pRB family proteins are required for H3K27 trimethylation and Polycomb repression complexes binding to and silencing p16^INK4a tumor suppressor gene

Abstract

Genetic studies have demonstrated that Bmi1 promotes cell proliferation and stem cell self-renewal with a correlative decrease of p16INK4a expression. Here, we demonstrate that Polycomb genes EZH2 and BMI1 repress p16 expression in human and mouse primary cells, but not in cells deficient for pRB protein function. The p16 locus is H3K27-methylated and bound by BMI1, RING2, and SUZ12. Inactivation of pRB family proteins abolishes H3K27 methylation and disrupts BMI1, RING2, and SUZ12 binding to the p16 locus. These results suggest a model in which pRB proteins recruit PRC2 to trimethylate p16, priming the BMI1-containing PRC1L ubiquitin ligase complex to silence p16. Received October 3, 2006; revised version accepted November 14, 2006. The mammalian pRB family proteins, pRB, p107, and p130 (also known as pocket proteins), play a key role in controlling the G1-to-S transition of the cell cycle and maintaining differentiated cells in a reversible quiescent or permanent senescent arrest state (Weinberg 1995;Cobrinik 2005). The pocket proteins are hypophosphorylated in cells exiting mitosis as well as in quiescent cells, where they bind to and negatively regulate the function of the E2F family transcription factors (Trimarchi and Lees 2002). In cells entering the cell cycle, extracellular mitogens first induce the expression of D-type cyclins, which bind to and activate CDK4 and CDK6, leading to the phosphorylation of pRB family proteins, causing functional inactivation by E2F dissociation, thereby promoting a G1-to-S transition. Inhibition of CDK4 and CDK6 by the INK4 family of CDK inhibitors (p16 INK4c, and p19 INK4d) retains pRB family proteins in their hypophosphorylated, growth-suppressive states and prevents G1-to-S progression. Disruption of the INK4-RB pathway, consisting of INK4-cyclinDs-CDK4/6-RB-E2Fs, deregulates G1-to-S control and represents a common event in the development of most, if not all, types of cancer (Sherr 1996).Among the major challenges toward a better understanding of G1 control by the INK4-pRB pathway is how different INK4 genes are regulated, thereby linking G1 control to different cellular pathways. INK4 proteins are relatively stable, and the primary regulation of INK4 is through transcriptional control. The expression of each of the INK4 genes is distinctly different during development, in different adult tissues, and in response to different cellular conditions (Roussel 1999). There have been only a few reports wherein a transcriptional regulator has been demonstrated to bind to an INK4 promoter by either gel shift or chromatin immunoprecipitation (ChIP) assay. Identification of factors that directly bind to INK4 promoters holds the key to linking different cellular pathways to G1 control by the INK4-pRB pathway, but these links remain disproportionately poorly understood in comparison with our knowledge of the function of the INK4-pRB pathway (Pei and Xiong 2005).To elucidate the molecular mechanisms regulating p16 expression, we tested whether p16 gene expression is directly reg...

E2f6 and Bmi1 cooperate in axial skeletal development

“…The neural stem cell defect can be partially rescued by disruption of the Ink4a-Arf locus (Molofsky et al, 2005). Although Bmi1 has been shown by chromatin immunoprecipitation to be present at the promoters of p16 INK4a and p19 ARF (Bracken et al, 2007;Kotake et al, 2007), Bmi1 is unable to directly bind to DNA in a sequence-specific manner (Tagawa et al, 1990;Alkema et al, 1997).…”

“…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.…”

Impact of nucleosome dynamics and histone modifications on cell proliferation during Arabidopsis development

“…One possibility is that PcGinteracting proteins facilitate PRC2 targeting. Interestingly, the tumor suppressor Retinoblastoma protein (Rb) is necessary to maintain H3K27 methylation and PRC2 and PRC1 binding to the promoter of the CDK inhibitor p16 in human cells (Kotake et al, 2007). The plant homolog of human Rb, the RETINOBLASTOMA-RE-LATED (RBR) protein, binds to FIE (Mosquna et al, 2004) and MSI1 (Ach et al, 1997;Rossi et al, 2001).…”

Epigenetic regulation of cancer growth by histone demethylases

“…Their oncogenic potential is mainly mediated through PcG-mediated H3K27 methylation and epigenetic inactivation of the INK4A-ARF locus. 39,40 The INK4A-ARF locus encodes tumor suppressor genes p16 INK4A and p14 ARF , which are key regulators of cellular senescence.…”