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.     

Genetic inactivation of the polycomb repressive complex 2 in T cell acute lymphoblastic leukemia

T cell acute lymphoblastic leukemia (T-ALL) is an immature hematopoietic malignancy driven mainly by oncogenic activation of NOTCH1 signaling. In this study we report the presence of loss-of-function mutations and deletions of the EZH2 and SUZ12 genes, which encode crucial components of the Polycomb repressive complex 2 (PRC2), in 25% of T-ALLs. To further study the role of PRC2 in T-ALL, we used NOTCH1-dependent mouse models of the disease, as well as human T-ALL samples, and combined locus-specific and global analysis of NOTCH1-driven epigenetic changes. These studies demonstrated that activation of NOTCH1 specifically induces loss of the repressive mark Lys27 trimethylation of histone 3 (H3K27me3) by antagonizing the activity of PRC2. These studies suggest a tumor suppressor role for PRC2 in human leukemia and suggest a hitherto unrecognized dynamic interplay between oncogenic NOTCH1 and PRC2 function for the regulation of gene expression and cell transformation.     

Enhancer of zeste homolog 2 promotes the proliferation and invasion of epithelial ovarian cancer cells

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2) that includes noncatalytic subunits suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED). When present in PRC2, EZH2 catalyzes trimethylation on lysine 27 residue of histone H3 (H3K27Me3), resulting in epigenetic silencing of gene expression. Here, we investigated the expression and function of EZH2 in epithelial ovarian cancer (EOC). When compared with primary human ovarian surface epithelial (pHOSE) cells, EZH2, SUZ12, and EED were expressed at higher levels in all 8 human EOC cell lines tested. Consistently, H3K27Me3 was also overexpressed in human EOC cell lines compared with pHOSE cells. EZH2 was significantly overexpressed in primary human EOCs (n = 134) when compared with normal ovarian surface epithelium (n = 46; P < 0.001). EZH2 expression positively correlated with expression of Ki67 (P < 0.001; a marker of cell proliferation) and tumor grade (P = 0.034) but not tumor stage (P = 0.908) in EOC. There was no correlation of EZH2 expression with overall (P = 0.3) or disease-free survival (P = 0.2) in high-grade serous histotype EOC patients (n = 98). Knockdown of EZH2 expression reduced the level of H3K27Me3 and suppressed the growth of human EOC cells both in vitro and in vivo in xenograft models. EZH2 knockdown induced apoptosis of human EOC cells. Finally, we showed that EZH2 knockdown suppressed the invasion of human EOC cells. Together, these data demonstrate that EZH2 is frequently overexpressed in human EOC cells and its overexpression promotes the proliferation and invasion of human EOC cells, suggesting that EZH2 is a potential target for developing EOC therapeutics.     

Structure of the Catalytic Domain of EZH2 Reveals Conformational Plasticity in Cofactor and Substrate Binding Sites and Explains Oncogenic Mutations

Polycomb repressive complex 2 (PRC2) is an important regulator of cellular differentiation and cell type identity. Overexpression or activating mutations of EZH2, the catalytic component of the PRC2 complex, are linked to hyper-trimethylation of lysine 27 of histone H3 (H3K27me3) in many cancers. Potent EZH2 inhibitors that reduce levels of H3K27me3 kill mutant lymphoma cells and are efficacious in a mouse xenograft model of malignant rhabdoid tumors. Unlike most SET domain methyltransferases, EZH2 requires PRC2 components, SUZ12 and EED, for activity, but the mechanism by which catalysis is promoted in the PRC2 complex is unknown. We solved the 2.0 Å crystal structure of the EZH2 methyltransferase domain revealing that most of the canonical structural features of SET domain methyltransferase structures are conserved. The site of methyl transfer is in a catalytically competent state, and the structure clarifies the structural mechanism underlying oncogenic hyper-trimethylation of H3K27 in tumors harboring mutations at Y641 or A677. On the other hand, the I-SET and post-SET domains occupy atypical positions relative to the core SET domain resulting in incomplete formation of the cofactor binding site and occlusion of the substrate binding groove. A novel CXC domain N-terminal to the SET domain may contribute to the apparent inactive conformation. We propose that protein interactions within the PRC2 complex modulate the trajectory of the post-SET and I-SET domains of EZH2 in favor of a catalytically competent conformation.     

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.     

The polycomb group protein EZH2 inhibits lung cancer cell growth by repressing the transcription factor Nrf2

EZH2 is a key component of the polycomb PRC2 complex and functions as a histone H3 Lys27 (H3K27) trimethyltransferase. Here we show that EZH2 is down-regulated in human non-small cell lung cancer and low EZH2 expression predicts poor survival. Further we demonstrate that EZH2 inhibits lung cancer cell proliferation and colony formation in vitro and growth in vivo. We found that EZH2 binds to the promoter of Nrf2, where it increases H3K27me3 and represses Nrf2 expression. Finally, Nrf2 seems to be essential for the hyper proliferation of lung cancer cells in the absence of EZH2.     

EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer

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.