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 ...
Organization of chromatin by epigenetic mechanisms is essential for establishing and maintaining cellular identity in developing and adult organisms. A key question that remains unresolved about this process is how epigenetic marks are transmitted to the next cell generation during cell division. Here we provide a model to explain how trimethylated Lys 27 of histone 3 (H3K27me3), which is catalysed by the EZH2-containing Polycomb Repressive Complex 2 (PRC2), is maintained in proliferating cells. We show that the PRC2 complex binds to the H3K27me3 mark and colocalizes with this mark in G1 phase and with sites of ongoing DNA replication. Efficient binding requires an intact trimeric PRC2 complex containing EZH2, EED and SUZ12, but is independent of the catalytic SET domain of EZH2. Using a heterologous reporter system, we show that transient recruitment of the PRC2 complex to chromatin, upstream of the transcriptional start site, is sufficient to maintain repression through endogenous PRC2 during subsequent cell divisions. Thus, we suggest that once the H3K27me3 is established, it recruits the PRC2 complex to maintain the mark at sites of DNA replication, leading to methylation of H3K27 on the daughter strands during incorporation of newly synthesized histones. This mechanism ensures maintenance of the H3K27me3 epigenetic mark in proliferating cells, not only during DNA replication when histones synthesized de novo are incorporated, but also outside S phase, thereby preserving chromatin structure and transcriptional programs.
Epigenetic regulation of chromatin structure is essential for the expression of genes determining cellular specification and function. The Polycomb repressive complex 2 (PRC2) di- and trimethylates histone H3 on lysine 27 (H3K27me2/me3) to establish repression of specific genes in embryonic stem cells and during differentiation. How the Polycomb group (PcG) target genes are regulated by environmental cues and signaling pathways is quite unexplored. Here, we show that the mitogen- and stress-activated kinases (MSK), through a mechanism that involves promoter recruitment, histone H3K27me3S28 phosphorylation, and displacement of PcG proteins, lead to gene activation. We present evidence that the H3K27me3S28 phosphorylation is functioning in response to stress signaling, mitogenic signaling, and retinoic acid (RA)-induced neuronal differentiation. We propose that MSK-mediated H3K27me3S28 phosphorylation serves as a mechanism to activate a subset of PcG target genes determined by the biological stimuli and thereby modulate the gene expression program determining cell fate.
The Polycomb group (PcG) proteins are essential for embryogenesis, and their expression is often found deregulated in human cancer. The PcGs form two major protein complexes, called polycomb repressive complexes 1 and 2 (PRC1 and PRC2) whose function is to maintain transcriptional repression. Here, we demonstrate that the chromodomain-containing protein, CBX8, which is part of one of the PRC1 complexes, regulates proliferation of diploid human and mouse fibroblasts through direct binding to the INK4A-ARF locus. Furthermore, we demonstrate that CBX8 is limiting for the regulation of INK4A-ARF, and that ectopic expression of CBX8 leads to repression of the Ink4a-Arf locus and bypass of senescence, leading to cellular immortalization. Gene expression and location analysis demonstrate that besides the INK4A-ARF locus, CBX8 also regulates a number of other genes important for cell growth and survival. On the basis of these results, we conclude that CBX8 is an essential component of one of the PRC1 complexes, which directly regulate the expression of numerous target genes, including the INK4A-ARF locus, involved in cell-fate decisions.
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