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.     

Role of Bmi-1 and Ring1A in H2A ubiquitylation and Hox gene silencing

Polycomb group (PcG) proteins exist in at least two biochemically distinct protein complexes, the EED-EZH2 complex and the PRC1 complex, that respectively possess H3-K27 methyltransferase and H2A-K119 ubiquitin E3 ligase activities. How the enzymatic activities are regulated and what their role is in Hox gene silencing are not clear. Here, we demonstrate that Bmi-1 and Ring1A, two components of the PRC1 complex, play important roles in H2A ubiquitylation and Hox gene silencing. We show that both proteins positively regulate H2A ubiquitylation. Chromatin immunoprecipitation (ChIP) assays demonstrate that Bmi-1 and other components of the two PcG complexes bind to the promoter of HoxC13. Knockout Bmi-1 results in significant loss of H2A ubiquitylation and upregulation of Hoxc13 expression, whereas EZH2-mediated H3-K27 methylation is not affected. Our results suggest that EZH2-mediated H3-K27 methylation functions upstream of PRC1 and establishes a critical role for Bmi-1 and Ring1A in H2A ubiquitylation and Hox gene silencing.     

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.     

Polycomb group proteins: remembering how to catch chromatin during replication

Polycomb group (PcG) proteins maintain the expression state of PcG‐responsive genes during development of multicellular organisms. Recent observations suggest that “the H3K27me3 modification” acts to maintain Polycomb repressive complex (PRC) 2, the enzyme that creates this modification, on replicating chromatin. This could in turn promote propagation of H3K27me3 on newly replicated daughter chromatin, and promote recruitment of PRC1. Other work suggests that PRC1‐class complexes can be maintained on replicating chromatin, at least in vitro, independently of H3K27me3. Thus, histone modifications and PcG proteins themselves may both be maintained through replication.     

Ezh2 requires PHF1 to efficiently catalyze H3 lysine 27 trimethylation in vivo

The mammalian Polycomblike protein PHF1 was previously shown to interact with the Polycomb group (PcG) protein Ezh2, a histone methyltransferase whose activity is pivotal in sustaining gene repression during development and in adulthood. As Ezh2 is active only when part of the Polycomb Repressive Complexes (PRC2-PRC4), we examined the functional role of its interaction with PHF1. Chromatin immunoprecipitation experiments revealed that PHF1 resides along with Ezh2 at Ezh2-regulated genes such as the HoxA loci and the non-Hox MYT1 and WNT1 genes. Knockdown of PHF1 or of Ezh2 led to up-regulated HoxA gene expression. Interestingly, depletion of PHF1 did correlate with reduced occupancy of Bmi-1, a PRC1 component. As expected, knockdown of Ezh2 led to reduced levels of its catalytic products H3K27me2/H3K27me3. However, reduced levels of PHF1 also led to decreased global levels of H3K27me3. Notably, the levels of H3K27me3 decreased while those of H3K27me2 increased at the up-regulated HoxA loci tested. Consistent with this, the addition of PHF1 specifically stimulated the ability of Ezh2 to catalyze H3K27me3 but not H3K27me1/H3K27me2 in vitro. We conclude that PHF1 modulates the activity of Ezh2 in favor of the repressive H3K27me3 mark. Thus, we propose that PHF1 is a determinant in PcG-mediated gene repression.     

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.     

PcG蛋白转录抑制复合物组份的功能及构成的时空特征

PcG蛋白主要以PRC1和PRC2两组复合物的形式存在,通过参与核小体组蛋白翻译后修饰等机制,发挥调控靶基因转录的功能. PRC1复合体中的RING1A/B具有使组蛋白H2AK119泛素化的活性;PRC2中的EZH2具有使组蛋白H3K27三甲基化的活性,形成PRC1锚定到核小体上的位点. PcG蛋白的表达特征具有发育阶段和细胞类型时空特异性. 长链非编码RNA等反式作用因子能募集PcG蛋白结合于靶基因,发挥靶向作用. 本文就PcG蛋白功能、构成的时空特异性、募集机制及其与疾病发生的关系研究进展做一综述.     

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 group protein-associated chromatin is reproduced in post-mitotic G1 phase and is required for S phase progression

Polycomb group (PcG) proteins form two distinct complexes, PRC1 and PRC2, to regulate developmental target genes by maintaining the epigenetic state in cells. PRC2 methylates histone H3 at lysine 27 (H3K27), and PRC1 then recognizes methyl-H3K27 to form repressive chromatin. However, it remains unknown how PcG proteins maintain stable and plastic chromatin during cell division. Here we report that PcG-associated chromatin is reproduced in the G(1) phase in post-mitotic cells and is required for subsequent S phase progression. In dividing cells, H3K27 trimethylation (H3K27Me(3)) marked mitotic chromosome arms where PRC2 (Suz12 and Ezh2) co-existed, whereas PRC1 (Bmi1 and Pc2) appeared in distinct foci in the pericentromeric regions. As each PRC complex was increasingly assembled from mitosis to G(1) phase, PRC1 formed H3K27Me(3)-based chromatin intensively during middle and late G(1) phase; this chromatin was highly resistant to in situ nuclease treatment. Thus, the transition from mitosis to G(1) phase is crucial for PcG-mediated chromatin inheritance. Knockdown of Suz12 markedly reduced the amount of H3K27Me(3) on mitotic chromosomes, and as a consequence, PRC1 foci were not fully transmitted to post-mitotic daughter cells. S phase progression was markedly delayed in these Suz12-knockdown cells. The fact that PcG-associated chromatin is reproduced during post-mitotic G(1) phase suggests the possibility that PcG proteins enable their target chromatin to be remodeled in response to stimuli in the G(1) phase.     

Dopamine Signaling Leads to Loss of Polycomb Repression and Aberrant Gene Activation in Experimental Parkinsonism

Polycomb group (PcG) proteins bind to and repress genes in embryonic stem cells through lineage commitment to the terminal differentiated state. PcG repressed genes are commonly characterized by the presence of the epigenetic histone mark H3K27me3, catalyzed by the Polycomb repressive complex 2. Here, we present in vivo evidence for a previously unrecognized plasticity of PcG-repressed genes in terminally differentiated brain neurons of parkisonian mice. We show that acute administration of the dopamine precursor, L-DOPA, induces a remarkable increase in H3K27me3S28 phosphorylation. The induction of the H3K27me3S28p histone mark specifically occurs in medium spiny neurons expressing dopamine D1 receptors and is dependent on Msk1 kinase activity and DARPP-32-mediated inhibition of protein phosphatase-1. Chromatin immunoprecipitation (ChIP) experiments showed that increased H3K27me3S28p was accompanied by reduced PcG binding to regulatory regions of genes. An analysis of the genome wide distribution of L-DOPA-induced H3K27me3S28 phosphorylation by ChIP sequencing (ChIP-seq) in combination with expression analysis by RNA-sequencing (RNA-seq) showed that the induction of H3K27me3S28p correlated with increased expression of a subset of PcG repressed genes. We found that induction of H3K27me3S28p persisted during chronic L-DOPA administration to parkisonian mice and correlated with aberrant gene expression. We propose that dopaminergic transmission can activate PcG repressed genes in the adult brain and thereby contribute to long-term maladaptive responses including the motor complications, or dyskinesia, caused by prolonged administration of L-DOPA in Parkinson''s disease.