Role Of Polycomb Repressive Complex Research Articles

Wnt/β-catenin and NFκB signaling synergize to trigger growth factor-free regeneration of adult primary human hepatocytes.

The liver has a remarkable capacity to regenerate, which is sustained by the ability of hepatocytes to act as facultative stem cells that, while normally quiescent, re-enter the cell cycle after injury. Growth factor signaling is indispensable in rodents, whereas Wnt/β-catenin is not required for effective tissue repair. However, the molecular networks that control human liver regeneration remain unclear. Organotypic 3D spheroid cultures of primary human or murine hepatocytes were used to identify the signaling network underlying cell cycle re-entry. Furthermore, we performed chemogenomic screening of a library enriched for epigenetic regulators and modulators of immune function to determine the importance of epigenomic control for human hepatocyte regeneration. Our results showed that, unlike in rodents, activation of Wnt/β-catenin signaling is the major mitogenic cue for adult primary human hepatocytes. Furthermore, we identified TGFβ inhibition and inflammatory signaling through NF-κB as essential steps for the quiescent-to-regenerative switch that allows Wnt/β-catenin-induced proliferation of human cells. In contrast, growth factors, but not Wnt/β-catenin signaling, triggered hyperplasia in murine hepatocytes. High-throughput screening in a human model confirmed the relevance of NFκB and revealed the critical roles of polycomb repressive complex 2, as well as of the bromodomain families I, II, and IV. This study revealed a network of NFκB, TGFβ, and Wnt/β-catenin that controls human hepatocyte regeneration in the absence of exogenous growth factors, identified novel regulators of hepatocyte proliferation, and highlighted the potential of organotypic culture systems for chemogenomic interrogation of complex physiological processes.

Polycomb repressive complex 2 regulates basal cell fate during adult olfactory neurogenesis

Adult neurogenesis occurs in the mammalian olfactory epithelium to maintain populations of neurons that are vulnerable to injury yet essential for olfaction. Multipotent olfactory basal stem cells are activated by damage, although mechanisms regulating lineage decisions are not understood. Using mouse lesion models, we focused on defining the role of Polycomb repressive complexes (PRCs) in olfactory neurogenesis. PRC2 has a well-established role in developing tissues, orchestrating transcriptional programs via chromatin modification. PRC2 proteins are expressed in olfactory globose basal cells (GBCs) and nascent neurons. Conditional PRC2 loss perturbs lesion-induced neuron production, accompanied by altered histone modifications and misexpression of lineage-specific transcription factors in GBCs. De-repression of Sox9 in PRC2-mutant GBCs is accompanied by increased Bowman's gland production, defining an unrecognized role for PRC2 in regulating gland versus neuron cell fate. Our findings support a model for PRC2-dependent mechanisms promoting sensory neuronal differentiation in an adult neurogenic niche.

Epigenetic cues regulating airway development in human lung organoids: Polycomb repressive complex 2 and altered chromatin accessibility determine cell fate.

Today, human organoids are becoming an integrated part of genomics and epigenomics, as they provide a platform that can be used for the definite study of molecular and cellular mechanisms occurring at different stages of development, particularly organogenesis, within the human body. Airway development is a complex process heavily influenced by epigenetic regulatory mechanisms in response to environmental changes, and as such, human lung organoids are an indispensable asset for further exploration of these mechanisms as a mode of transition from human in vitro to human ex vivo studies. Cultured primarily in compounds mimicking the extracellular matrix, such as Matrigel, these lung organoids have helped us to come to a better understanding of the role of polycomb repressive complex 2 (PRC2) and enhancer of zeste homolog 2 (EZH2) in lung epithelial cell differentiation and airway development, which was first reported in the FASEB journal in 2019. The following is an extended account of how the histone methylation-regulating PRC2 comes to play in the molding of the human bronchial tree, along with further epigenetic insights based on more recently developed human lung organoids.

Oncogenic Roles of Polycomb Repressive Complex 2 in Bladder Cancer and Upper Tract Urothelial Carcinoma.

Cancers of the urinary tract are one of the most common malignancies worldwide, causing high morbidity and mortality, and representing a social burden. Upper tract urothelial carcinoma (UTUC) accounts for 5-10% of urinary tract cancers, and its oncogenic mechanisms remain elusive. We postulated that cancers of the lower and the upper urinary tract may share some important oncogenic mechanisms. Therefore, the oncogenic mechanisms discovered in the lower urinary tract may guide the investigation of molecular mechanisms in the upper urinary tract. Based on this strategy, we revisited a high-quality transcriptome dataset of 510 patients with non-muscle invasive bladder cancer (NMIBC), and performed an innovative gene set enrichment analysis of the transcriptome. We discovered that the epigenetic regulation of polycomb repressive complex 2 (PRC2) is responsible for the recurrence and progression of lower-track urinary cancers. Additionally, a PRC2-related gene signature model was discovered to be effective in classifying bladder cancer patients with distinct susceptibility of subsequent recurrence and progression (log-rank p < 0.001 and = 0.001, respectively). We continued to discover that the same model can differentiate stage T3 UTUC patients from stage Ta/T1 patients (p = 0.026). Immunohistochemical staining revealed the presence of PRC2 components (EZH2, EED, and SUZ12) and methylated PRC2 substrates (H3K27me3) in the archived UTUC tissues. The H3K27me3 exhibited higher intensity and area intensity product in stage T3 UTUC tissues than in stage Ta/T1 tissues (p = 0.006 and 0.015, respectively), implicating stronger PRC2 activity in advanced UTUC. The relationship between H3K27 methylation and gene expression is examined using correlations. The H3K27me3 abundance is positively correlated with the expression levels of CDC26, RP11-2B6, MAPK1IP1L, SFR1, RP11-196B3, CDK5RAP2, ANXA5, STX11, PSMD5, and FGFRL1. It is also negatively correlated with CNPY2, KB-1208A12, RP11-175B9, ZNF692, RANP8, RP11-245C17, TMEM266, FBXW9, SUGT1P2, and PRH1. In conclusion, PRC2 and its epigenetic effects are major oncogenic mechanisms underlying both bladder cancer and UTUC. The epigenetically regulated genes of PRC2 in urothelial carcinoma were also elucidated using correlation statistics.

Role of Polycomb Repressive Complex 2 in Regulation of Human Transcription Factor Gene Expression

For instance, this was the case with 16 of 20 TF families. The results are interpreted to indicate that while individual TFs such as EZH2 may be specific for broadly expressed or tissue targeted genes, this is not a property of most TF families. Most have both broadly expressed and tissue targeted members

An essential role for Polycomb Repressive Complex 2 in the mouse ovary.

Polycomb repressive complex 2 (PRC2) catalyses the repressive epigenetic modification of histone 3 lysine 27 tri-methylation (H3K27me3) and functions as a key epigenetic regulator during embryonic development. PRC2 is known to regulate the development of a range of tissues by transcriptional silencing of genes that control cell differentiation, but its roles in female germline and ovarian development remain unknown. Using a mouse model with hypomorphic embryonic ectoderm development (EED) function that reduced H3K27me3 in somatic and germ cells, we found that PRC2 was required for survival, with more than 95% of female animals dying before birth. Although surviving adult EED hypomorphic females appeared morphologically similar to controls and were fertile, Eedhypo/hypo adult ovaries were abnormal, with altered morphology characterised by abnormal follicles. Early Eedhypo/hypo and control fetal ovaries were morphologically similar, and germ cells entered meiosis normally. Immunofluorescent analyses of somatic and germline markers indicated that ovarian development in Eedhypo/hypo ovaries was similar to heterozygous and WT controls. However, TUNEL analyses revealed higher rates of apoptosis in the ovarian surface epithelium, and transcriptional analyses revealed changes in genes regulating epithelial and steroidogenic cell differentiation, possibly foreshadowing the defects observed in adult ovaries of hypomorphic females. While it was possible to analyse early-mid fetal ovarian development, postnatal stages were inaccessible due to the high level of lethality during late fetal stages. Despite this limitation, the data we were able to obtain reveal a novel role for EED in the ovary that is likely to alter ovarian development and ovarian function in adult animals.

Role of Polycomb Repressive Complex 2 in Regulation of Human Transcription Factor Gene Expression

Control of gene expression is now recognized as a central issue in the field of molecular biology. We now know the sequences of many genomes including that of the human genome, and we know the nature of many pathways involved in control of gene expression. It remains difficult, however, to look at the DNA sequences surrounding a particular gene and tell which methods of regulatory control are in use. I have been pursuing the idea that progress might be made by comparing the regulatory regions of paired gene populations in which one population is strongly expressed and the other weakly. Here I report the results obtained with human genes encoding transcription factors (TF). In this population, broadly expressed genes are strongly expressed while tissue targeted TF expression is suppressed in most tissues. The results demonstrated that the promoter region of broadly expressed TF genes is enriched in binding sites for POLR2A, a component of RNA polymerase II while promoters of tissue targeted genes are enriched in EZH2, a subunit of polycomb repressive complex 2 (PRC2). It was rare to observe promoters with binding sites for both POLR2A and EZH2. The findings are interpreted to indicate that strong expression of broadly expressed TF genes is due to the presence of RNA polymerase II at the promoter while weak expression of tissue targeted promoters results from the presence of PRC2. Finally, transcription factor families were compared in the proportion of broadly expressed and tissue targeted genes they contain. The results demonstrated that most families possess both broadly expressed and tissue targeted members. For instance, this was the case with 16 of 20 TF families examined. The results are interpreted to indicate that while individual TFs such as EZH2 may be specific for broadly expressed or tissue targeted genes, this is not a property of most TF families.

Polycomb Repressive Complex 2 Regulates Genes Necessary for Intestinal Microfold Cell (M Cell) Development.

Background & AimsMicrofold cells (M cells) are immunosurveillance epithelial cells located in the Peyer’s patches (PPs) in the intestine and are responsible for monitoring and transcytosis of antigens, microorganisms, and pathogens. Mature M cells use the receptor glycoprotein 2 (GP2) to aid in transcytosis. Recent studies have shown transcription factors, Spi-B and SRY-Box Transcription Factor 8 (Sox8). are necessary for M-cell differentiation, but not sufficient. An exhaustive set of factors sufficient for differentiation and development of a mature GP2+ M cell remains elusive. Our aim was to understand the role of polycomb repressive complex 2 (PRC2) as an epigenetic regulator of M-cell development. Estrogen-related–receptor γ (Esrrg), identified as a PRC2-regulated gene, was studied in depth, in addition to its relationship with Spi-B and Sox8.MethodsComparative chromatin immunoprecipitation and global run-on sequencing analysis of mouse intestinal organoids were performed in stem condition, enterocyte conditions, and receptor activator of nuclear factor κ B ligand–induced M-cell condition. Esrrg, which was identified as one of the PRC2-regulated transcription factors, was studied in wild-type mice and knocked out in intestinal organoids using guide RNA's. Sox8 null mice were used to study Esrrg and its relation to Sox8.Resultschromatin immunoprecipitation and global run-on sequencing analysis showed 12 novel PRC2 regulated transcription factors, PRC2-regulated Esrrg is a novel M-cell–specific transcription factor acting on a receptor activator of nuclear factor κB ligand–receptor activator of nuclear factor κB–induced nuclear factor-κB pathway, upstream of Sox8, and necessary but not sufficient for a mature M-cell marker of Gp2 expression.ConclusionsPRC2 regulates a significant set of genes in M cells including Esrrg, which is critical for M-cell development and differentiation. Loss of Esrrg led to an immature M-cell phenotype lacking in Sox8 and Gp2 expression. Transcript profiling: the data have been deposited in the NCBI Gene Expression Omnibus database (GSE157629).

NSD2-E1099K Mutation Leads to Glucocorticoid-Resistant B Cell Lymphocytic Leukemia in Mice

Background: NSD2 (nuclear receptor binding SET domain protein 2) is a histone methyltransferase specific for dimethylation of histone H3 lysine 36 (H3K36me2), a modification associated with gene activation. In pediatric acute lymphoblastic leukemia (ALL), particularly at relapse, a gain of function mutation (E1099K) of NSD2 is found in 10-15% of cases. The NSD2 mutation is found in addition to fusion proteins such as E2A-PBX and ETV6-RUNX1. The mutation can be subclonal at diagnosis and dominant at relapse, suggesting a link to therapeutic resistance. The NSD2-E1099K mutation affects gene expression through an increase in H3K36me2 and a decrease in H3K27me3. Using CRISPR/Cas9-edited isogenic ALL cell lines, we found that NSD2-E1099K mutation drove oncogenic programming by altering chromatin architecture, gene expression and enhancing cell growth, migration and infiltration to the central neural system (CNS). NSD2 mutation caused resistance of ALL cells to glucocorticoids (GC) by blocking genome wide binding of the glucocorticoid receptor (GR, encoded by NR3C1 gene) preventing GC-mediated induction of pro-apoptotic genes. NR3C1 levels were depressed in NSD2-E1099K cells and GC failed to induce autoactivation of NR3C1. While H3K27me3 was globally decreased by NSD2-E1099K, increased H3K27me3 was noted at the promoter of NR3C1, suggesting a novel role of polycomb repressive complex 2 as a therapeutic target for relapsed ALL with NSD2 mutation. While NSD2 is highly expressed in B cells and NSD2 knockout causes defects in B cell development, how the NSD2 mutation affects B cell development and leukemia occurrence in vivo is uncertain. Aims: To determine the role of NSD2 mutation in the pathogenesis of lymphocytic malignancies and GC resistance in a mouse model. Methods: We generated a conditional NSD2-E1099K knock-in mouse model in which the NSD2-E1099K allele was placed in the Rosa26 locus and expressed in B cells under the control of Cd19-Cre (Cd19+/-NSD2E1099K/WT). The resulting phenotype was characterized through peripheral blood counts, cellular morphology and histology of blood smears, bone marrow (BM), spleen and liver, flow cytometric analysis, germinal center B cells (GCB) immunization, BM transplantation, and hematopoiesis analysis in a CD3-/- background. We further established mouse leukemia cell lines with NSD2 mutation for functional analysis. RNA-Seq, real time PCR, immunoblotting, and apoptosis analysis (Annexin V/PI staining) following GC treatment were performed to demonstrate the effects of NSD2 mutation on histone modifications, transcriptome and GC resistance. Results: The NSD2-E1099K mutation increased H3K36me2 and decreased H3K27me3 in isolated B cells from mouse BM and spleen. Mice were aged and did not develop signs of malignancy and RNA-sequencing showed few differences between B cells with or without the NSD2 mutation. However, after immunizing the mice with sheep red blood cells (SRBC), more GCBs were seen in the spleen of NSD2 mutant mice, suggesting mutant NSD2 stimulated germinal center hyperplasia. Transplantation of BM cells from mice expressing NSD2-E1099K into lethally irradiated recipients lead to an expansion of B cells while myeloid and T cells and life span of the recipients impaired. The NSD2 knock-in mouse model was crossed with Cd3-/- mice to create Cd19+/-Cd3-/-NSD2E1099K/WT mice, which within 2 months of birth developed a disease resembling an immature B lymphocytic leukemia (B220+CD19+IgM+IgD-CD5-) with infiltration of the spleen, liver and CNS and a median survival of 4.8 months. These tumors could be transplanted into immunodeficient mice but not immunocompetent mice. RNA seq analysis of these cells revealed 6,815 genes (3,295 upregulated and 3,520 downregulated) differentially expressed in NSD2 mutant B cells compared to normal B cells. The upregulated genes were related to abnormal immunoglobulin level , B cell activation, T-helper 1 physiology, and decreased B cell apoptosis. Importantly, the NSD2 mutant leukemic cells displayed depressed level of NR3C1 gene expression and GC resistance. Conclusions: The NSD2 mutation alters B cell development, particularly in an immunodeficient background and causes B cells to become resistant to glucocorticoids. The inability of the mutation to generate disease on its own except in an immunodeficient background suggests genes that collaborate with NSD2 in ALL may play a role in immune escape. Disclosures No relevant conflicts of interest to declare.

The Role of Polycomb Repressive Complex in Malignant Peripheral Nerve Sheath Tumor.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive soft tissue sarcomas that can arise most frequently in patients with neurofibromatosis type 1 (NF1). Despite an increasing understanding of the molecular mechanisms that underlie these tumors, there remains limited therapeutic options for this aggressive disease. One potentially critical finding is that a significant proportion of MPNSTs exhibit recurrent mutations in the genes EED or SUZ12, which are key components of the polycomb repressive complex 2 (PRC2). Tumors harboring these genetic lesions lose the marker of transcriptional repression, trimethylation of lysine residue 27 on histone H3 (H3K27me3) and have dysregulated oncogenic signaling. Given the recurrence of PRC2 alterations, intensive research efforts are now underway with a focus on detailing the epigenetic and transcriptomic consequences of PRC2 loss as well as development of novel therapeutic strategies for targeting these lesions. In this review article, we will summarize the recent findings of PRC2 in MPNST tumorigenesis, including highlighting the functions of PRC2 in normal Schwann cell development and nerve injury repair, as well as provide commentary on the potential therapeutic vulnerabilities of a PRC2 deficient tumor cell.

PRC2-mediated MHC-I silencing drives immune evasion.

Burr et al. report an evolutionarily conserved, tumour-intrinsic role for Polycomb repressive complex 2 (PRC2) in the epigenetic silencing of MHC class I antigen presentation pathway genes, illustrating that cancer cells can co-opt the immunomodulatory functions of PRC2 to evade immune surveillance.

Mo1872 – The Regulatory Role of Polycomb Repressive Complex 1 (PRC1) in Inflammatory Bowel Disease

Mo1872 – The Regulatory Role of Polycomb Repressive Complex 1 (PRC1) in Inflammatory Bowel Disease

Deregulated Polycomb functions in myeloproliferative neoplasms.

Polycomb proteins function in the maintenance of gene silencing via post-translational modifications of histones and chromatin compaction. Genetic and biochemical studies have revealed that the repressive function of Polycomb repressive complexes (PRCs) in transcription is counteracted by the activating function of Trithorax-group complexes; this balance fine-tunes the expression of genes critical for development and tissue homeostasis. The function of PRCs is frequently dysregulated in various cancer cells due to altered expression or recurrent somatic mutations in PRC genes. The tumor suppressive functions of EZH2-containing PRC2 and a PRC2-related protein ASXL1 have been investigated extensively in the pathogenesis of hematological malignancies, including myeloproliferative neoplasms (MPN). BCOR, a component of non-canonical PRC1, suppresses various hematological malignancies including MPN. In this review, we focus on recent findings on the role of PRCs in the pathogenesis of MPN and the therapeutic impact of targeting the pathological functions of PRCs in MPN.

Requirement for PRC1 subunit BMI1 in host gene activation by Epstein-Barr virus protein EBNA3C.

Epstein–Barr virus proteins EBNA3A, EBNA3B and EBNA3C control hundreds of host genes after infection. Changes in epigenetic marks around EBNA3-regulated genes suggest that they exert transcriptional control in collaboration with epigenetic factors. The roles of polycomb repressive complex (PRC)2 subunit SUZ12 and of PRC1 subunit BMI1 were assessed for their importance in EBNA3-mediated repression and activation. ChIP-seq experiments for SUZ12 and BMI1 were performed to determine their global localization on chromatin and analysis offered further insight into polycomb protein distribution in differentiated cells. Their localization was compared to that of each EBNA3 to resolve longstanding questions about the EBNA3–polycomb relationship. SUZ12 did not co-localize with any EBNA3, whereas EBNA3C co-localized significantly and co-immunoprecipitated with BMI1. In cells expressing a conditional EBNA3C, BMI1 was sequestered to EBNA3C-binding sites after EBNA3C activation. When SUZ12 or BMI1 was knocked down in the same cells, SUZ12 did not contribute to EBNA3C-mediated regulation. Surprisingly, after BMI1 knockdown, EBNA3C repressed equally efficiently but host gene activation by EBNA3C was impaired. This overturns previous assumptions about BMI1/PRC1 functions during EBNA3C-mediated regulation, for the first time identifies directly a host factor involved in EBNA3-mediated activation and provides a new insight into how PRC1 can be involved in gene activation.

Low socioeconomic status and unhealthy lifestyle habits are associated with premature occurrence of stochastic epigenetic mutations: A meta-analysis within the LIFEPATH project

Introduction Low socioeconomic status (SES) is associated with earlier onset of age-related chronic conditions and reduced life expectancy. Epigenetic variability by SES is one of the proposed molecular mechanisms mediating this association. In a recent study, we found that low SES, in both childhood and adulthood is associated with accelerated epigenetic ageing, independent from other risk factors for non-communicable diseases, and that the effect may be partially reversible. DNA methylation is gradually deregulated during ageing by errors in methylation maintenance, a process referred to as epigenetic drift. The number of stochastic epigenetic mutations (SEMs) increases exponentially with age, most likely because of an accumulation of exposure-related damage. Epigenetic deregulation may play a role in the development of age-related diseases. This study, within the LIFEPATH project, aims to disentangle the biomolecular mechanisms mediating health inequalities by SES. We hypothesized that low education level, a marker of low socioeconomic status, and unhealthy lifestyle habits are associated with an acceleration of the occurrence of SEMs and, as a consequence, with an increased risk of age-related diseases. Methods SEMs are defined as extreme outliers, exceeding three times interquartile ranges the first quartile (lower than Q1 − 3 × IQR) or the third quartile higher than (Q3 + 3 × IQR), in the distribution of DNA methylation values for a CpG across samples. We conducted a meta-analysis including thirteen cohorts with more than 15,000 individuals to investigate the association of low SES, smoking, obesity, high alcohol intake, and low physical activity with the total number of SEMs. Additionally, in three prospective case-control studies nested in cohorts, we investigated whether the total number of SEMs is associated with higher risk of different cancers. Finally, we examined whether SEMs are randomly distributed across the genome, or are enriched in functional genomic regions. Results We found that low SES, smoking, obesity, high alcohol intake, and low physical activity modulate the association of the total number of SEMs with ageing. The effect of risk factors is more pronounced at older ages, supporting the hypothesis of an accumulation of SEMs as a biomarker of accumulation of exposure-related genomic damage. In nested case-control studies, a higher number of SEMs was associated with higher risk of lung and breast cancer, suggesting accumulation of SEMs belongs to the causal pathway linking low SES and unhealthy lifestyle with age-related diseases and longevity. Enrichment analyses revealed that the genomic distribution of SEMs is highly non-random, as they are enriched in transcription factor binding sites (TFBS). The most significantly enriched TF is a member of the Polycomb Repressive Complex 2 (PRC2), EZH2, a crucial regulatory element in cancer and other age-related diseases like mental disorders. Conclusions We found that low SES and unhealthy lifestyle habits are associated with premature accumulation of age-related epigenetic mutations and that DNA methylation dysregulation is associated with the development of cancer and other age-related diseases. We have identified novel biomolecular mechanisms mediating health inequalities, suggesting a crucial role of Polycomb Repressive Complex 2 associated genomic regions.

The role of polycomb repressive complex 2 in early T-cell precursor acute lymphoblastic leukemia

ABSTRACTGenetic lesions affecting polycomb repressive complex 2 (PRC2) have been found in more than 40% of pediatric cases of early T-cell precursor acute lymphoblastic leukemia. The functional role of these PRC2 alterations has been obscure. Our recent data suggest that compromise of PRC2 blocks differentiation and accentuates growth and survival signaling.

Polycomb repression complex 2 is required for the maintenance of retinal progenitor cells and balanced retinal differentiation

Polycomb repressive complexes maintain transcriptional repression of genes encoding crucial developmental regulators through chromatin modification. Here we investigated the role of Polycomb repressive complex 2 (PRC2) in retinal development by inactivating its key components Eed and Ezh2. Conditional deletion of Ezh2 resulted in a partial loss of PRC2 function and accelerated differentiation of Müller glial cells. In contrast, inactivation of Eed led to the ablation of PRC2 function at early postnatal stage. Cell proliferation was reduced and retinal progenitor cells were significantly decreased in this mutant, which subsequently caused depletion of Müller glia, bipolar, and rod photoreceptor cells, primarily generated from postnatal retinal progenitor cells. Interestingly, the proportion of amacrine cells was dramatically increased at postnatal stages in the Eed-deficient retina. In accordance, multiple transcription factors controlling amacrine cell differentiation were upregulated. Furthermore, ChIP-seq analysis showed that these deregulated genes contained bivalent chromatin (H3K27me3+ H3K4me3+). Our results suggest that PRC2 is required for proliferation in order to maintain the retinal progenitor cells at postnatal stages and for retinal differentiation by controlling amacrine cell generation.

PRC2 specifies ectoderm lineages and maintains pluripotency in primed but not na\xefve ESCs

Polycomb repressive complex 2 and the epigenetic mark that it deposits, H3K27me3, are evolutionarily conserved and play critical roles in development and cancer. However, their roles in cell fate decisions in early embryonic development remain poorly understood. Here we report that knockout of polycomb repressive complex 2 genes in human embryonic stem cells causes pluripotency loss and spontaneous differentiation toward a meso-endoderm fate, owing to de-repression of BMP signalling. Moreover, human embryonic stem cells with deletion of EZH1 or EZH2 fail to differentiate into ectoderm lineages. We further show that polycomb repressive complex 2-deficient mouse embryonic stem cells also release Bmp4 but retain their pluripotency. However, when converted into a primed state, they undergo spontaneous differentiation similar to that of hESCs. In contrast, polycomb repressive complex 2 is dispensable for pluripotency when human embryonic stem cells are converted into the naive state. Our studies reveal both lineage- and pluripotent state-specific roles of polycomb repressive complex 2 in cell fate decisions.

Polycomb repressive complex 1 modifies transcription of active genes.

This study examines the role of Polycomb repressive complex 1 (PRC1) at active genes. The PRC1 and PRC2 complexes are crucial for epigenetic silencing during development of an organism. They are recruited to Polycomb response elements (PREs) and establish silenced domains over several kilobases. Recent studies show that PRC1 is also directly recruited to active genes by the cohesin complex. Cohesin participates broadly in control of gene transcription, but it is unknown whether cohesin-recruited PRC1 also plays a role in transcriptional control of active genes. We address this question using genome-wide RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq). The results show that PRC1 influences transcription of active genes, and a significant fraction of its effects are likely direct. The roles of different PRC1 subunits can also vary depending on the gene. Depletion of PRC1 subunits by RNA interference alters phosphorylation of RNA polymerase II (Pol II) and occupancy by the Spt5 pausing-elongation factor at most active genes. These effects on Pol II phosphorylation and Spt5 are likely linked to changes in elongation and RNA processing detected by nascent RNA-seq, although the mechanisms remain unresolved. The experiments also reveal that PRC1 facilitates association of Spt5 with enhancers and PREs. Reduced Spt5 levels at these regulatory sequences upon PRC1 depletion coincide with changes in Pol II occupancy and phosphorylation. Our findings indicate that, in addition to its repressive roles in epigenetic gene silencing, PRC1 broadly influences transcription of active genes and may suppress transcription of nonpromoter regulatory sequences.

Flipping between Polycomb repressed and active transcriptional states introduces noise in gene expression

Polycomb repressive complexes (PRCs) are important histone modifiers, which silence gene expression; yet, there exists a subset of PRC-bound genes actively transcribed by RNA polymerase II (RNAPII). It is likely that the role of Polycomb repressive complex is to dampen expression of these PRC-active genes. However, it is unclear how this flipping between chromatin states alters the kinetics of transcription. Here, we integrate histone modifications and RNAPII states derived from bulk ChIP-seq data with single-cell RNA-sequencing data. We find that Polycomb repressive complex-active genes have greater cell-to-cell variation in expression than active genes, and these results are validated by knockout experiments. We also show that PRC-active genes are clustered on chromosomes in both two and three dimensions, and interactions with active enhancers promote a stabilization of gene expression noise. These findings provide new insights into how chromatin regulation modulates stochastic gene expression and transcriptional bursting, with implications for regulation of pluripotency and development.