controlling transcription initiation and burst frequency.

The Polycomb repressive system plays a fundamental role in controlling gene expression during mammalian development. To achieve this, Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) bind target genes and use histone modification-dependent feedback mechanisms to form Polycomb chromatin domains and repress transcription. The inter-relatedness of PRC1 and PRC2 activity at these sites has made it difficult to discover the specific components of Polycomb chromatin domains that drive gene repression and to understand mechanistically how this is achieved. Here, by exploiting rapid degron-based approaches and time-resolved genomics, we kinetically dissect Polycomb-mediated repression and discover that PRC1 functions independently of PRC2 to counteract RNA polymerase II binding and transcription initiation. Using single-cell gene expression analysis, we reveal that PRC1 acts uniformly within the cell population and that repression is achieved by controlling transcriptional burst frequency. These important new discoveries provide a mechanistic and conceptual framework for Polycomb-dependent transcriptional control.

Removal of H2Aub1 by ubiquitin-specific proteases 12 and 13 is required for stable Polycomb-mediated gene repression in Arabidopsis

BackgroundStable gene repression is essential for normal growth and development. Polycomb repressive complexes 1 and 2 (PRC1&2) are involved in this process by establishing monoubiquitination of histone 2A (H2Aub1) and subsequent trimethylation of lysine 27 of histone 3 (H3K27me3). Previous work proposed that H2Aub1 removal by the ubiquitin-specific proteases 12 and 13 (UBP12 and UBP13) is part of the repressive PRC1&2 system, but its functional role remains elusive.ResultsWe show that UBP12 and UBP13 work together with PRC1, PRC2, and EMF1 to repress genes involved in stimulus response. We find that PRC1-mediated H2Aub1 is associated with gene responsiveness, and its repressive function requires PRC2 recruitment. We further show that the requirement of PRC1 for PRC2 recruitment depends on the initial expression status of genes. Lastly, we demonstrate that removal of H2Aub1 by UBP12/13 prevents loss of H3K27me3, consistent with our finding that the H3K27me3 demethylase REF6 is positively associated with H2Aub1.ConclusionsOur data allow us to propose a model in which deposition of H2Aub1 permits genes to switch between repression and activation by H3K27me3 deposition and removal. Removal of H2Aub1 by UBP12/13 is required to achieve stable PRC2-mediated repression.

If You Like It Then You Shoulda Put Two “RINGs” on It: Delineating the Roles of vPRC1 and cPRC1

To delineate the roles of variant (vPRC1) and canonical (cPRC1) Polycomb repressive complex 1, Blackledge etal. (2020) and Tamburri etal. (2020) elegantly disrupt RING1A/B catalytic activity without affecting stability of either complex and then explore the precise contribution of vPRC1-mediated H2AK119ub1 to Polycomb-mediated gene repression.

PRC1 Catalytic Activity Is Central to Polycomb System Function.

SummaryThe Polycomb repressive system is an essential chromatin-based regulator of gene expression. Despite being extensively studied, how the Polycomb system selects its target genes is poorly understood, and whether its histone-modifying activities are required for transcriptional repression remains controversial. Here, we directly test the requirement for PRC1 catalytic activity in Polycomb system function. To achieve this, we develop a conditional mutation system in embryonic stem cells that completely removes PRC1 catalytic activity. Using this system, we demonstrate that catalysis by PRC1 drives Polycomb chromatin domain formation and long-range chromatin interactions. Furthermore, we show that variant PRC1 complexes with DNA-binding activities occupy target sites independently of PRC1 catalytic activity, providing a putative mechanism for Polycomb target site selection. Finally, we discover that Polycomb-mediated gene repression requires PRC1 catalytic activity. Together these discoveries provide compelling evidence that PRC1 catalysis is central to Polycomb system function and gene regulation.

Synergy between Variant PRC1 Complexes Defines Polycomb-Mediated Gene Repression.

SummaryThe Polycomb system modifies chromatin and plays an essential role in repressing gene expression to control normal mammalian development. However, the components and mechanisms that define how Polycomb protein complexes achieve this remain enigmatic. Here, we use combinatorial genetic perturbation coupled with quantitative genomics to discover the central determinants of Polycomb-mediated gene repression in mouse embryonic stem cells. We demonstrate that canonical Polycomb repressive complex 1 (PRC1), which mediates higher-order chromatin structures, contributes little to gene repression. Instead, we uncover an unexpectedly high degree of synergy between variant PRC1 complexes, which is fundamental to gene repression. We further demonstrate that variant PRC1 complexes are responsible for distinct pools of H2A monoubiquitylation that are associated with repression of Polycomb target genes and silencing during X chromosome inactivation. Together, these discoveries reveal a new variant PRC1-dependent logic for Polycomb-mediated gene repression.

Maintenance of spatial gene expression by Polycomb-mediated repression after formation of a vertebrate body plan.

ABSTRACTPolycomb group (PcG) proteins are transcriptional repressors that are important regulators of cell fate during embryonic development. Among them, Ezh2 is responsible for catalyzing the epigenetic repressive mark H3K27me3 and is essential for animal development. The ability of zebrafish embryos lacking both maternal and zygotic ezh2 to form a normal body plan provides a unique model for comprehensively studying Ezh2 function during early development in vertebrates. By using a multi-omics approach, we found that Ezh2 is required for the deposition of H3K27me3 and is essential for proper recruitment of Polycomb group protein Rnf2. However, despite the complete absence of PcG-associated epigenetic mark and proteins, only minor changes in H3K4me3 deposition and gene and protein expression occur. These changes were mainly due to local dysregulation of transcription factors outside their normal expression boundaries. Altogether, our results in zebrafish show that Polycomb-mediated gene repression is important immediately after the body plan is formed to maintain spatially restricted expression profiles of transcription factors, and we highlight the differences that exist in the timing of PcG protein action between vertebrate species.

The SERTAD protein Taranis plays a role in Polycomb-mediated gene repression.

The Polycomb group (PcG) proteins have been implicated in epigenetic transcriptional repression in development, stem cell maintenance and in cancer. The chromodomain protein Polycomb (Pc) is a key member of the PcG. Pc binds to the histone mark, trimethylated histone 3 lysine 27 (H3K27me3), to initiate transcriptional repression. How PcG proteins are recruited to target loci is not fully understood. Here we show that the Drosophila SERTA domain protein Taranis (Tara) is involved in transcriptional regulation of Pc target genes. Embryos lacking Tara exhibit a partial homeotic transformation of cuticular the segments, a phenotype associated with the loss of Pc function. Moreover, Drosophila embryos homozygous for a tara hypomorphic allele also misexpress engrailed, a Pc-regulated gene, and this phenotype is associated with the loss of Pc binding to the cis response element in the engrailed enhancer. In relation to that, Pc recruitment is reduced on the salivary gland polytene chromosomes and specifically at the engrailed locus. These results suggest that Tara might be required for positioning Pc to a subset of its target genes.

PP11. RNA-SEQ ANALYSIS OF PRIMARY AND RECURRENT GLIOBLASTOMA IDENTIFIES NOVEL THERAPEUTIC STRATEGIES

AbstractGlioblastoma multiforme (GBM) is the most common and aggressive primary brain tumour. High patient mortality arising from a high propensity for tumour recurrence has driven a need to characterize the genetic changes associated with recurrent GBM. Here, we present a comprehensive landscape of transcriptome profiles of 10 primary and recurrent GBM cancers using RNA-seq, revealing extensive recurrent-specific gene expression changes and somatic mutations. Perturbation of the extracellular matrix, and enhanced ECM degradation, emerge as important changes accompanying GBM recurrence; this perturbation is multimodal and implicates diverse functions including enhanced matrix metalloproteinase (MMP) mediated ECM degradation, silencing of collagen encoding genes (e.g. COL1A1, COL1A2, COL3A1, COL6A3) or their mutation (e.g. COL7A1), and enhanced ECM degradation via dysregulation of SAPK/JNK signaling. Mutational signature analysis revealed striking enrichment of C to T transitions, accounting for 40 to 50% of all base-substitution mutations, implicating CT transition enhancing mutagenic processes in the etiology of GBM. Aberrant epigenetic regulation consistent with dysfunctional polycomb-mediated gene repression manifests in inappropriate activation of putative tumour promoter genes in recurrent tumours. Of 188 genes identified as differentially expressed in our GBM cohort, 38 genes were shared in a separate validation cohort of tumours from the TCGA GBM database. This includes seven genes (ADM, HPCAL4, SNCB, SV2B, TMEM130, H19, VSNL1) with correlations between gene expression and patient survival according to Kaplan-Meier plots generated from TCGA data. This study has highlighted new insights into GBM tumour recurrence and revealed potential novel candidates as prognostic biomarkers and rational therapeutic targets.

Histone H3 Serine 28 Is Essential for Efficient Polycomb-Mediated Gene Repression in Drosophila.

Trimethylation at histone H3K27 is central to the polycomb repression system. Juxtaposed to H3K27 is a widely conserved phosphorylatable serine residue (H3S28) whose function is unclear. To assess the importance of H3S28, we generated a Drosophila H3 histone mutant with a serine-to-alanine mutation at position 28. H3S28A mutant cells lack H3S28ph on mitotic chromosomes but support normal mitosis. Strikingly, all methylation states of H3K27 drop in H3S28A cells, leading to Hox gene derepression and to homeotic transformations in adult tissues. These defects are not caused by active H3K27 demethylation nor by the loss of H3S28ph. Biochemical assays show that H3S28A nucleosomes are a suboptimal substrate for PRC2, suggesting that the unphosphorylated state of serine 28 is important for assisting in the function of polycomb complexes. Collectively, our data indicate that the conserved H3S28 residue in metazoans has a role in supporting PRC2 catalysis.

Synovial sarcoma: recent discoveries as a roadmap to new avenues for therapy.

Oncogenesis in synovial sarcoma is driven by the chromosomal translocation t(X,18; p11,q11), which generates an in-frame fusion of the SWI/SNF subunit SS18 to the C-terminal repression domains of SSX1 or SSX2. Proteomic studies have identified an integral role of SS18-SSX in the SWI/SNF complex, and provide new evidence for mistargeting of polycomb repression in synovial sarcoma. Two recent in vivo studies are highlighted, providing additional support for the importance of WNT signaling in synovial sarcoma: One used a conditional mouse model in which knockout of β-catenin prevents tumor formation, and the other used a small-molecule inhibitor of β-catenin in xenograft models. Synovial sarcoma appears to arise from still poorly characterized immature mesenchymal progenitor cells through the action of its primary oncogenic driver, the SS18-SSX fusion gene, which encodes a multifaceted disruptor of epigenetic control. The effects of SS18-SSX on polycomb-mediated gene repression and SWI/SNF chromatin remodeling have recently come into focus and may offer new insights into the basic function of these processes. A central role for deregulation of WNT-β-catenin signaling in synovial sarcoma has also been strengthened by recent in vivo studies. These new insights into the the biology of synovial sarcoma are guiding novel preclinical and clinical studies in this aggressive cancer.

DISTINCT AND SEPARABLE ROLES FOR HISTONE METHYLTRANSFERASE EZH2 IN NEUROGENIC ASTROCYTES

BACKGROUND: The histone methyltransferase EZH2 is overexpressed in many tumors including gliomas. EZH2 catalyzes H3K27me3, which underlies Polycomb-mediated gene repression, and EZH2-inhibitors have been identified as potential therapeutic agents. In the adult brain subventricular zone (SVZ), a specialized population of astrocytes serves as neural stem cells (NSCs), generating new neurons throughout life. Understanding the epigenetic mechanisms that enable lifelong neurogenesis from SVZ NSCs may help inform therapeutic strategies for gliomas. METHODS: We studied the expression and function of EZH2 in postnatal mouse brain with genetic strategies, gene expression, and chromatin analysis. The expression of EZH2 in the human brain SVZ was also analyzed. RESULTS: In SVZ astroglia, EZH2 was distinctly required for both NSC self-renewal and neuronal lineage specification. By deleting the EZH2 target Ink4a/Arf, we were able to rescue NSC proliferation, but not neuronal differentiation. During adult neurogenesis from mouse SVZ astroglia, EZH2 directly targets Olig2, and repression of this bHLH transcription factor is required for neuronal differentiation. Genome-wide transcript and chromatin analyses also indicate that EZH2 prevents the inappropriate expression of transcription factors involved in non-SVZ neurogenesis. Analysis of human brain specimens indicated EZH2 expression in SVZ astrocytes as well as young migratory neurons. The cell-type expression and correlation with neurogenesis in the human SVZ was remarkably similar to that of the mouse, suggesting a conserved epigenetic mechanism for neurogenesis from this specialized population of astrocytes. CONCLUSIONS: Our finding that EZH2 plays distinct and separable roles in this astroglial population provides novel insights into the complex role of Polycomb gene repression in both normal brain development and brain tumors. Our results, while counterintuitive, indicate that EZH2 is critical for both self-renewal proliferation via Ink4a/Arf repression and differentiation by repressing key developmental regulators, including Olig2, which is required for glioma cell proliferation. The development of EZH2 inhibitors for cancer chemotherapy may benefit from this new understanding of the multiple, distinct roles that EZH2 plays in neural cell differentiation. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

Shall we crosstalk? – The relationship between DNA methylation and histone H3 lysine 27 trimethylation (comment on DOI 10.1002/bies.201300130)

Cancer cells usurp silencing mechanisms to extinguish functional pathways by epigenetic processes. There are two important epigenetic silencing mechanisms, polycomb repressive complex 2 (PRC2)-based histone H3 lysine 27 trimethylation (H3K27me3), and DNA methylation, which are associated with a variety of cellular functions. Studies have shown that these two epigenetic marks seem to be dependent on one another, although the precise mechanistic link between them remains to be fully characterized 1. This crosstalk between DNA methylation and PRC2-H3K27me3 has implications for understanding stem cell differentiation, somatic cell reprogramming, and tumorigenesis as well: dysregulation of these two epigenetic mechanisms has frequently been observed in many types of cancers. It is well appreciated that stem cell PRC2 targets – which are generally associated with embryonic development – are more likely to have cancer-specific promoter DNA methylation than non-targets 1. During tumorigenesis, these vulnerable genes undergo replacement of dynamic H3K27me3 modification with static DNA methylation. By contrast, DNA methylation in CpG sequences interferes with PRC2 recruitment 2. H3K27me3 and DNA methylation are mutually exclusive in CpG islands (CGIs), as ascertained via precise genome-wide analyses 3. These data lead to an idea that during tumorigenesis, aberrant DNA methylation patterns affect the affinity of PRC2 binding and drive redistribution of the PRC2 pattern. Recently, Meehan and coworkers 4, 5 reported that DNA methylation could be a mark for appropriate polycomb-mediated gene repression by constraining PRC2 targeting. Since dense DNA methylation in CGIs interferes with the binding of polycomb protein components, altered DNA methylation patterns in cancer cells influence the PRC2 binding landscape. Meehan et al. uncovered an unappreciated plausible role of DNA methylation in gene regulation. Consistently, studies have shown that artificial removal of DNA methylation induces the accumulation of PRC2 in those genomic loci 3. Fascinatingly, data from these studies evoke another plausible feature of PRC2. PRC2 is mostly enriched in CGIs, although precise mechanisms of its recruitment are not fully understood so far. It is possible that PRC2 stochastically and preferentially modifies DNA at certain loci with low transcription activity but lack of DNA methylation. Since DNA methylated CGIs generally confer low transcription activity, removal of DNA methylation in the CGIs may provide preferential targets for PRC2 binding. Consequently, high levels of DNA methylation and H3K27me3 are rarely found at the same silenced loci, as was reported in whole epigenome analyses. Hence, a direct mechanistic link between PRC2 and DNA methylation may not be required in this regard. An aberrant epigenome is a hallmark of cancer. Understanding such complex epigenetic abnormalities may open the door for developing better therapeutic strategies for cancer treatment.

Redistribution of H3K27me3 upon DNA hypomethylation results in de-repression of Polycomb target genes.

BackgroundDNA methylation and the Polycomb repression system are epigenetic mechanisms that play important roles in maintaining transcriptional repression. Recent evidence suggests that DNA methylation can attenuate the binding of Polycomb protein components to chromatin and thus plays a role in determining their genomic targeting. However, whether this role of DNA methylation is important in the context of transcriptional regulation is unclear.ResultsBy genome-wide mapping of the Polycomb Repressive Complex 2-signature histone mark, H3K27me3, in severely DNA hypomethylated mouse somatic cells, we show that hypomethylation leads to widespread H3K27me3 redistribution, in a manner that reflects the local DNA methylation status in wild-type cells. Unexpectedly, we observe striking loss of H3K27me3 and Polycomb Repressive Complex 2 from Polycomb target gene promoters in DNA hypomethylated cells, including Hox gene clusters. Importantly, we show that many of these genes become ectopically expressed in DNA hypomethylated cells, consistent with loss of Polycomb-mediated repression.ConclusionsAn intact DNA methylome is required for appropriate Polycomb-mediated gene repression by constraining Polycomb Repressive Complex 2 targeting. These observations identify a previously unappreciated role for DNA methylation in gene regulation and therefore influence our understanding of how this epigenetic mechanism contributes to normal development and disease.

Ring1b bookmarks genes in pancreatic embryonic progenitors for repression in adult β cells

Polycomb-mediated gene repression is essential for embryonic development, yet its precise role in lineage-specific programming is poorly understood. Here we inactivated Ring1b, encoding a polycomb-repressive complex 1 subunit, in pancreatic multipotent progenitors (Ring1b(progKO)). This caused transcriptional derepression of a subset of direct Ring1b target genes in differentiated pancreatic islet cells. Unexpectedly, Ring1b inactivation in differentiated islet β cells (Ring1b(βKO)) did not cause derepression, even after multiple rounds of cell division, suggesting a role for Ring1b in the establishment but not the maintenance of repression. Consistent with this notion, derepression in Ring1b(progKO) islets occurred preferentially in genes that were targeted de novo by Ring1b during pancreas development. The results support a model in which Ring1b bookmarks its target genes during embryonic development, and these genes are maintained in a repressed state through Ring1b-independent mechanisms in terminally differentiated cells. This work provides novel insights into how epigenetic mechanisms contribute to shaping the transcriptional identity of differentiated lineages.

RING1B ubiquitination and stability are regulated by ARF

The activity and stability of the E3 ubiquitin ligase RING1B are controlled by the ubiquitin system. Self-ubiquitination of RING1B, generating K6, K27 and K48-based mixed polyubiquitin chains, is a prerequisite for its activity as an E3 ligase for histone H2A. Monoubiquitination of histone H2A is one of the hallmarks of Polycomb-mediated gene silencing. The destruction of RING1B however, is mediated through K48 polyubiquitination catalyzed by the ubiquitin ligase E6-AP. Both forms of ubiquitination of RING1B are mutually exclusive and therefore the balance between them may constitute a point of regulation of Polycomb-mediated gene repression. Here we identify ARF as a regulator of RING1B ubiquitination. ARF appears to selectively prevent RING1B self-ubiquitination, probably allowing more efficient E6-AP-mediated ubiquitination and subsequent degradation of RING1B. By binding to the RING domain of RING1B, ARF disrupts RING1B homodimerization, providing a potential mechanism for its effect on RING1B self-ubiquitination.

Histone Deacetylase Inhibitors Reverse SS18-SSX–Mediated Polycomb Silencing of the Tumor Suppressor Early Growth Response 1 in Synovial Sarcoma

Synovial sarcoma is a soft tissue malignancy characterized by the fusion of SS18 to either SSX1, SSX2, or SSX4 genes. SS18 and SSX are transcriptional cofactors involved in activation and repression of gene transcription, respectively. SS18 interacts with SWI/SNF, whereas SSX associates with the polycomb chromatin remodeling complex. Thus, fusion of these two proteins brings together two opposing effects on gene expression and chromatin structure. Recent studies have shown that a significant number of genes are down-regulated by the SS18-SSX fusion protein and that the clinically applicable histone deacetylase (HDAC) inhibitor romidepsin inhibits synovial sarcoma growth. Therefore, we set out to identify direct targets of SS18-SSX among genes down-regulated in synovial sarcoma and investigated if romidepsin can specifically counteract SS18-SSX-mediated transcriptional dysregulation. Here, we report that the tumor suppressor early growth response 1 (EGR1) is repressed by the SS18-SSX protein through a direct association with the EGR1 promoter. This SS18-SSX binding correlates with trimethylation of Lys(27) of histone H3 (H3K27-M3) and recruitment of polycomb group proteins to this promoter. In addition, we found that romidepsin treatment reverts these modifications and reactivates EGR1 expression in synovial sarcoma cell models. Our data implicate polycomb-mediated epigenetic gene repression as a mechanism of oncogenesis in synovial sarcoma. Furthermore, our work highlights a possible mechanism behind the efficacy of a clinically applicable HDAC inhibitor in synovial sarcoma treatment.

Biochemical Analysis of Mammalian Polycomb Group Protein Complexes and the Identification of Genetic Elements that Block Polycomb-Mediated Gene Repression

Biochemical Analysis of Mammalian Polycomb Group Protein Complexes and the Identification of Genetic Elements that Block Polycomb-Mediated Gene Repression