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.     

Polycomb repressive complex 1: Regulators of neurogenesis from embryonic to adult stage

Development of vertebrate nervous system is a complex process which involves differential gene expression and disruptions in this process or in the mature brain, may lead to neurological disorders and diseases. Extensive work that spanned several decades using rodent models and recent work on stem cells have helped uncover the intricate process of neuronal differentiation and maturation. There are various morphological changes, genetic and epigenetic modifications which occur during normal mammalian neural development, one of the chromatin modifications that controls vital gene expression are the posttranslational modifications on histone proteins, that controls accessibility of translational machinery. Among the histone modifiers, polycomb group proteins (PcGs), such as Ezh2, Eed and Suz12 form large protein complexes—polycomb repressive complex 2 (PRC2); while Ring1b and Bmi1 proteins form core of PRC1 along with accessory proteins such as Cbx, Hph, Rybp and Pcgfs catalyse histone modifications such as H3K27me3 and H2AK119ub1. PRC1 proteins are known to play critical role in X chromosome inactivation in females but they also repress the expression of key developmental genes and tightly regulate the mammalian neuronal development. In this review we have discussed the signalling pathways, morphogens and nuclear factors that initiate, regulate and maintain cells of the nervous system. Further, we have extensively reviewed the recent literature on the role of Ring1b and Bmi1 in mammalian neuronal development and differentiation; as well as highlighted questions that are still unanswered.     

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.     

The Polycomb Repressive Complex 1 Protein BMI1 Is Required for Constitutive Heterochromatin Formation and Silencing in Mammalian Somatic Cells

The polycomb repressive complex 1 (PRC1), containing the core BMI1 and RING1A/B proteins, mono-ubiquitinylates histone H2A (H2A^ub) and is associated with silenced developmental genes at facultative heterochromatin. It is, however, assumed that the PRC1 is excluded from constitutive heterochromatin in somatic cells based on work performed on mouse embryonic stem cells and oocytes. We show here that BMI1 is required for constitutive heterochromatin formation and silencing in human and mouse somatic cells. BMI1 was highly enriched at intergenic and pericentric heterochromatin, co-immunoprecipitated with the architectural heterochromatin proteins HP1, DEK1, and ATRx, and was required for their localization. In contrast, BRCA1 localization was BMI1-independent and partially redundant with that of BMI1 for H2A^ub deposition, constitutive heterochromatin formation, and silencing. These observations suggest a dynamic and developmentally regulated model of PRC1 occupancy at constitutive heterochromatin, and where BMI1 function in somatic cells is to stabilize the repetitive genome.     

A phosphorylated form of Mel-18 targets the Ring1B histone H2A ubiquitin ligase to chromatin

Recent studies have shown that PRC1-like Polycomb repressor complexes monoubiquity-late chromatin on histone H2A at lysine residue 119. Here we have analyzed the function of the polycomb protein Mel-18. Using affinity-tagged human MEL-18, we identify a polycomb-like complex, melPRC1, containing the core PRC1 proteins, RING1/2, HPH2, and CBX8. We show that, in ES cells, melPRC1 can functionally substitute for other PRC1-like complexes in Hox gene repression. A reconstituted subcomplex containing only Ring1B and Mel-18 functions as an efficient ubiquitin E3 ligase. This complex ubiquitylates free histone substrates nonspecifically but is highly specific for histone H2A lysine 119 in the context of nucleosomes. Mutational analysis demonstrates that while Ring1B is required for E3 function, Mel-18 directs this activity to H2A lysine 119 in chromatin. Moreover, this substrate-targeting function of Mel-18 is dependent on its prior phosphorylation at multiple residues, providing a direct link between chromatin modification and cell signaling pathways.     

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.     

USP7 Cooperates with SCML2 To Regulate the Activity of PRC1

USP7 is a protein deubiquitinase with an essential role in development. Here, we provide evidence that USP7 regulates the activity of Polycomb repressive complex 1 (PRC1) in coordination with SCML2. There are six versions of PRC1 defined by the association of one of the PCGF homologues (PCGF1 to PCGF6) with the common catalytic subunit RING1B. First, we show that SCML2, a Polycomb group protein that associates with PRC1.2 (containing PCGF2/MEL18) and PRC1.4 (containing PCGF4/BMI1), modulates the localization of USP7 and bridges USP7 with PRC1.4, allowing for the stabilization of BMI1. Chromatin immunoprecipitation (ChIP) experiments demonstrate that USP7 is found at SCML2 and BMI1 target genes. Second, inhibition of USP7 leads to a reduction in the level of ubiquitinated histone H2A (H2Aub), the catalytic product of PRC1 and key for its repressive activity. USP7 regulates the posttranslational status of RING1B and BMI1, a specific component of PRC1.4. Thus, not only does USP7 stabilize PRC1 components, its catalytic activity is also necessary to maintain a functional PRC1, thereby ensuring appropriate levels of repressive H2Aub.     

Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity

SUZ12 is a recently identified Polycomb group (PcG) protein, which together with EZH2 and EED forms different Polycomb repressive complexes (PRC2/3). These complexes contain histone H3 lysine (K) 27/9 and histone H1 K26 methyltransferase activity specified by the EZH2 SET domain. Here we show that mice lacking Suz12, like Ezh2 and Eed mutant mice, are not viable and die during early postimplantation stages displaying severe developmental and proliferative defects. Consistent with this, we demonstrate that SUZ12 is required for proliferation of cells in tissue culture. Furthermore, we demonstrate that SUZ12 is essential for the activity and stability of the PRC2/3 complexes in mouse embryos, in tissue culture cells and in vitro. Strikingly, Suz12-deficient embryos show a specific loss of di- and trimethylated H3K27, demonstrating that Suz12 is indeed essential for EZH2 activity in vivo. In conclusion, our data demonstrate an essential role of SUZ12 in regulating the activity of the PRC2/3 complexes, which are required for regulating proliferation and embryogenesis.     

Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation

In many higher organisms, 5%-15% of histone H2A is ubiquitylated at lysine 119 (uH2A). The function of this modification and the factors involved in its establishment, however, are unknown. Here we demonstrate that uH2A occurs on the inactive X chromosome in female mammals and that this correlates with recruitment of Polycomb group (PcG) proteins belonging to Polycomb repressor complex 1 (PRC1). Based on our observations, we tested the role of the PRC1 protein Ring1B and its closely related homolog Ring1A in H2A ubiquitylation. Analysis of Ring1B null embryonic stem (ES) cells revealed extensive depletion of global uH2A levels. On the inactive X chromosome, uH2A was maintained in Ring1A or Ring1B null cells, but not in double knockout cells, demonstrating an overlapping function for these proteins in development. These observations link H2A ubiquitylation, X inactivation, and PRC1 PcG function, suggesting an unanticipated and novel mechanism for chromatin-mediated heritable gene silencing.