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

Abstract

The p16INK4A and p14ARF 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 Polycomb-Repressive 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.

Figure 1.

PcG proteins bind throughout the INK4A-ARF locus. (A) ChIP analysis of the INK4B and INK4A-ARF loci in TIG3-TERT HEFs. The precipitated DNA was amplified by real-time qPCR using primers specific for the regions indicated by red bars in the top of the panel. Enrichments are presented as percentages of total input. (B) ChIP analysis of the INK4B and INK4A-ARF loci in CD34-positive BM cells.

Figure 2.

Ectopically expressed BMI1 is recruited to the INK4A-ARF locus. (A) Western blot analysis of LZRS- and LZRSBMI1-infected TIG3-TERT HEFs. (B) qPCR analysis of the mRNA levels of ARF and INK4A in cells from A. (C) ChIP analysis of BMI1, CBX8, and RNA polymerase II enrichments in LZRS- and LZRSBMI1-infected TIG3-TERT HEFs. (D) ChIP analysis at the human “peak” PcG-binding region (primer set 7 in C) and the ARF promoter in LZRS- and LZRSBMI1-infected TIG3-TERT HEFs.

Figure 3.

PcGs and H3K27me3 are lost from the Ink4a-Arf locus in cells undergoing senescence. (A) Western blots of cell lysates prepared from MEFs at increasing passage numbers were probed with the indicated antibodies. (B) qPCR analysis of the indicated genes on mRNA prepared from the cells shown in A. (C) Representation of the mouse Ink4a-Arf gene locus. Amplified regions in D are indicated as red bars. (D) ChIP analysis using the indicated antibodies and the cells shown in A on the Ink4a-Arf locus.

Figure 4.

Targeted depletion of PRC2 displaces BMI1 from the INK4A-ARF locus, increases the expression of INK4A, and leads to senescence. (A) Western blot analysis of cell lysates prepared from TIG3-TERT HEFs infected with pRS, pRSSUZ12, or pRSEZH2, 7 d after selection with puromycin. (B) qPCR analysis of INK4A and ARF mRNA levels from cells from A. (C) ChIP analysis of PcGs and H3K27me3 enrichments on the INK4A and ARF promoters in pRS- and pRSSUZ12-infected cells. (D) β-galactosidase staining to detect senescent cells in TIG3-TERT cells infected with pRS, pRSSUZ12, pRSEZH2, or pRSBMI1 constructs.

Figure 5.

Model for how PcG proteins regulate cellular senescence in vivo. (A) In young proliferating normal diploid cells, the PRC2 complex is highly abundant and maintains the levels of H3K27me3 along the INK4B and INK4A-ARF loci. This in turn ensures the continued association of the BMI1-containing PRC1 complex and repression of the INK4A, INK4B, and ARF genes. (B) In older or stressed cells the levels of EZH2 are down-regulated, leading to the disruption of the PRC2 complex and loss of H3K27me3 along the INK4B and INK4A-ARF loci. Without H3K27me3, the BMI1-containing PRC1 complex is displaced. We propose a model in which cumulative stress leads to the progressive alteration of the chromatin state around the INK4B and INK4A-ARF loci, thereby conferring accessibility to specific transcriptional activators (X, Y and Z).