Polycomblike 2 facilitates the recruitment of PRC2 Polycomb group complexes to the inactive X chromosome and to target loci in embryonic stem cells

Polycomb group (PcG) proteins play an important role in the control of developmental gene expression in higher organisms. In mammalian systems, PcG proteins participate in the control of pluripotency, cell fate, cell cycle regulation, X chromosome inactivation and parental imprinting. In this study we have analysed the function of the mouse PcG protein polycomblike 2 (Pcl2), one of three homologues of the Drosophila Polycomblike (Pcl) protein. We show that Pcl2 is expressed at high levels during early embryogenesis and in embryonic stem (ES) cells. At the biochemical level, Pcl2 interacts with core components of the histone H3K27 methyltransferase complex Polycomb repressive complex 2 (PRC2), to form a distinct substoichiometric biochemical complex, Pcl2-PRC2. Functional analysis using RNAi knockdown demonstrates that Pcl2-PRC2 facilitates both PRC2 recruitment to the inactive X chromosome in differentiating XX ES cells and PRC2 recruitment to target genes in undifferentiated ES cells. The role of Pcl2 in PRC2 targeting in ES cells is critically dependent on a conserved PHD finger domain, suggesting that Pcl2 might function through the recognition of a specific chromatin configuration.     

The Arginine Methyltransferase PRMT6 Cooperates with Polycomb Proteins in Regulating HOXA Gene Expression

Protein arginine methyltransferase 6 (PRMT6) catalyses asymmetric dimethylation of histone H3 at arginine 2 (H3R2me2a), which has been shown to impede the deposition of histone H3 lysine 4 trimethylation (H3K4me3) by blocking the binding and activity of the MLL1 complex. Importantly, the genomic occurrence of H3R2me2a has been found to coincide with histone H3 lysine 27 trimethylation (H3K27me3), a repressive histone mark generated by the Polycomb repressive complex 2 (PRC2). Therefore, we investigate here a putative crosstalk between PRMT6- and PRC-mediated repression in a cellular model of neuronal differentiation. We show that PRMT6 and subunits of PRC2 as well as PRC1 are bound to the same regulatory regions of rostral HOXA genes and that they control the differentiation-associated activation of these genes. Furthermore, we find that PRMT6 interacts with subunits of PRC1 and PRC2 and that depletion of PRMT6 results in diminished PRC1/PRC2 and H3K27me3 occupancy and in increased H3K4me3 levels at these target genes. Taken together, our data uncover a novel, additional mechanism of how PRMT6 contributes to gene repression by cooperating with Polycomb proteins.     

Polycomb-like 3 promotes polycomb repressive complex 2 binding to CpG islands and embryonic stem cell self-renewal

Polycomb repressive complex 2 (PRC2) trimethylates lysine 27 of histone H3 (H3K27me3) to regulate gene expression during diverse biological transitions in development, embryonic stem cell (ESC) differentiation, and cancer. Here, we show that Polycomb-like 3 (Pcl3) is a component of PRC2 that promotes ESC self-renewal. Using mass spectrometry, we identified Pcl3 as a Suz12 binding partner and confirmed Pcl3 interactions with core PRC2 components by co-immunoprecipitation. Knockdown of Pcl3 in ESCs increases spontaneous differentiation, yet does not affect early differentiation decisions as assessed in teratomas and embryoid bodies, indicating that Pcl3 has a specific role in regulating ESC self-renewal. Consistent with Pcl3 promoting PRC2 function, decreasing Pcl3 levels reduces H3K27me3 levels while overexpressing Pcl3 increases H3K27me3 levels. Furthermore, chromatin immunoprecipitation and sequencing (ChIP-seq) reveal that Pcl3 co-localizes with PRC2 core component, Suz12, and depletion of Pcl3 decreases Suz12 binding at over 60% of PRC2 targets. Mutation of conserved residues within the Pcl3 Tudor domain, a domain implicated in recognizing methylated histones, compromises H3K27me3 formation, suggesting that the Tudor domain of Pcl3 is essential for function. We also show that Pcl3 and its paralog, Pcl2, exist in different PRC2 complexes but bind many of the same PRC2 targets, particularly CpG islands regulated by Pcl3. Thus, Pcl3 is a component of PRC2 critical for ESC self-renewal, histone methylation, and recruitment of PRC2 to a subset of its genomic sites.     

Histone H2A Mono-Ubiquitination Is a Crucial Step to Mediate PRC1-Dependent Repression of Developmental Genes to Maintain ES Cell Identity

Two distinct Polycomb complexes, PRC1 and PRC2, collaborate to maintain epigenetic repression of key developmental loci in embryonic stem cells (ESCs). PRC1 and PRC2 have histone modifying activities, catalyzing mono-ubiquitination of histone H2A (H2AK119u1) and trimethylation of H3 lysine 27 (H3K27me3), respectively. Compared to H3K27me3, localization and the role of H2AK119u1 are not fully understood in ESCs. Here we present genome-wide H2AK119u1 maps in ESCs and identify a group of genes at which H2AK119u1 is deposited in a Ring1-dependent manner. These genes are a distinctive subset of genes with H3K27me3 enrichment and are the central targets of Polycomb silencing that are required to maintain ESC identity. We further show that the H2A ubiquitination activity of PRC1 is dispensable for its target binding and its activity to compact chromatin at Hox loci, but is indispensable for efficient repression of target genes and thereby ESC maintenance. These data demonstrate that multiple effector mechanisms including H2A ubiquitination and chromatin compaction combine to mediate PRC1-dependent repression of genes that are crucial for the maintenance of ESC identity. Utilization of these diverse effector mechanisms might provide a means to maintain a repressive state that is robust yet highly responsive to developmental cues during ES cell self-renewal and differentiation.     

Partitioning and plasticity of repressive histone methylation states in mammalian chromatin

Methylation of position-specific lysine residues in histone N termini is a central modification for regulating epigenetic transitions in chromatin. Each methylatable lysine residue can exist in a mono-, di-, or trimethylated state, thereby extending the indexing potential of this particular modification. Here, we examine all possible methylation states for histone H3 lysine 9 (H3-K9) and lysine 27 (H3-K27) in mammalian chromatin. Using highly specific antibodies together with quantitative mass spectrometry, we demonstrate that pericentric heterochromatin is selectively enriched for H3-K27 monomethylation and H3-K9 trimethylation. This heterochromatic methylation profile is dependent on the Suv39h histone methyltransferases (HMTases) but independent of the euchromatic G9a HMTase. In Suv39h double null cells, pericentric heterochromatin is converted to alternative methylation imprints and accumulates H3-K27 trimethylation and H3-K9 monomethylation. Our data underscore the selective presence of distinct histone lysine methylation states in partitioning chromosomal subdomains but also reveal a surprising plasticity in propagating methylation patterns in eukaryotic chromatin.     

Dominant alleles identify SET domain residues required for histone methyltransferase of Polycomb repressive complex 2

Polycomb gene silencing requires histone methyltransferase activity of Polycomb repressive complex 2 (PRC2), which methylates lysine 27 of histone H3. Information on how PRC2 works is limited by lack of structural data on the catalytic subunit, Enhancer of zeste (E(Z)), and the paucity of E(z) mutant alleles that alter its SET domain. Here we analyze missense alleles of Drosophila E(z), selected for molecular study because of their dominant genetic effects. Four missense alleles identify key E(Z) SET domain residues, and a fifth is located in the adjacent CXC domain. Analysis of mutant PRC2 complexes in vitro, and H3-K27 methylation in vivo, shows that each SET domain mutation disrupts PRC2 histone methyltransferase. Based on known SET domain structures, the mutations likely affect either the lysine-substrate binding pocket, the binding site for the adenosylmethionine methyl donor, or a critical tyrosine predicted to interact with the substrate lysine epsilon-amino group. In contrast, the CXC mutant retains catalytic activity, Lys-27 specificity, and trimethylation capacity. Deletion analysis also reveals a functional requirement for a conserved E(Z) domain N-terminal to CXC and SET. These results identify critical SET domain residues needed for PRC2 enzyme function, and they also emphasize functional inputs from outside the SET domain.     

The polycomb group mutant esc leads to augmented levels of paused Pol II in the Drosophila embryo

Many developmental control genes contain paused RNA polymerase II (Pol II) and are thereby "poised" for rapid and synchronous activation in the early Drosophila embryo. Evidence is presented that Polycomb group (PcG) repressors can influence paused Pol II. ChIP-Seq and GRO-Seq assays were used to determine the genome-wide distributions of Pol II, H3K27me3, and H3K4me3 in extra sex combs (esc) mutant embryos. ESC is a key component of the Polycomb repressive complex 2 (PRC2), which mediates H3K27me3 modification. Enhanced Pol II occupancy is observed for thousands of genes in esc mutant embryos, including genes not directly regulated by PRC2. Thus, it would appear that silent genes lacking promoter-associated paused Pol II in wild-type embryos are converted into "poised" genes with paused Pol II in esc mutants. We suggest that this conversion of silent genes into poised genes might render differentiated cell types susceptible to switches in identity in PcG mutants.