pRB family proteins are required for H3K27 trimethylation and Polycomb repression complexes binding to and silencing p16INK4alpha tumor suppressor gene

Abstract

Genetic studies have demonstrated that Bmi1 promotes cell proliferation and stem cell self-renewal with a correlative decrease of p16(INK4a) 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.

Figure 1.

pRB family proteins negatively regulate p16 gene expression. The levels of p16, p18, and p21 proteins (A,C) and mRNA (B,D) were determined in WI38 and VA13 cells by direct immunoblotting or Q-RT–PCR. Data are expressed relative to the corresponding values for WI38 (B) or WI-38/Mock (D) cells, and mean values and standard deviations were calculated from triplicates of a representative experiment. (E) WI38 cells were infected with an empty lentivirus vector (Mock) or lentivirus vectors encoding shRNA silencing individually human RB, p107, or p130 genes. The efficiency of silencing and the effect of silencing on p16 expression were determined by direct immunoblotting.

Figure 2.

The oncogene BMI1 represses p16 gene expression. (A,B) WI38 cells were infected with empty (Mock) or BMI1-expressing retroviruses and selected by puromycin treatment. The levels of individual mRNA (A) and protein (B) levels were determined by Q-RT–PCR and direct immunoblotting, respectively. Q-RT–PCR results are expressed relative to the corresponding values for WI38/Mock cells, and mean values and standard deviations were calculated from triplicates of a representative experiment. (C) WI38 cells were infected with a retrovirus vector encoding shRNA against either GFP or BMI1 and selected by puromycin treatment. The efficiency of BMI1 silencing and the effect of BMI1 silencing on p16 expression were determined by direct immunoblotting. (D) The effects of BMI1 silencing on the expression of the four INK4 and ARF genes were determined by Q-RT–PCR, and results are expressed relative to the corresponding values for WI38/GFP-i cells. The mean values and standard deviations were calculated from triplicates of a representative experiment. (E) The growth curves of WI38 cells infected with a retrovirus vector encoding shRNA against either GFP or BMI1. Viable cells were counted by Trypan Blue staining at indicated days after initial seeding of 2 × 10⁵ cells.

Figure 3.

BMI1-mediated p16 repression requires the function of pRB proteins. (A) WI38, VA13, 293, and Saos-2 cells were infected with empty or BMI1-expressing retroviruses and selected with puromycin. The levels of individual proteins were determined by Western blotting. (B) The growth curve of WI38, VA13, and Saos-2 cells infected with empty or BMI1-expressing retroviruses were determined by Trypan Blue staining at indicated days after initial seeding of 2 × 10⁵ cells (WI38) or 1 × 10⁵ cells (VA13 and Saos-2). (C,D) WI38/Mock and WI38/E7 (G418 resistant) were reinfected with empty or BMI1-expressing retroviruses and selected by puromycin treatment. The levels of individual proteins (C) and mRNA (D) were determined by direct immunoblotting and Q-RT–PCR (D).

Figure 4.

BMI1 directly associates with the p16 genomic region through pRB protein function. (A) The passage-dependent expression of the p16 gene in MEFs was determined by Q-RT–PCR, and results are expressed relative to the corresponding values for MEFs (pass 2). (Left panel) The mean values and standard deviations were calculated from triplicates of a representative experiment. (Middle panel) The levels of Bmi1 protein in MEFs were determined by Western blotting. (Right panel) ChIP assays using antibodies against Bmi1 and IgG control in multiply passed MEFs. (B, left panel) The levels of pRB, p107, p130, and p16 proteins in wild-type (WT) and RB^−/−; p107^−/−; p130^−/− (TKO) MEFs were determined by Western blotting. (Right panel) ChIP assays were done using antibodies against Bmi1 and IgG control in wild-type or TKO MEFs. (C) WI38 cells were infected with a retrovirus vector encoding shRNA against either GFP or EZH2 and selected by puromycin treatment. (Top left panel) The efficiency of EZH2 silencing was determined by RT–PCR. The effect of EZH2 silencing on p16 expression was determined by Western blotting (bottom left panel) and by Q-RT–PCR (right panel). The results are expressed relative to the corresponding values for WI38/GFP-i cells. The mean values and standard deviations were calculated from triplicates of a representative experiment. (D) A schematic representation of the human p16 and GAPDH gene loci and amplicons (a, b, c, and d) used for ChIP assays. WI38/Mock and WI38/E7 cells were reinfected with BMI1-expressing retroviruses. Antibodies against BMI1, RING2, SUZ12, and trimethyl-H3K27 and IgG control were used in the ChIP assays. PCR was carried out using primers for each amplicon.