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.     

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.     

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.     

RYBP represses endogenous retroviruses and preimplantation- and germ line-specific genes in mouse embryonic stem cells

Polycomb repressive complexes (PRCs) are important chromatin regulators of embryonic stem (ES) cell function. RYBP binds Polycomb H2A monoubiquitin ligases Ring1A and Ring1B and has been suggested to assist PRC localization to their targets. Moreover, constitutive inactivation of RYBP precludes ES cell formation. Using ES cells conditionally deficient in RYBP, we found that RYBP is not required for maintenance of the ES cell state, although mutant cells differentiate abnormally. Genome-wide chromatin association studies showed RYBP binding to promoters of Polycomb targets, although its presence is dispensable for gene repression. We discovered, using Eed-knockout (KO) ES cells, that RYBP binding to promoters was independent of H3K27me3. However, recruiting of PRC1 subunits Ring1B and Mel18 to their targets was not altered in the absence of RYBP. In contrast, we have found that RYBP efficiently represses endogenous retroviruses (murine endogenous retrovirus [MuERV] class) and preimplantation (including zygotic genome activation stage)- and germ line-specific genes. These observations support a selective repressor activity for RYBP that is dispensable for Polycomb function in the ES cell state. Also, they suggest a role for RYBP in epigenetic resetting during preimplantation development through repression of germ line genes and PcG targets before formation of pluripotent epiblast cells.     

LANA-Mediated Recruitment of Host Polycomb Repressive Complexes onto the KSHV Genome during De Novo Infection

One of the hallmarks of the latent phase of Kaposi’s sarcoma-associated herpesvirus (KSHV) infection is the global repression of lytic viral gene expression. Following de novo KSHV infection, the establishment of latency involves the chromatinization of the incoming viral genomes and recruitment of the host Polycomb repressive complexes (PRC1 and PRC2) to the promoters of lytic genes, which is accompanied by the inhibition of lytic genes. However, the mechanism of how PRCs are recruited to the KSHV episome is still unknown. Utilizing a genetic screen of latent genes in the context of KSHV genome, we identified the latency-associated nuclear antigen (LANA) to be responsible for the genome-wide recruitment of PRCs onto the lytic promoters following infection. We found that LANA initially bound to the KSHV genome right after infection and subsequently recruited PRCs onto the viral lytic promoters, thereby repressing lytic gene expression. Furthermore, both the DNA and chromatin binding activities of LANA were required for the binding of LANA to the KSHV promoters, which was necessary for the recruitment of PRC2 to the lytic promoters during de novo KSHV infection. Consequently, the LANA-knockout KSHV could not recruit PRCs to its viral genome upon de novo infection, resulting in aberrant lytic gene expression and dysregulation of expression of host genes involved in cell cycle and proliferation pathways. In this report, we demonstrate that KSHV LANA recruits host PRCs onto the lytic promoters to suppress lytic gene expression following de novo infection.     

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.     

RYBP-PRC1 complexes mediate H2A ubiquitylation at polycomb target sites independently of PRC2 and H3K27me3

Polycomb-repressive complex 1 (PRC1) has a central role in the regulation of heritable gene silencing during differentiation and development. PRC1 recruitment is generally attributed to interaction of the chromodomain of the core protein Polycomb with trimethyl histone H3K27 (H3K27me3), catalyzed by a second complex, PRC2. Unexpectedly we find that RING1B, the catalytic subunit of PRC1, and associated monoubiquitylation of histone H2A are targeted to closely overlapping sites in wild-type and PRC2-deficient mouse embryonic stem cells (mESCs), demonstrating an H3K27me3-independent pathway for recruitment of PRC1 activity. We show that this pathway is mediated by RYBP-PRC1, a complex comprising catalytic subunits of PRC1 and the protein RYBP. RYBP-PRC1 is recruited to target loci in mESCs and is also involved in Xist RNA-mediated silencing, the latter suggesting a wider role in Polycomb silencing. We discuss the implications of these findings for understanding recruitment and function of Polycomb repressors.     

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.     

NAR Breakthrough Article: A dimeric state for PRC2

Polycomb repressive complex-2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. Long non-coding RNAs (lncRNAs) can recruit PRC2 to chromatin. Previous studies identified PRC2 subunits in a complex with the apparent molecular weight of a dimer, which might be accounted for by the incorporation of additional protein subunits or RNA rather than PRC2 dimerization. Here we show that reconstituted human PRC2 is in fact a dimer, using multiple independent approaches including analytical size exclusion chromatography (SEC), SEC combined with multi-angle light scattering and co-immunoprecipitation of differentially tagged subunits. Even though it contains at least two RNA-binding subunits, each PRC2 dimer binds only one RNA molecule. Yet, multiple PRC2 dimers bind a single RNA molecule cooperatively. These observations suggest a model in which the first RNA binding event promotes the recruitment of multiple PRC2 complexes to chromatin, thereby nucleating repression.