Genomewide analysis of PRC1 and PRC2 occupancy identifies two classes of bivalent domains

In embryonic stem (ES) cells, bivalent chromatin domains with overlapping repressive (H3 lysine 27 tri-methylation) and activating (H3 lysine 4 tri-methylation) histone modifications mark the promoters of more than 2,000 genes. To gain insight into the structure and function of bivalent domains, we mapped key histone modifications and subunits of Polycomb-repressive complexes 1 and 2 (PRC1 and PRC2) genomewide in human and mouse ES cells by chromatin immunoprecipitation, followed by ultra high-throughput sequencing. We find that bivalent domains can be segregated into two classes -- the first occupied by both PRC2 and PRC1 (PRC1-positive) and the second specifically bound by PRC2 (PRC2-only). PRC1-positive bivalent domains appear functionally distinct as they more efficiently retain lysine 27 tri-methylation upon differentiation, show stringent conservation of chromatin state, and associate with an overwhelming number of developmental regulator gene promoters. We also used computational genomics to search for sequence determinants of Polycomb binding. This analysis revealed that the genomewide locations of PRC2 and PRC1 can be largely predicted from the locations, sizes, and underlying motif contents of CpG islands. We propose that large CpG islands depleted of activating motifs confer epigenetic memory by recruiting the full repertoire of Polycomb complexes in pluripotent cells.     

Robertsonian translocations modify genomic distribution of γH2AFX and H3.3 in mouse germ cells

Heterozygosity for Robertsonian translocations hampers pairing and synapsis between the translocated chromosome and its normal homologs during meiotic prophase I. This causes meiotic silencing of unsynapsed chromatin in pericentromeric regions. Several lines of evidence suggest that autosomal asynapsis leads to meiotic arrest in males and two underlying mechanisms have been proposed: (1) reactivation of the X and Y chromosomes due to competition for silencing factors and (2) meiotic silencing of genes that are located in the unsynapsed regions and are essential for meiotic progression. The latter mechanism requires that asynapsis and meiotic silencing spread beyond the p-arms of the normal homologs into gene-rich regions. We used chromatin immunoprecipitation assays to determine whether histones γH2AFX and H3.3, both marks of asynapsis and meiotic silencing, are enriched in gene-rich regions of the translocated chromosomes and their homologs in the spermatocytes of heterozygous carriers of Robertsonian translocations. We also asked if γH2AFX and H3.3 enrichment was reduced at the X chromosome and if γH2AFX and H3.3 enrichment was higher on the normal homolog. Our data show that γH2AFX enrichment extends as far as 9–15 Mb of the annotated genomic sequence of the q-arms of the translocated chromosomal trivalents and that both γH2AFX and H3.3 levels are reduced over the X chromosome. Our data are also suggestive of an asymmetry in γH2AFX and H3.3 enrichment with a bias toward the non-translocated homolog.     

The recruitment of chromatin modifiers by long noncoding RNAs: lessons from PRC2

Polycomb repressive complex-2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. Among chromatin modifying factors shown to be recruited and regulated by long noncoding RNAs (lncRNAs), PRC2 is one of the most studied. Mammalian PRC2 binds thousands of RNAs in vivo, and it is becoming a model system for the recruitment of chromatin modifying factors by RNA. Yet, well-defined PRC2-binding motifs within target RNAs have been elusive. From the protein side, PRC2 RNA-binding subunits contain no known RNA-binding domains, complicating functional studies. Here we provide a critical review of existing models for the recruitment of PRC2 to chromatin by RNAs. This discussion may also serve researchers who are studying the recruitment of other chromatin modifiers by lncRNAs.     

Mitotic phosphorylation of PRC1 at Thr470 is required for PRC1 oligomerization and proper central spindle organization

During cell division, chromosome segregation is orchestrated by the interaction of spindle microtubules with thecentromere. A dramatic remodeling of interpolar microtubules into an organized central spindle between the separatingchromatids is required for the initiation and execution of cytokinesis. Central spindle organization requires mitotic kine-sins, the chromosomal passenger protein complex, and microtubule bundling protein PRC1. PRC1 is phosphorylated byCdc2 at Thr470 and Thr481 during mitosis. However, the functional relevance of PRC1 phosphorylation at Thr470 hasremained elusive. Here we show that expression of the non-phosphorylatable mutant PRC1~(T470A) but not the phospho-mimi-cking mutant PRC1~(T470E) causes aberrant organization of the central spindle. Immunoprecipitation experiment indicatesthat both PRC1~(T470A) and PRC1~(T470E) mutant proteins associate with wild-type PRC1, suggesting that phosphorylationof Thr470 does not alter PRC1 self-association. In addition, in vitro co-sedimentation experiment showed that PRC1binds to microtubule independent of the phosphorylation state of Thr470. Gel-filtration experiment suggested that phos-phorylation of Thr470 promotes oligomerization of PRC1. Given the fact that prevention of the Thr470 phosphorylationinhibits PRC1 oligomerization in vitro and causes an aberrant organization of central spindle in vivo, we propose thatthis phosphorylation-dependent PRC1 oligomerization ensures that central spindle assembly occurs at the appropriatetime in the cell cycle.     

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.     

Interdependence of PRC1 and PRC2 for recruitment to Polycomb Response Elements

Polycomb Group (PcG) proteins are epigenetic repressors essential for control of development and cell differentiation. They form multiple complexes of which PRC1 and PRC2 are evolutionary conserved and obligatory for repression. The targeting of PRC1 and PRC2 is poorly understood and was proposed to be hierarchical and involve tri-methylation of histone H3 (H3K27me3) and/or monoubiquitylation of histone H2A (H2AK118ub). Here, we present a strict test of this hypothesis using the Drosophila model. We discover that neither H3K27me3 nor H2AK118ub is required for targeting PRC complexes to Polycomb Response Elements (PREs). We find that PRC1 can bind PREs in the absence of PRC2 but at many PREs PRC2 requires PRC1 to be targeted. We show that one role of H3K27me3 is to allow PcG complexes anchored at PREs to interact with surrounding chromatin. In contrast, the bulk of H2AK118ub is unrelated to PcG repression. These findings radically change our view of how PcG repression is targeted and suggest that PRC1 and PRC2 can communicate independently of histone modifications.     

PRC1 is a microtubule binding and bundling protein essential to maintain the mitotic spindle midzone

Midzone microtubules of mammalian cells play an essential role in the induction of cell cleavage, serving as a platform for a number of proteins that play a part in cytokinesis. We demonstrate that PRC1, a mitotic spindle-associated Cdk substrate that is essential to cell cleavage, is a microtubule binding and bundling protein both in vivo and in vitro. Overexpression of PRC1 extensively bundles interphase microtubules, but does not affect early mitotic spindle organization. PRC1 contains two Cdk phosphorylation motifs, and phosphorylation is possibly important to mitotic suppression of bundling, as a Cdk phosphorylation-null mutant causes extensive bundling of the prometaphase spindle. Complete suppression of PRC1 by siRNA causes failure of microtubule interdigitation between half spindles and the absence of a spindle midzone. Truncation mutants demonstrate that the NH2-terminal region of PRC1, rich in alpha-helical sequence, is important for localization to the cleavage furrow and to the center of the midbody, whereas the central region, with the highest sequence homology between species, is required for microtubule binding and bundling activity. We conclude that PRC1 is a microtubule-associated protein required to maintain the spindle midzone, and that distinct functions are associated with modular elements of the primary sequence.     

Independent Mechanisms Target SMCHD1 to Trimethylated Histone H3 Lysine 9-Modified Chromatin and the Inactive X Chromosome

The chromosomal protein SMCHD1 plays an important role in epigenetic silencing at diverse loci, including the inactive X chromosome, imprinted genes, and the facioscapulohumeral muscular dystrophy locus. Although homology with canonical SMC family proteins suggests a role in chromosome organization, the mechanisms underlying SMCHD1 function and target site selection remain poorly understood. Here we show that SMCHD1 forms an active GHKL-ATPase homodimer, contrasting with canonical SMC complexes, which exist as tripartite ring structures. Electron microscopy analysis demonstrates that SMCHD1 homodimers structurally resemble prokaryotic condensins. We further show that the principal mechanism for chromatin loading of SMCHD1 involves an LRIF1-mediated interaction with HP1γ at trimethylated histone H3 lysine 9 (H3K9me3)-modified chromatin sites on the chromosome arms. A parallel pathway accounts for chromatin loading at a minority of sites, notably the inactive X chromosome. Together, our results provide key insights into SMCHD1 function and target site selection.     

Recruitment of PRC1 function at the initiation of X inactivation independent of PRC2 and silencing

In mammals X inactivation is initiated by expression of Xist RNA and involves the recruitment of Polycomb repressive complex 1 (PRC1) and 2 (PRC2), which mediate chromosome-wide ubiquitination of histone H2A and methylation of histone H3, respectively. Here, we show that PRC1 recruitment by Xist RNA is independent of gene silencing. We find that Eed is required for the recruitment of the canonical PRC1 proteins Mph1 and Mph2 by Xist. However, functional Ring1b is recruited by Xist and mediates ubiquitination of histone H2A in Eed deficient embryonic stem (ES) cells, which lack histone H3 lysine 27 tri-methylation. Xist expression early in ES cell differentiation establishes a chromosomal memory, which allows efficient H2A ubiquitination in differentiated cells and is independent of silencing and PRC2. Our data show that Xist recruits PRC1 components by both PRC2 dependent and independent modes and in the absence of PRC2 function is sufficient for the establishment of Polycomb-based memory systems in X inactivation.     

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.