The Polycomb group proteins bind throughout the INK4A-ARF locus and are disassociated in senescent cells

Abstract

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 ...

E2f6 and Bmi1 cooperate in axial skeletal development

“…It has previously been shown that repression of Hox genes and other key developmental regulators is established in embryonic stem (ES) cells (Boyer et al, 2006). Thus, we used murine ES cells to perform chromatin immunoprecipitation on four genes, HoxA7, HoxA10, HoxA11, and Arf, which have previously been shown to be directly regulated by Bmi1 (Cao et al, 2005;Bracken et al, 2007;Kotake et al, 2007;Xi et al, 2007). We found that Bmi1 was directly bound to the promoters of HoxA7, HoxA10, HoxAll, and Arf in mouse embryonic stem cells (Fig.…”

Epigenetic regulation of aging stem cells

“…Whether BMI1 directly binds to the p16 INK4a /p19 ARF genomic locus has not yet been shown in adult NSC populations. However, in mouse and human fibroblasts, direct binding of BMI1 to the p16 INK4a /p19 ARF promoter triggers an increase in the repressive mark H3K27me3 at this locus (Bracken et al, 2007), suggesting that BMI1 may affect changes in chromatin state in a similar manner in adult NSCs (Bracken et al, 2007).…”

“…The decrease in Ezh2 expression in HSC does not seem to elicit changes in the histone marks H3K4me3 and H3K27me3 on p16 INK4a /p19 ARF locus in a subset of HSCs (Attema et al, 2009). Nevertheless, Ezh2 downregulation in mouse and human fibroblasts leads to decreased binding of BMI1 and other PcG members at the p16 INK4a /p19 ARF locus in response to stress stimuli or senescence (Bracken et al, 2007). EZH2 may thus orchestrate chromatin control at additional repressed targets in HSCs and other adult stem cell types.…”

Attributes of adult stem cells

“…On the other hand, trimethylation of lysine 9 and lysine 27 of histone 3 (H3K9Me3, H3K27Me3) are so-called 'repressive histone marks' associated with inactive regions of chromatin, whereas H3K4Me3 and acetylation of H3 and H4 are associated with active transcription. H3K27Me3 serves as a docking site for the bulky Bmi1-containing polycomb repressive complex-1 (PRC-1), whose presence at this site blocks the accessibility to many gene loci, including the Ink4a/Arf locus [75]. DNA methylation is expected to be associated with the stable repression of pluripotency genes, such as Oct4 and Nanog, observed in differentiating ES cells [76].…”