The Polycomb group proteins foster gene repression profiles required for proper development and unimpaired adulthood, and comprise the components of the Polycomb-Repressive Complex 2 (PRC2) including the histone H3 Lys 27 (H3K27) methyltransferase Ezh2. How mammalian PRC2 accesses chromatin is unclear. This study found Jarid2 associates with PRC2 and stimulates its enzymatic activity in vitro. Jarid2 contains a Jumonji C domain, but is devoid of detectable histone demethylase activity. Instead, its artificial recruitment to a promoter in vivo resulted in corecruitment of PRC2 with resultant increased levels of di- and trimethylation of H3K27 (H3K27me2/3). Jarid2 colocalizes with Ezh2 and MTF2, a homolog of Drosophila Pcl, at endogenous genes in embryonic stem (ES) cells. Jarid2 can bind DNA and its recruitment in ES cells is interdependent with that of PRC2, as Jarid2 knockdown reduced PRC2 at its target promoters, and ES cells devoid of the PRC2 component EED are deficient in Jarid2 promoter access. In addition to the well-documented defects in embryonic viability upon down-regulation of Jarid2, ES cell differentiation is impaired, as is Oct4 silencing (Li, 2010).

Since the first characterization of the PRC2 core complex, the subsequent, persuasive evidence supports that PRC2 is actually a family of complexes whose composition varies during development, as a function of cell type, or even from one promoter to another. This study identified two new components that interact with PRC2: MTF2 and Jarid2. These analyses of the proteins that interact with the PRC2 complex initiated with transformed cells. Yet it has become clear that interactions observed using transformed cells might be specific to such cells, and not a determinant to the integrity of a normal organism. Thus, studies of a developmentally relevant process was incorporated and it was confirmed that the interactions observed between PRC2 and Jarid2 were of consequence to the developmental program (Li, 2010).

MTF2 is a paralog of Drosophila Pcl. PHF1, another mammalian paralog of Pcl, is required for efficient H3K27me3 and gene silencing in HeLa cells. Although PHF1 appears dispensable for PRC2 recruitment in HeLa cells, work in Drosophila has suggested that the absence of Pcl could impair PRC2 gene targeting. It is possible that the other paralogs of Pcl (MTF2 and PHF19) exhibit a role that is partially redundant with PHF1 function and thereby maintain PRC2 recruitment upon its knockdown. Pcl and its mammalian paralogs contain two PHD domains and a tudor domain, domains reported to potentially recognize methylated histones. Although the ability of Pcl to specifically bind modified histone has not been elucidated to date, it is tempting to speculate that the PHD and tudor domains could target Pcl to specific chromatin regions. Its presence would then stabilize PRC2 recruitment and promote its enzymatic activity. In support of this hypothesis, it was observed that, whereas Ezh2 targeting is severely impaired in Eed-/- ES cells, MTF2 recruitment is affected in a promoter-dependent manner and to a lesser extent than that of Ezh2. This observation suggests that MTF2 gene targeting could be partially independent of PRC2 (Li, 2010).

The exact function of Jarid2 is more enigmatic. Indeed, Jarid2 is a member of a family of enzymes capable of demethylating histones. However, Jarid2 is devoid of the amino acids required for iron and αKG binding, and consequently is unable to catalyze this reaction. It is considered that Jarid2 could act as a dominant negative and inhibit the activity of other histone demethylases; however, coexpression of Jarid2 with, for instance, SMCX did not affect H3K4me3 demethylation. Jarid2 has two domains that could potentially bind DNA: the ARID domain and a zinc finger. Although the ARID domain of Jarid2 was reported to bind DNA, band shift assay suggests that other parts of the Jarid2 C terminus (potentially a zinc finger) are also important for binding to DNA. The SELEX experiment performed with the full-length Jarid2 did not allow identification of any sequence-specific DNA binding, but did result in a slight enrichment of GC-rich DNA sequences. Importantly, it was found that the N-terminal part of Jarid2 could robustly stimulate PRC2-Ezh2 enzymatic activity on nucleosomes. A knockdown of Jarid2 decreased the enrichment of PRC2 at its target genes. Conversely, overexpression of a Gal4-Jarid2 chimera recruited PRC2 at a stably integrated reporter and increased PRC2 enrichment at its target genes, supporting the hypothesis that Jarid2 contributes to PRC2 recruitment (Li, 2010).

In the case of Drosophila, PRE (Polycomb group response element) sequences have been described, and PRC2 access to chromatin is expected to involve the concerted action of several distinct and specific DNA-binding proteins that interact directly or indirectly with PRC2. However, these same DNA-binding factors, or even a combination thereof, are also found at active genes devoid of PRC2. What distinguishes PRE sequences harboring PRC2 from active genes is still not clear. During the evolution from Drosophila to mammals, only a few of the DNA-binding factors that bind PREs (Dsp1 and Pho) are conserved. Either PRC2 recruitment in mammals involves other mechanisms, or distinct transcription factors have emerged to stabilize PRC2 at its target genes. A recent study has identified a presumed mammalian PRE; however, the role of this putative PRE at the endogenous locus that is enriched for PRC2 is not reproduced when the element is integrated upstream of a transgene, as PRC2 is absent. Of note, whereas DNA-binding proteins are likely to play an important role for PRC2 recruitment in mammals, some studies have now suggested that long noncoding RNA could also be involved in this process. These observations together suggest that the recruitment of PRC2 to target genes is complex and requires more than one factor. These findings suggest that the DNA-binding activity of Jarid2 is one such factor, but its affinity for DNA is low and likely requires the help of other factors (Li, 2010).

A critical issue at this juncture is whether or not the composition of PRC2 changes during development. This study reports that Jarid2 interacts with PRC2, but its expression, unlike the PRC2 core components, seems to be restricted to some cell lines. In agreement with previous gene expression profiles that monitored mRNA levels during the reprogramming of mouse embryonic fibroblast cells into ES cells, it is observed that Jarid2 expression is higher in undifferentiated ES cells and decreases upon differentiation. Polycomb target genes are enriched with the H2A variant H2A.Z in undifferentiated ES cells; furthermore, H2A.Z and PRC2 targeting are interdependent in these cells. This result suggests that PRC2 recruitment might involve distinct mechanisms in ES cells and differentiated cells. It is possible that Jarid2 somehow contributes to this specificity (Li, 2010).

Knockdown of Jarid2 in undifferentiated ES cells does not give rise to an obvious phenotype; gene expression patterns appear to be only moderately affected, and cell proliferation is unchanged. In contrast, when cells are induced to differentiate, a process that entails dramatic changes in gene expression, impairments were observed as a function of Jarid2 knockdown. Interference with Jarid2 resulted in a failure to accurately coordinate the expression of genes required for the differentiation process, consistent with the previous report on Suz12 knockout cells. Instead of the requisite silencing of OCT4 and Nanog loci that occurs upon normal differentiation, each of which become enriched in H3K27me3, Jarid2 knockdown prevented such H3K27 methylation at these genes, and this correlated with their delayed repression. Thus, the Jumonji family of proteins that usually exhibits demethylase activity that might function in opposition to the role mediated by PRC2 contains the member Jarid2 that is devoid of such activity and instead facilitates the action of PRC2 through enabling its access to chromatin (Li, 2010).

Ezh2 functions as a histone H3 Lys 27 (H3K27) methyltransferase when comprising the Polycomb-Repressive Complex 2 (PRC2). Trimethylation of H3K27 (H3K27me3) correlates with transcriptionally repressed chromatin. The means by which PRC2 targets specific chromatin regions is currently unclear, but noncoding RNAs (ncRNAs) have been shown to interact with PRC2 and may facilitate its recruitment to some target genes. This study, carried out in mammalian cells, shows that Ezh2 interacts with HOTAIR and Xist. Ezh2 is phosphorylated by cyclin-dependent kinase 1 (CDK1) at threonine residues 345 and 487 in a cell cycle-dependent manner. A phospho-mimic at residue 345 increased HOTAIR ncRNA binding to Ezh2, while the phospho-mimic at residue 487 was ineffectual. An Ezh2 domain comprising T345 was found to be important for binding to HOTAIR and the 5' end of Xist (Kaneko, 2010).

The results presented here demonstrate that PRC2 binding to HOTAIR (expressed from the HOXC cluster) and RepA (repeats found in Xist) ncRNAs is mediated through its Ezh2 component, and that phosphorylation of Ezh2-T345 up-regulates HOTAIR-binding activity. Given that phosphorylation at this site is cell cycle-regulated, it is speculated that PRC2 recruitment to chromatin, mediated through Ezh2 interaction with HOTAIR or RepA ncRNAs and presumably other ncRNAs, must be restricted to a tightly defined interval during the cell cycle (G2/M). Most importantly, the results establish that there are at least two populations of PRC2 complexes during the G2-M stages of the cell cycle. This is consistent with a model whereby PRC2 is recruited to specific genes to initiate repression as a function of its Ezh2 component being phosphorylated at T345, after which other PRC2 complexes then spread the repressing signature (H3K27me2/3). Of note, a recent study has also documented that human Ezh2 is phosphorylated at Thr 350 (murine T345) by CDK1, and, in agreement with the current findings, the report shows that this modification is ineffectual with respect to the integrity of the PRC2 complex and PRC2-mediated histone lysine methyltransferase activity. Instead, this study shows that mutant Ezh2 that is not subject to T350 phosphorylation results in down-regulated PRC2 recruitment, such that appropriate gene repression is thwarted. This report demonstrates that abrogation of this phosphorylation site within Ezh2 compromises Ezh2 interaction with ncRNAs, and this may bear directly on the mechanism by which PRC2 recruitment is impaired (Kaneko, 2010).

It has been demonstrated previously that the Eed component of PRC2 binds to trimethylated histone-repressive marks, but its binding to H3K27me3 in particular results in an allosteric effect that markedly increases the histone methyltransferase activity of its partner, Ezh2. Thus, PRC2 binding to the product of its activity increases its production of this mark. It is postulated that HOTAIR and RepA ncRNAs (and other ncRNAs) recruit PRC2 to initiate repression of target genes, and that this recruitment is enhanced by Ezh2 phosphorylation at T345. This proposed mechanism is consistent with only a small percentage of Ezh2 being phosphorylated at T345. If ncRNA species are responsible for targeting PRC2 to chromatin during G2/M, it is postulated that the recruited PRC2 would set the initial H3K27me3 mark. A larger number of PRC2 complexes, independent of their Ezh2 component being phosphorylated, would then propagate this mark upon their Eed component binding to the initial H3K27me3, with resultant allosteric activation of their Ezh2 activity (Kaneko, 2010).

An important question remaining is whether Ezh2 is the only component of PRC2 that binds to ncRNAs. These studies established that a 30-amino-acid domain of the PRC2-associated protein Jarid2 also binds to ncRNAs. Additionally, a recent study suggested that the Suz12 subunit of PRC2 binds to nascent transcripts. It was postulated that this binding results in the halting of RNA polymerase II. Interestingly, it was postulated that Suz12 binding to nascent transcripts requires a unique stem-loop structure on the RNA. This structure is similar to the structure of RepA, and computer analysis of the amino acid sequence of Suz12 revealed a putative domain at its N terminus with a predicted RNA-binding domain. These observations collectively suggest that PRC2 recruitment to its target genes is mediated by ncRNA, different species of which likely bind to different PRC2 subunits. Whether specificity or affinity of PRC2 for its target genes is regulated by PRC2 binding through its component(s) to one family of ncRNA (specificity), or whether multiple subunits of PRC2 simultaneously bind different ncRNAs or domains within a ncRNA (affinity), remains to be established. Regardless, the studies described here are beginning to shed light on the role of ncRNAs in mediating the recruitment of mammalian PRC2 to its target genes (Kaneko, 2010).

A key step in gene repression by Polycomb is trimethylation of histone H3 K27 by PCR2 to form H3K27me3. H3K27me3 provides a binding surface for PRC1. This study shows that monoubiquitination of histone H2A by PRC1-type complexes to form H2Aub creates a binding site for Jarid2-Aebp2-containing PRC2 and promotes H3K27 trimethylation on H2Aub nucleosomes. Jarid2, Aebp2 and H2Aub thus constitute components of a positive feedback loop establishing H3K27me3 chromatin domains (Kalb, 2014).

Nucleosomes constitute the building blocks of eukaryotic chromosomes. They consist of a core of histone proteins around which DNA is wrapped in two helical turns. The post-translational modification of histones is a key step for the regulation of diverse processes that occur on nucleosomal DNA. Specific histone modifications often decorate arrays of nucleosomes that comprise many kilobases of DNA, but how such extended stretches of chromatin become modified is not well understood. A paradigm for a long-range chromatin-modification mechanism is transcriptional repression by Polycomb protein complexes. The Polycomb system generates two distinct histone modifications: methylation of K27 in histone H3 and monoubiquitination of K119 in histone H2A in vertebrates and of the corresponding K118 in Drosophila H2A. Polycomb repressive complex 2 (PRC2) catalyzes mono-, di- and trimethylation at H3 K27. At inactive Polycomb-target genes, H3 K27 trimethyl marks typically decorate nucleosomes across the entire upstream, promoter and coding region and are essential for repression of these genes. The H3K27me3 modification is recognized by Polycomb, a subunit of the canonical Polycomb repressive complex 1 (PRC1), and is thought to promote PRC1 interaction with chromatin across the entire length of repressed genes. PRC1 has been proposed to repress transcription through chromatin compaction and also through its ubiquitin-ligase activity for H2Amonoubiquitination. To gain insight into the function of H2Aub, this study set out to identify interactors of this modification (Kalb, 2014).

Arrays of four nucleosomes (referred to as oligonucleosomes) were reconstituted with recombinant Drosophila or Xenopus histones and monoubiquitinated H2A in these templates, using appropriate recombinant enzymes. Drosophila monoubiquitinated H2AK118 (H2AK118ub) oligonucleosomes and the corresponding unmodified oligonucleosome control template were used for affinity purification of H2AK118ub-binding proteins from Drosophila embryo nuclear extracts. In parallel, Xenopus monoubiquitinated H2A K119 (H2AK119ub) and unmodified control oligonucleosomes were used to identify vertebrate H2AK119ub interactors in nuclear extracts from mouse embryonic stem cells. In both experiments, quantitative MS analyses identified PRC2 subunits as being among the most highly enriched H2Aub interactors. Jarid2 and Aebp2 were the PRC2 subunits showing highest enrichment in both cases (Kalb, 2014).

The identification of PRC2 as an H2Aub interactor in both flies and vertebrates prompted an analysis of PRC2 histone methyltransferase (HMTase) activity on H2Aub nucleosomes. Recombinant human PRC2 containing EED, EZH2, SUZ12 and RBBP4 (referred to as PRC2) and assemblies of the same complex that in addition contained AEBP2 (AEBP2-PRC2), JARID2 (JARID2-PRC2) or both JARID2 and AEBP2 (JARID2-AEBP2-PRC2) were reconstituted. For substrates, Xenopus mononucleosomes were used that were either unmodified or monoubiquitinated at H2A K119, and in all cases western blot analyses were used with antibodies against either monomethylated H3 K27 (H3K27me1) or H3K27me3 to monitor PRC2 activity. A time-course experiment was performed to compare the activity of PRC2 and JARID2-AEBP2-PRC2 on H2A and H2Aub nucleosomes. It was found that, consistently with earlier reports, the catalytic activity of PRC2 alone is largely unchanged on H2Aub nucleosome templates. As expected, inclusion of JARID2 and AEBP2 in PRC2 resulted in stronger activity for H3 K27 methylation on unmodified nucleosome templates. However, a much stronger increase was used in H3K27me3 formation when JARID2-AEBP2-PRC2 was used for HMTase reactions on H2Aub nucleosomes. It was estimated that JARID2-AEBP2-PRC2 trimethylates H3K27 in H2Aub nucleosomes with an efficiency 25-fold higher than that of PRC2. To assess the contributions of JARID2 and AEBP2 to this stimulation of HMTase activity, the catalytic activity was compared of all four forms of PRC2 on H2A and H2Aub nucleosome substrates. JARID2-PRC2 showed higher H3K27 methyltransferase activity than did PRC2 on unmodified nucleosomes, as previously reported, but this was not further increased on H2Aub nucleosomes. In contrast, AEBP2-PRC2 methylated H3K27 in H2Aub nucleosomes with considerably higher efficiency than in unmodified nucleosomes. This suggests that AEBP2 is critical for the specific activation of PRC2 by H2Aub, whereas JARID2 has a more general function in boosting PRC2 HMTase activity, independently of the H2A modification state (Kalb, 2014).

The work reported in this study reveals that Jarid2-Aebp2-containing PRC2 binds to H2Aub nucleosomes and demonstrates that H3K27 trimethylation by this complex is strongly enhanced on H2Aub nucleosomes. This establishes H2Aub, Aebp2 and Jarid2 as components of a positive feedback loop in which H2Aub promotes PRC2 binding and H3K27 trimethylation, and H3K27me3 in turn promotes binding of the canonical PRC1 via the chromodomain of Polycomb. It is currently not clear whether canonical PRC1 indeed has E3 ligase activity for H2Amonoubiquitination or whether this modification is generated only by forms of PRC1 lacking Polycomb. Intriguingly, in embryonic stem cells, the PRC1-type complexes PRC1.1 and PRC1.6 were also identified as H2Aub interactors, results suggesting an additional feedback loop for H2A ubiquitination in vertebrates. The positive feedback loop for H3K27me3 formation by H2Aub uncovered in this study provides a rationale for how extended domains of Polycomb-repressed chromatin could be generated in both Drosophila and vertebrates. These findings could explain why H3K27me3 levels at Polycomb-target genes are reduced in mouse embryonic stem cells in which H2AK119ub has been depleted. However, it was previously found that bulk H3K27me3 levels were undiminished in late-stage Drosophila larvae in which bulk H2Aub levels had been depleted, thus suggesting that maintenance of H3K27me3-containing chromatin domains does not strictly depend on H2Aub. The H2Aub-mediated feedback loop may thus primarily be required for the initial formation of H3K27me3 chromatin domains when Polycomb repression is first established during the early stages of embryogenesis (Kalb, 2014).