A.P.Bracken and D.Pasini contributed equally to this workRecent 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 nontransformed human cells. In addition, the pRB-E2F pathway tightly regulates their expression and, consistent with this, we ®nd 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. Signi®cantly, we propose that EZH2 is a bona ®de oncogene, since we ®nd that ectopic expression of EZH2 is capable of providing a proliferative advantage to primary cells and, in addition, its gene locus is speci®cally ampli®ed in several primary tumors. Keywords: E2F/EED/EZH2/Polycomb/pRB IntroductionThe retinoblastoma protein (pRB) pathway plays a critical role in regulating progression through the mammalian cell cycle. The high frequency with which alterations have been found in the core members of this pathway in human tumors has led to the suggestion that its inactivation is an obligatory event for the development of all human cancers (Hanahan and Weinberg, 2000). The most studied and best understood targets for pRB are members of the E2F transcription factor family (Dyson, 1998). The E2Fs are essential for the transcriptional regulation of a number of genes whose products control cell cycle progression. These include both genes essential for the entry into the S phase of the cell cycle, such as CCNE1 (Cyclin E1) and CCNA2 (Cyclin A2), and genes that are involved in the regulation of DNA replication, such as CDC6, DHFR and TK1 (Helin, 1998;Trimarchi and Lees, 2002).Recently, a number of laboratories, including ours, have used gene expression pro®ling and ChIP assays to identify novel E2F-regulated genes (Mu Èller et al., 2001;Weinmann et al., 2001Weinmann et al., , 2002Ren et al., 2002). Due to the critical role of the E2Fs in controlling the expression of key regulators of cell proliferation, it is very likely that several novel key regulators of cell proliferation are amongst these many identi®ed genes. The characterization of these will be critical for both the understanding of normal proliferation control and its deregulation in cancer.The identi®cation of a number of Polycomb group (PcG) genes, Enhancer of Zeste Homolog 2 (EZH2), Embryonic Ectoderm Development (EED) and Suppressor of Zeste 12 (SUZ12) as potential E2F targets was one of the most interesting ®ndings in our screens (Mu Èller et al., 2001). PcG genes are best known for their role in maintaining the repression of HOX genes during development (for reviews, see Brock and van Lohuizen, 2001;Jacobs and van Lohuizen, 2002). The PcG proteins are b...

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.[Keywords: Polycomb; chromatin; epigenetics; stem cells; differentiation] Supplemental material is available at http://www.genesdev.org.

The p16INK4A and p14 ARF proteins, encoded by the INK4A-ARF locus, are key regulators of cellular senescence, yet the mechanisms triggering their up-regulation are not well understood. Here, we show that the ability of the oncogene BMI1 to repress the INK4A-ARF locus requires its direct association and is dependent on the continued presence of the EZH2-containing PolycombRepressive Complex 2 (PRC2) complex. Significantly, EZH2 is down-regulated in stressed and senescing populations of cells, coinciding with decreased levels of associated H3K27me3, displacement of BMI1, and activation of transcription. These results provide a model for how the INK4A-ARF locus is activated and how Polycombs contribute to cancer. Cellular senescence is an irreversible growth arrest triggered by several types of stress, including DNA damage, telomere shortening, and oncogene activation (Dimri 2005). Recently, its relevance as a bona fide tumor-suppressive mechanism in vivo has been highlighted (for review, see Narita and Lowe 2005). The Polycomb group (PcG) proteins BMI1, CBX7, and CBX8 are capable of delaying the onset of senescence in mouse and human embryonic fibroblasts (MEFs and HEFs) (Jacobs et al. 1999;Gil et al. 2004;Dietrich et al. 2007). This has been shown to correlate with a decrease in the levels of p16 INK4A and, in some cases, p14 ARF (p19 Arf in mice). Both of these proteins are encoded by the INK4A-ARF locus and are tumor suppressors that act upstream of the pRB and p53 pathways, respectively (Lowe and Sherr 2003).The BMI1-containing Polycomb-Repressive Complex 1 (PRC1), of which many variants are thought to exist, also contains the CBX (CBX2, CBX4, CBX6, CBX7, and CBX8), PHC1-3, RNF1-2, and SCML1-2 proteins (Levine et al. 2004). A second complex, PRC2, contains the histone methyltransferase EZH2, which together with EED and SUZ12 trimethylates histone H3 on Lys 27 (H3K27me3) (Cao and Zhang 2004;Pasini et al. 2004b). The ability of PRC1 to bind to chromatin is dependent on PRC2 function, and it has been proposed that this is primarily achieved via binding to the H3K27me3 mark (Rastelli et al. 1993;Hernandez-Munoz et al. 2005).In this study, we address several outstanding questions concerning the regulation of the INK4A-ARF locus by BMI1. We establish that BMI1 together with other PcGs and the associated H3K27me3 mark "blanket" the locus both in vivo and in vitro (tissue culture) in both mouse and human cells. We show that the repression of the locus by BMI1 is dependent on the continued association of the EZH2-containing PRC2 complex and that the levels of EZH2 are down-regulated in stressed and senescent cells. This down-regulation leads to the loss of H3K27me3, displacement of BMI1, and activation of INK4A transcription, resulting in senescence. Taken together, our results provide a model for how the INK4A-ARF locus is regulated in response to multiple cellular signals and how increased expression of the PcGs contributes to cancer. Results and Discussion PcGs and associated H3K27me3 'blanket' the INK4A-ARF locus ...

SUZ12 is a recently identified Polycomb group (PcG) protein, which together with EZH2 and EED forms different Polycomb repressive complexes (PRC2/3). These complexes contain histone H3 lysine (K) 27/9 and histone H1 K26 methyltransferase activity specified by the EZH2 SET domain. Here we show that mice lacking Suz12, like Ezh2 and Eed mutant mice, are not viable and die during early postimplantation stages displaying severe developmental and proliferative defects. Consistent with this, we demonstrate that SUZ12 is required for proliferation of cells in tissue culture. Furthermore, we demonstrate that SUZ12 is essential for the activity and stability of the PRC2/3 complexes in mouse embryos, in tissue culture cells and in vitro. Strikingly, Suz12-deficient embryos show a specific loss of di- and trimethylated H3K27, demonstrating that Suz12 is indeed essential for EZH2 activity in vivo. In conclusion, our data demonstrate an essential role of SUZ12 in regulating the activity of the PRC2/3 complexes, which are required for regulating proliferation and embryogenesis