Polycomb Repressive Complex 2 Binding Research Articles

DNA elements tether canonical Polycomb Repressive Complex 1 to human genes.

Development of multicellular animals requires epigenetic repression by Polycomb group proteins. The latter assemble in multi-subunit complexes, of which two kinds, Polycomb Repressive Complex 1 (PRC1) and Polycomb Repressive Complex 2 (PRC2), act together to repress key developmental genes. How PRC1 and PRC2 recognize specific genes remains an open question. Here we report the identification of several hundreds of DNA elements that tether canonical PRC1 to human developmental genes. We use the term tether to describe a process leading to a prominent presence of canonical PRC1 at certain genomic sites, although the complex is unlikely to interact with DNA directly. Detailed analysis indicates that sequence features associated with PRC1 tethering differ from those that favour PRC2 binding. Throughout the genome, the two kinds of sequence features mix in different proportions to yield a gamut of DNA elements that range from those tethering predominantly PRC1 or PRC2 to ones capable of tethering both complexes. The emerging picture is similar to the paradigmatic targeting of Polycomb complexes by Polycomb Response Elements (PREs) of Drosophila but providing for greater plasticity.

CK2-mediated phosphorylation of SUZ12 promotes PRC2 function by stabilizing enzyme active site

Polycomb repressive complex 2 (PRC2) plays a key role in maintaining cell identity during differentiation. Methyltransferase activity of PRC2 on histone H3 lysine 27 is regulated by diverse cellular mechanisms, including posttranslational modification. Here, we report a unique phosphorylation-dependent mechanism stimulating PRC2 enzymatic activity. Residue S583 of SUZ12 is phosphorylated by casein kinase 2 (CK2) in cells. A crystal structure captures phosphorylation in action: the flexible phosphorylation-dependent stimulation loop harboring S583 becomes engaged with the catalytic SET domain through a phosphoserine-centered interaction network, stabilizing the enzyme active site and in particular S-adenosyl-methionine (SAM)-binding pocket. CK2-mediated S583 phosphorylation promotes catalysis by enhancing PRC2 binding to SAM and nucleosomal substrates and facilitates reporter gene repression. Loss of S583 phosphorylation impedes PRC2 recruitment and H3K27me3 deposition in pluripotent mESCs and compromises the ability of PRC2 to maintain differentiated cell identity.

Abstract 14259: Novel Long Noncoding RNA, Ppp1r1b-LncRNA , Epigenetically Promotes Myogenic Differentiation Program in Cardiac and Skeletal Myocytes

Introduction: Emerged as critical regulators of the transcriptome, the lncRNAs play important roles in cardiac development and might be targeted to treat human cardiomyocyte dysfunction in congenital heart disease. In our work, we identified a novel lncRNA, Ppp1r1b-lncRNA , as an essential, functionally conserved, positive regulator of myogenic differentiation. Hypothesis: Ppp1r1b-lncRNA promotes myogenic differentiation program of cardiac and skeletal myocytes via epigenic mechanisms. Methods: Antisense oligonucleotides (GapmeR) were used to suppress Ppp1r1b-lncRNA . Chromatin immunoprecipitation (CHIP) and chromatin isolation by RNA purification (CHIRP) were used to analyze gene specific histone modification level and lncRNA: chromatin interaction, respectively. RNA pull-down and RNA immunoprecipitation (RIP) were used to identify Ppp1r1b-lncRNA : protein interactome. Results: By silencing Ppp1r1b-lncRNA, skeletal myoblasts and human-induced pluripotent stem cell derived cardiomyocytes (hiPSCs-CMs) failed to develop fully differentiated myotubes. Analysis of GapmeR injected neonatal mouse heart further confirmed the role of Ppp1r1b-lncRNA in normal myogenesis. Members of muscle specific transcription factors, including MyoD1 and TBX5, were not induced during myogenic differentiation in the Ppp1r1b-lncRNA depleted cardiac and skeletal muscle precursor cells. Histone modification analysis revealed enrichment of H3K27 tri-methylation at MyoD1 and TBX5 promoters in GapmeR treated skeletal myoblasts. Furthermore, Ppp1r1b-lncRNA was found to bind to Ezh2, and ChIRP analysis revealed enriched interaction of Ppp1r1b-lncRNA with Myod1 and Tbx5 promoters, suggesting that Ppp1r1b-lncRNA induces transcription of myogenic regulatory factors by interacting with the polycomb repressive complex 2 (PRC2) at the chromatin interface. Correspondingly, the silencing of Ppp1r1b-lncRNA increased EZH2 binding at promoter regions of myogenic transcription factors. Conclusions: Ppp1r1b-lncRNA promotes myogenic differentiation by inhibiting histone methylation on the promotors of myogenic master regulators via competing for PRC2 binding with chromatin during heart and skeletal muscle development.

A cryptic transactivation domain of EZH2 binds AR and AR's splice variant, promoting oncogene activation and tumorous transformation.

Enhancer of Zeste Homolog 2 (EZH2) and androgen receptor (AR) are crucial chromatin/gene regulators involved in the development and/or progression of prostate cancer, including advanced castration-resistant prostate cancer (CRPC). To sustain prostate tumorigenicity, EZH2 establishes non-canonical biochemical interaction with AR for mediating oncogene activation, in addition to its canonical role as a transcriptional repressor and enzymatic subunit of Polycomb Repressive Complex 2 (PRC2). However, the molecular basis underlying non-canonical activities of EZH2 in prostate cancer remains elusive, and a therapeutic strategy for targeting EZH2:AR-mediated oncogene activation is also lacking. Here, we report that a cryptic transactivation domain of EZH2 (EZH2TAD) binds both AR and AR spliced variant 7 (AR-V7), a constitutively active AR variant enriched in CRPC, mediating assembly and/or recruitment of transactivation-related machineries at genomic sites that lack PRC2 binding. Such non-canonical targets of EZH2:AR/AR-V7:(co-)activators are enriched for the clinically relevant oncogenes. We also show that EZH2TAD is required for the chromatin recruitment of EZH2 to oncogenes, for EZH2-mediated oncogene activation and for CRPC growth in vitro and in vivo. To completely block EZH2's multifaceted oncogenic activities in prostate cancer, we employed MS177, a recently developed proteolysis-targeting chimera (PROTAC) of EZH2. Strikingly, MS177 achieved on-target depletion of bothEZH2's canonical (EZH2:PRC2) and non-canonical (EZH2TAD:AR/AR-V7:co-activators) complexes in prostate cancer cells, eliciting far more potent antitumor effects than the catalytic inhibitors of EZH2. Overall, this study reports a previously unappreciated requirement for EZH2TAD for mediating EZH2's non-canonical (co-)activator recruitment and gene activation functions in prostate cancer and suggests EZH2-targeting PROTACs as a potentially attractive therapeutic for the treatment of aggressive prostate cancerthat rely on the circuits wired by EZH2 and AR.

ATRX histone binding and helicase activities have distinct roles in neuronal differentiation.

ATRX is a chromatin remodeler, which is mutated in ATRX syndrome, a neurodevelopmental disorder. ATRX mutations that alter histone binding or chromatin remodeling activities cluster in the PHD finger or the helicase domain respectively. Using engineered mouse embryonic stem cells that exclusively express ATRX protein with mutations in the PHD finger (PHDmut) or helicase domains (K1584R), we examine how specific ATRX mutations affect neurodifferentiation. ATRX PHDmut and K1584R proteins interact with the DAXX histone chaperone but show reduced localization to pericentromeres. Neurodifferentiation is both delayed and compromised in PHDmut and K1584R, and manifest differently from complete ATRX loss. We observe reduced enrichment of PHDmut protein to ATRX targets, while K1584R accumulates at these sites. Interestingly, ATRX mutations have distinct effects on the genome-wide localization of the polycomb repressive complex 2 (PRC2), with PHDmut and ATRX knockout showing reduced PRC2 binding at polycomb targets and K1584R showing loss at some sites and gains at others. Notably, each mutation associated with unique gene signatures, suggesting distinct pathways leading to impaired neurodifferentiation. Our results indicate that the histone binding and chromatin remodeling functions of ATRX play non-redundant roles in neurodevelopment, and when mutated lead to ATRX syndrome through separate regulatory pathways.

A TET1-PSPC1-Neat1 molecular axis modulates PRC2 functions in controlling stem cell bivalency.

SUMMARYTET1 maintains hypomethylation at bivalent promoters through its catalytic activity in embryonic stem cells (ESCs). However, TET1 catalytic activity-independent function in regulating bivalent genes is not well understood. Using a proteomics approach, we map the TET1 interactome in ESCs and identify PSPC1 as a TET1 partner. Genome-wide location analysis reveals that PSPC1 functionally associates with TET1 and Polycomb repressive complex-2 (PRC2). We establish that PSPC1 and TET1 repress, and the lncRNA Neat1 activates, bivalent gene expression. In ESCs, Neat1 is preferentially bound to PSPC1 alongside its PRC2 association at bivalent promoters. During the ESC-to-epiblast-like stem cell (EpiLC) transition, PSPC1 and TET1 maintain PRC2 chromatin occupancy at bivalent gene promoters, while Neat1 facilitates the activation of certain bivalent genes by promoting PRC2 binding to their mRNAs. Our study demonstrates a TET1-PSPC1-Neat1 molecular axis that modulates PRC2-binding affinity to chromatin and bivalent gene transcripts in controlling stem cell bivalency

Simultaneous disruption of PRC2 and enhancer function underlies histone H3.3-K27M oncogenic activity in human hindbrain neural stem cells.

Driver mutations in genes encoding histone H3 proteins resulting in p.Lys27Met substitutions (H3-K27M) are frequent in pediatric midline brain tumors. However, the precise mechanisms by which H3-K27M causes tumor initiation remain unclear. Here, we use human hindbrain neural stem cells to model the consequences of H3.3-K27M on the epigenomic landscape in a relevant developmental context. Genome-wide mapping of epitope-tagged histone H3.3 revealed that both the wild type and the K27M mutant incorporate abundantly at pre-existing active enhancers and promoters, and to a lesser extent at Polycomb repressive complex 2 (PRC2)-bound regions. At active enhancers, H3.3-K27M leads to focal H3K27ac loss, decreased chromatin accessibility and reduced transcriptional expression of nearby neurodevelopmental genes. In addition, H3.3-K27M deposition at a subset of PRC2 target genes leads to increased PRC2 and PRC1 binding and augmented transcriptional repression that can be partially reversed by PRC2 inhibitors. Our work suggests that, rather than imposing de novo transcriptional circuits, H3.3-K27M drives tumorigenesis by locking initiating cells in their pre-existing, immature epigenomic state, via disruption of PRC2 and enhancer functions.

Motif-driven interactions between RNA and PRC2 are rheostats that regulate transcription elongation.

Although Polycomb repressive complex 2 (PRC2) is now recognized as an RNA-binding complex, the full range of binding motifs and why PRC2-RNA complexes often associate with active genes have not been elucidated. Here we identify high-affinity RNA motifs whose mutations weaken PRC2 binding and attenuate its repressive function in mouse embryonic stem cells. Interactions occur at promoter-proximal regions and frequently coincide with pausing of RNA Polymerase II (POL-II). Surprisingly, while PRC2-associated nascent transcripts are highly expressed, ablating PRC2 further upregulates expression via loss of pausing and enhanced transcription elongation. Thus, PRC2-nascent RNA complexes operate as rheostats to fine-tune transcription by regulating transitions between pausing and elongation, explaining why PRC2-RNA complexes frequently occur within active genes. Nascent RNA also targets PRC2 in cis and downregulates neighboring genes. We propose a unifying model in which RNA specifically recruits PRC2 to repress genes through POL-II pausing and, more classically, H3K27-trimethylation.

Single-molecule and in silico dissection of the interaction between Polycomb repressive complex 2 and chromatin

Polycomb repressive complex 2 (PRC2) installs and spreads repressive histone methylation marks on eukaryotic chromosomes. Because of the key roles that PRC2 plays in development and disease, how this epigenetic machinery interacts with DNA and nucleosomes is of major interest. Nonetheless, the mechanism by which PRC2 engages with native-like chromatin remains incompletely understood. In this work, we employ single-molecule force spectroscopy and molecular dynamics simulations to dissect the behavior of PRC2 on polynucleosome arrays. Our results reveal an unexpectedly diverse repertoire of PRC2 binding configurations on chromatin. Besides reproducing known binding modes in which PRC2 interacts with bare DNA, mononucleosomes, and adjacent nucleosome pairs, our data also provide direct evidence that PRC2 can bridge pairs of distal nucleosomes. In particular, the "1-3" bridging mode, in which PRC2 engages two nucleosomes separated by one spacer nucleosome, is a preferred low-energy configuration. Moreover, we show that the distribution and stability of different PRC2-chromatin interaction modes are modulated by accessory subunits, oncogenic histone mutations, and the methylation state of chromatin. Overall, these findings have implications for the mechanism by which PRC2 spreads histone modifications and compacts chromatin. The experimental and computational platforms developed here provide a framework for understanding the molecular basis of epigenetic maintenance mediated by Polycomb-group proteins.

Ybx1 fine-tunes PRC2 activities to control embryonic brain development

Chromatin modifiers affect spatiotemporal gene expression programs that underlie organismal development. The Polycomb repressive complex 2 (PRC2) is a crucial chromatin modifier in executing neurodevelopmental programs. Here, we find that PRC2 interacts with the nucleic acid–binding protein Ybx1. In the mouse embryo in vivo, Ybx1 is required for forebrain specification and restricting mid-hindbrain growth. In neural progenitor cells (NPCs), Ybx1 controls self-renewal and neuronal differentiation. Mechanistically, Ybx1 highly overlaps PRC2 binding genome-wide, controls PRC2 distribution, and inhibits H3K27me3 levels. These functions are consistent with Ybx1-mediated promotion of genes involved in forebrain specification, cell proliferation, or neuronal differentiation. In Ybx1-knockout NPCs, H3K27me3 reduction by PRC2 enzymatic inhibitor or genetic depletion partially rescues gene expression and NPC functions. Our findings suggest that Ybx1 fine-tunes PRC2 activities to regulate spatiotemporal gene expression in embryonic neural development and uncover a crucial epigenetic mechanism balancing forebrain–hindbrain lineages and self-renewal–differentiation choices in NPCs.

PRC2 Components Maintain DNA Hypermethylation of the Upstream Promoter and Regulate Robo4 Expression in Endothelial Cells.

Roundabout4 (Robo4) is an endothelial cell-specific protein that stabilizes the vasculature in pathological angiogenesis and inflammation. We previously determined a 3-kb Robo4 promoter and demonstrated the importance of the upstream region for nuclear factor-kappaB (NF-κB)-mediated promoter activation induced by tumor necrosis factor α (TNFα). This region contains unique genomic features, including promoter region-specific DNA hypermethylation and chromatin condensation; however, the function of the region remains poorly understood. In this study, we analyzed the DNA sequences of the region and identified a motif for polycomb repressive complex 2 (PRC2). Chromatin immunoprecipitation assay indicates the binding of the PRC2 component, SUZ12, to the motif. A mutation in the motif decreased DNA methylation in embryonic stem cells and increased Robo4 promoter activity in endothelial cells. An inhibitor for the PRC2 component, EZH2, induced the promoter activity and expression of Robo4 in endothelial cells treated with or without TNFα. Taken together, these results indicate that the PRC2 components maintain DNA hypermethylation and suppress Robo4 expression via the PRC2 binding motif in the upstream promoter.

Ppp1r1b-lncRNA inhibits PRC2 at myogenic regulatory genes to promote cardiac and skeletal muscle development in mouse and human

Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators and play important roles in cardiac development and congenital heart disease. In a previous study, we identified a novel lncRNA, Ppp1r1b, with expression highly correlated with myogenesis. However, the molecular mechanism that underlies Ppp1r1b-lncRNA function in myogenic regulation is unknown. By silencing Ppp1r1b-lncRNA, mouse C2C12 and human skeletal myoblasts failed to develop fully differentiated myotubes. Myogenic differentiation was also impaired in PPP1R1B-lncRNA deficient human-induced pluripotent stem cell-derived cardiomyocytes (hiPSCs-CMs). The expression of myogenic transcription factors, including MyoD, Myogenin, and Tbx5, as well as sarcomere proteins, was significantly suppressed in Ppp1r1b-lncRNA inhibited myoblast cells and neonatal mouse heart. Histone modification analysis revealed increased H3K27 tri-methylation at MyoD1 and Myogenin promoters in GapmeR treated C2C12 cells. Furthermore, Ppp1r1b-lncRNA was found to bind to Ezh2, and chromatin isolation by RNA purification (ChIRP) assay revealed enriched interaction of Ppp1r1b-lncRNA with Myod1 and Tbx5 promoters, suggesting that Ppp1r1b-lncRNA induces transcription of myogenic transcription factors by interacting with the polycomb repressive complex 2 (PRC2) at the chromatin interface. Correspondingly, the silencing of Ppp1r1b-lncRNA increased EZH2 binding at promoter regions of myogenic transcription factors. Therefore, our results suggest that Ppp1r1b-lncRNA promotes myogenic differentiation through competing for PRC2 binding with chromatin of myogenic master regulators during heart and skeletal muscle development.

DEAD-Box Helicase 18 Counteracts PRC2 to Safeguard Ribosomal DNA in Pluripotency Regulation

Embryonic stem cells (ESCs) exhibit high levels of ribosomal RNA (rRNA) transcription and ribosome biogenesis. Here, we reveal an unexpected role for an essential DEAD-box helicase, DDX18, in antagonizing the polycomb repressive complex 2 (PRC2) to prevent deposition of the repressive H3K27me3 mark onto rDNA in pluripotent cells. DDX18 binds and sequesters PRC2 in the outer layer of the nucleolus and counteracts PRC2 complex formation invivo and invitro. DDX18 knockdown leads to increased occupancy of PRC2 and H3K27me3 at rDNA loci, accompanied by drastically decreased rRNA transcription and reduced ribosomal protein expression and translation. Auxin-induced rapid degradation ofDDX18 enhances PRC2 binding at rDNA. The inhibitionof PRC2 partially rescues the effects of DDX18 depletion on rRNA transcription and ESC self-renewal. These results demonstrate a critical role for DDX18 in safeguarding the chromatin and transcriptional integrity of rDNA by counteracting the epigenetic silencing machinery to promote pluripotency.

A Functional Link between Nuclear RNA Decay and Transcriptional Control Mediated by the Polycomb Repressive Complex 2

SummaryPluripotent embryonic stem cells (ESCs) constitute an essential cellular niche sustained by epigenomic and transcriptional regulation. Any role of post-transcriptional processes remains less explored. Here, we identify a link between nuclear RNA levels, regulated by the poly(A) RNA exosome targeting (PAXT) connection, and transcriptional control by the polycomb repressive complex 2 (PRC2). Knockout of the PAXT component ZFC3H1 impairs mouse ESC differentiation. In addition to the upregulation of bona fide PAXT substrates, Zfc3h1−/− cells abnormally express developmental genes usually repressed by PRC2. Such de-repression is paralleled by decreased PRC2 binding to chromatin and low PRC2-directed H3K27 methylation. PRC2 complex stability is compromised in Zfc3h1−/− cells with elevated levels of unspecific RNA bound to PRC2 components. We propose that excess RNA hampers PRC2 function through its sequestration from DNA. Our results highlight the importance of balancing nuclear RNA levels and demonstrate the capacity of bulk RNA to regulate chromatin-associated proteins.

A drug repurposing screening reveals a novel epigenetic activity of hydroxychloroquine

Multiple myeloma (MM) is an incurable hematological malignancy driven by several genetic and epigenetic alterations. The hyperactivation of the Polycomb repressive complex 2 (PRC2), a multi-subunit oncogenic histone methyltransferase, has been implicated in the pathogenesis of this malignancy. Upon protein-protein interaction (PPI) between the catalytic subunit EZH2 and EED, PRC2 primarily methylates lysine 27 of histone H3 (H3K27me3), thus modulating the chromatin structure and inducing transcriptional repression. Herein, we highlight a new mechanism of action that can contribute to explain the anti-tumor activity of hydroxychloroquine (HCQ), an anti-malaric agent also known as autophagy inhibitor. By structural studies, we demonstrate that HCQ inhibits the allosteric binding of PRC2 to EED within the H3K27me3-binding pocket, thus antagonizing the PRC2 catalytic activity. In silico results are compatible with the significant reduction of the H3K27me3 levels in MM cells exerted by HCQ. Overall, these findings disclose a novel epigenetic activity of HCQ with potential implications for its clinical repositioning.

The Complexity of PRC2 Subcomplexes.

Polycomb repressive complex 2 (PRC2) is a multisubunit protein complex essential for the development of multicellular organisms. Recruitment of PRC2 to target genes, followed by deposition and propagation of its catalytic product histone H3 lysine 27 trimethylation (H3K27me3), are key to the spatiotemporal control of developmental gene expression. Recent breakthrough studies have uncovered unexpected roles for substoichiometric PRC2 subunits in these processes. Here, we elaborate on how the facultative PRC2 subunits regulate catalytic activity, locus-specific PRC2 binding, and propagation of H3K27me3, and how this affects chromatin structure, gene expression, and cell fate.

C9orf72 and triplet repeat disorder RNAs: G-quadruplex formation, binding to PRC2 and implications for disease mechanisms.

Some neurological disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), fragile X syndrome, Huntington's disease, myotonic dystrophy, and various ataxias, can be caused by expansions of short nucleic acid sequence repeats in specific genes. A possible disease mechanism involves the transcribed repeat RNA binding an RNA-binding protein (RBP), resulting in its sequestration and thus dysfunction. Polycomb repressive complex 2 (PRC2), the histone methyltransferase that deposits the H3K27me3 mark of epigenetically silenced chromatin, binds G-rich RNAs and has especially high affinity for G-quadruplex (G-Q) structures. Here, we find that PRC2 target genes are derepressed and the RNA binding subunit EZH2 largely insoluble in postmortem brain samples from ALS/FTD patients with C9ORF72 (C9) repeat expansions, leading to the hypothesis that the (G4C2)n repeat RNA might be sequestering PRC2. Contrary to this expectation, we found that C9 repeat RNAs (n = 6 or 10) bind weakly to purified PRC2, and studies with the G-Q specific BG4 antibody and circular dichroism studies both indicated that these C9 RNAs have little propensity to form G-Qs in vitro. Several GC-rich triplet-repeat expansion RNAs also have low affinity for PRC2 and do not appreciably form G-Qs in vitro. The results are consistent with these sequences forming hairpin structures that outcompete G-Q folding when the repeat length is sufficiently large. We suggest that binding of PRC2 to these GC-rich RNAs is fundamentally weak but may be modulated in vivo by protein factors that affect secondary structure, such as helicases and other RBPs.

SCARNA10, a nuclear-retained long non-coding RNA, promotes liver fibrosis and serves as a potential biomarker.

Long non-coding RNAs (lncRNAs) are involved in numerous biological functions and pathological processes. However, the clinical significance of lncRNAs and their functions in liver fibrosis remain largely unclear.Methods: The transcript of lncRNA SCARNA10 in serum and liver samples from patients with advanced hepatic fibrosis, liver tissues from two fibrosis mouse models, and cultured hepatic stellate cells (HSCs) was determined by real-time RT-PCR. The effects of lentivirus-mediated knockdown or over-expression of SCARNA10 in liver fibrosis were examined in vitro and in vivo. Moreover, the effects and mechanisms of down-regulation or over-expression of SCARNA10 on the expression of the genes involved in TGFβ pathway were determined.Results: It was found lncRNA ENSMUST00000158992, named as Scarna10, was remarkably up-regulated in mouse fibrotic livers according to the microarray data. We observed that the transcript of SCARNA10 was increased in the serum and liver from patients with advanced hepatic fibrosis. Furthermore, we found that SCARNA10 promoted liver fibrosis both in vitro and in vivo through inducing hepatocytes (HCs) apoptosis and HSCs activation. Mechanistically, RNA immunoprecipitation (RIP) assays demonstrated that SCARNA10 physically associated with polycomb repressive complex 2 (PRC2). Additionally, our results demonstrated that SCARNA10 functioned as a novel positive regulator of TGFβ signaling in hepatic fibrogenesis by inhibiting the binding of PRC2 to the promoters of the genes associated with ECM and TGFβ pathway, thus promoting the transcription of these genes.Conclusions: Our study identified a crucial role of SCARNA10 in liver fibrosis, providing a proof of this molecule as a potential diagnostic marker and a possible therapeutic target against liver fibrosis.

Live-cell imaging reveals the dynamics of PRC2 and recruitment to chromatin by SUZ12-associated subunits.

Polycomb-repressive complex 2 (PRC2) is a histone methyltransferase that promotes epigenetic gene silencing, but the dynamics of its interactions with chromatin are largely unknown. Here we quantitatively measured the binding of PRC2 to chromatin in human cancer cells. Genome editing of a HaloTag into the endogenous EZH2 and SUZ12 loci and single-particle tracking revealed that ∼80% of PRC2 rapidly diffuses through the nucleus, while ∼20% is chromatin-bound. Short-term treatment with a small molecule inhibitor of the EED-H3K27me3 interaction had no immediate effect on the chromatin residence time of PRC2. In contrast, separation-of-function mutants of SUZ12, which still form the core PRC2 complex but cannot bind accessory proteins, revealed a major contribution of AEBP2 and PCL homolog proteins to chromatin binding. We therefore quantified the dynamics of this chromatin-modifying complex in living cells and separated the contributions of H3K27me3 histone marks and various PRC2 subunits to recruitment of PRC2 to chromatin.

Accurate H3K27 methylation can be established de novo by SUZ12-directed PRC2

The Polycomb repressive complex 2 (PRC2) catalyzes H3K27 methylation and is required for maintaining transcriptional patterns and cellular identity, but the specification and maintenance of genomic PRC2 binding and H3K27 methylation patterns remain incompletely understood. Epigenetic mechanisms have been proposed, wherein pre-existing H3K27 methylation directs recruitment and regulates the catalytic activity of PRC2 to support its own maintenance. Here we investigate if such mechanisms are required for specifying H3K27 methylation patterns in mouse embryonic stem cells (mESCs). Through re-expression of PRC2 subunits in genetic knockouts that have lost all H3K27 methylation, we demonstrate that methylation patterns can be accurately established de novo. We find that regional methylation kinetics correlate with original methylation patterns even in their absence, and specification of the genomic PRC2 binding pattern is retained and specifically dependent on the PRC2 core-subunit SUZ12. Thus, the H3K27 methylation patterns in mESCs are not dependent on self-autonomous epigenetic inheritance.