Polycomb Mediated Epigenetic Silencing and Replication Timing at the INK4a/ARF Locus during Senescence

Abstract

BackgroundThe INK4/ARF locus encodes three tumor suppressor genes (p15Ink4b, Arf and p16Ink4a) and is frequently inactivated in a large number of human cancers. Mechanisms regulating INK4/ARF expression are not fully characterized.Principal FindingsHere we show that in young proliferating embryonic fibroblasts (MEFs) the Polycomb Repressive Complex 2 (PRC2) member EZH2 together with PRC1 members BMI1 and M33 are strongly expressed and localized at the INK4/ARF regulatory domain (RD) identified as a DNA replication origin. When cells enter senescence the binding to RD of both PRC1 and PRC2 complexes is lost leading to a decreased level of histone H3K27 trimethylation (H3K27me3). This loss is accompanied with an increased expression of the histone demethylase Jmjd3 and with the recruitment of the MLL1 protein, and correlates with the expression of the Ink4a/Arf genes. Moreover, we show that the Polycomb protein BMI1 interacts with CDC6, an essential regulator of DNA replication in eukaryotic cells. Finally, we demonstrate that Polycomb proteins and associated epigenetic marks are crucial for the control of the replication timing of the INK4a/ARF locus during senescence.ConclusionsWe identified the replication licencing factor CDC6 as a new partner of the Polycomb group member BMI1. Our results suggest that in young cells Polycomb proteins are recruited to the INK4/ARF locus through CDC6 and the resulting silent locus is replicated during late S-phase. Upon senescence, Jmjd3 is overexpressed and the MLL1 protein is recruited to the locus provoking the dissociation of Polycomb from the INK4/ARF locus, its transcriptional activation and its replication during early S-phase. Together, these results provide a unified model that integrates replication, transcription and epigenetics at the INK4/ARF locus.

βTrCP regulates BMI1 protein turnover via ubiquitination and degradation

“…2,28 PcG proteins are also key determinants of cellular senescence. 12,26,29,30 Consistent with their pro-proliferative role, expression of two key PcG proteins BMI1 and EZH2 has been shown to be downregulated in senescent human cells, 12,30 and knockdown of BMI1 and/or EZH2 inhibits growth of tumor cells via induction of senescence or apoptosis. Thus, the PcG proteins are attractive molecular targets for cancer therapy.…”

Identification and Characterization of Bmi-1-responding Element within the Human p16 Promoter*

“…Recently, Bmi-1 was demonstrated to be recruited to the RD region through CDC6 (36). The RD region resides in a ϳ35 kbp upstream translation start site of p16 in human genome (37), while BRE locates at about 700-bp upstream that of p16.…”

“…Intriguingly, the alignment of BRE and human RD regions exhibits an aligned G-rich area, where the human RD region is similar to the BRE in the motif-2 cluster. Given that Bmi-1 is recruited to the RD region by CDC6 (36), whether the motif-2 region of RD will strengthen the association of Bmi-1-CDC6 to RD region for Ink4a/Arf regulation needs further study. Further work is also needed to determine whether and how BRE and RD regions co-operate in Bmi-1-mediated regulation of the Ink4a/ Arf locus.…”

The H3K27me3 demethylase, KDM6B, is induced by Epstein–Barr virus and over-expressed in Hodgkin's Lymphoma

“…KDM6B is one of only two demethylases known to remove the tri-methyl mark from H3K27, the other is UTX (Agger et al, 2007;Hong et al, 2007). These demethylases reverse polycomb group-mediated transcriptional repression by demethylating H3K27me3 and dissociating polycomb group complexes (Agger et al, 2009;Agherbi et al, 2009;Barradas et al, 2009). Both KDM6B and UTX are necessary for the lineage commitment of embryonic stem cells and for the terminal differentiation of progenitor cells.…”