05-1320 Sigma-AldrichAnti-EED Antibody, clone AA19

Polycomb repressive complexes 1 and 2 (PRC1 and 2) play a critical role in the epigenetic regulation of transcription during cellular differentiation, stem cell pluripotency and neoplastic progression. Here we show that the polycomb group protein EED, a core component of PRC2, physically interacts with and functions as part of PRC1. Components of PRC1 and PRC2 compete for EED binding. EED functions to recruit PRC1 to H3K27me3 loci and enhances PRC1-mediated H2A ubiquitin E3 ligase activity. Taken together, we suggest an integral role for EED as an epigenetic exchange factor coordinating the activities of PRC1 and 2.

DNA methylation and the Polycomb repression system are epigenetic mechanisms that play important roles in maintaining transcriptional repression. Recent evidence suggests that DNA methylation can attenuate the binding of Polycomb protein components to chromatin and thus plays a role in determining their genomic targeting. However, whether this role of DNA methylation is important in the context of transcriptional regulation is unclear.By genome-wide mapping of the Polycomb Repressive Complex 2-signature histone mark, H3K27me3, in severely DNA hypomethylated mouse somatic cells, we show that hypomethylation leads to widespread H3K27me3 redistribution, in a manner that reflects the local DNA methylation status in wild-type cells. Unexpectedly, we observe striking loss of H3K27me3 and Polycomb Repressive Complex 2 from Polycomb target gene promoters in DNA hypomethylated cells, including Hox gene clusters. Importantly, we show that many of these genes become ectopically expressed in DNA hypomethylated cells, consistent with loss of Polycomb-mediated repression.An intact DNA methylome is required for appropriate Polycomb-mediated gene repression by constraining Polycomb Repressive Complex 2 targeting. These observations identify a previously unappreciated role for DNA methylation in gene regulation and therefore influence our understanding of how this epigenetic mechanism contributes to normal development and disease.

Myc family members are critical to maintain embryonic stem cells (ESC) in the undifferentiated state. However, the mechanism by which they perform this task has not yet been elucidated. Here we show that Myc directly upregulates the transcription of all core components of the Polycomb repressive complex 2 (PRC2) as well as the ESC-specific PRC2-associated factors. By expressing Myc protein fused with the estrogen receptor (Myc-ER) in fibroblasts, we observed that Myc, binding to the regulatory elements of Suz12, Ezh2, and Eed, induces the acetylation of histones H3 and H4 and the recruitment of elongating RNA polymerase II at their promoters. The silencing of both c-Myc and N-Myc in ESC results in reduced expression of PRC2 and H3K27me3 at Polycomb target developmental regulators and upregulation of genes involved in primitive endoderm differentiation. The ectopic expression of PRC2 in ESC, either silenced for c-Myc and N-Myc or induced to differentiate by leukemia inhibitory factor (LIF) withdrawal, is sufficient to maintain the H3K27me3 mark at genes with bivalent histone modifications and keep repressed the genes involved in ESC differentiation. Thus, Myc proteins control the expression of developmental regulators via the upregulation of the Polycomb PRC2 complex.

Polycomb group proteins play essential roles in transcriptional regulation of multiple gene families involved in various pathophysiological processes. It is believed that Polycomb Repressive Complex 2 (PRC2) is targeted to chromatin by the EED subunit to methylate histone H3 lysine 27 (H3K27), leading to a repressive chromatin state that inhibits gene expression. Here we report that the chromodomain-containing protein CDYL specifically recognizes di- and tri-methylated H3K27 (H3K27me2 and H3K27me3) and directly interacts with EZH2, the catalytic subunit of PRC2. We show that CDYL dramatically enhances the methyltransferase activity of PRC2 toward oligonucleosome substrates in vitro. Genome-wide analysis of CDYL targets by ChIP sequencing revealed that CDYL and PRC2 share a number of genomic targets. CDYL is required for chromatin targeting and maximal enzymatic activity of PRC2 at their common target sites. Our experiments indicate that CDYL functions as a molecular bridge between PRC2 and the repressive chromatin mark H3K27me3, forming a positive feedback loop to facilitate the establishment and propagation of H3K27me3 modifications along the chromatin.

Polycomb group (PcG) proteins form multiprotein complexes, called Polycomb repressive complexes (PRCs). PRC2 contains the PcG proteins EZH2, SUZ12, and EED and represses transcription through methylation of lysine (K) 27 of histone H3 (H3). Suz12 is essential for PRC2 activity and its inactivation results in early lethality of mouse embryos. Here, we demonstrate that Suz12(-/-) mouse embryonic stem (ES) cells can be established and expanded in tissue culture. The Suz12(-/-) ES cells are characterized by global loss of H3K27 trimethylation (H3K27me3) and higher expression levels of differentiation-specific genes. Moreover, Suz12(-/-) ES cells are impaired in proper differentiation, resulting in a lack of repression of ES cell markers as well as activation of differentiation-specific genes. Finally, we demonstrate that the PcGs are actively recruited to several genes during ES cell differentiation, which despite an increase in H3K27me3 levels is not always sufficient to prevent transcriptional activation. In summary, we demonstrate that Suz12 is required for the establishment of specific expression programs required for ES cell differentiation. Furthermore, we provide evidence that PcGs have different mechanisms to regulate transcription during cellular differentiation.

The Polycomb group (PcG) proteins form chromatin-modifying complexes that are essential for embryonic development and stem cell renewal and are commonly deregulated in cancer. Here, we identify their target genes using genome-wide location analysis in human embryonic fibroblasts. We find that Polycomb-Repressive Complex 1 (PRC1), PRC2, and tri-methylated histone H3K27 co-occupy >1000 silenced genes with a strong functional bias for embryonic development and cell fate decisions. We functionally identify 40 genes derepressed in human embryonic fibroblasts depleted of the PRC2 components (EZH2, EED, SUZ12) and the PRC1 component, BMI-1. Interestingly, several markers of osteogenesis, adipogenesis, and chrondrogenesis are among these genes, consistent with the mesenchymal origin of fibroblasts. Using a neuronal model of differentiation, we delineate two different mechanisms for regulating PcG target genes. For genes activated during differentiation, PcGs are displaced. However, for genes repressed during differentiation, we paradoxically find that they are already bound by the PcGs in nondifferentiated cells despite being actively transcribed. Our results are consistent with the hypothesis that PcGs are part of a preprogrammed memory system established during embryogenesis marking certain key genes for repressive signals during subsequent developmental and differentiation processes.

Recent experiments have demonstrated that the Polycomb group (PcG) gene EZH2 is highly expressed in metastatic prostate cancer and in lymphomas. EZH2 is a component of the PRC2 histone methyltransferase complex, which also contains EED and SUZ12 and is required for the silencing of HOX gene expression during embryonic development. Here we demonstrate that both EZH2 and EED are essential for the proliferation of both transformed and non-transformed human cells. In addition, the pRB-E2F pathway tightly regulates their expression and, consistent with this, we find that EZH2 is highly expressed in a large set of human tumors. These results raise the question whether EZH2 is a marker of proliferation or if it is actually contributing to tumor formation. Significantly, we propose that EZH2 is a bona fide oncogene, since we find that ectopic expression of EZH2 is capable of providing a proliferative advantage to primary cells and, in addition, its gene locus is specifically amplified in several primary tumors.