Figure 5

EZH2 is required for looping between INK4a and INK4b. (A-D) Chromatin conformation capture-quantitative polymerase chain reaction (PCR) analysis on neonatal human diploid fibroblasts (A, B) and malignant rhabdoid tumour (MRT) cells (C, D) transduced with lentiviruses expressing either shRNAs targeting E(z)h2 mRNA (red) or a scrambled control (blue). Procedures were as described in the legend to Figure 4. (E) Parsimonious model for the coordinate regulation of human INK4a and INK4b by PcG repressive complexes (PRCs). In multipotent progenitor cells, young fibroblasts and highly proliferating MRT cancer cells, PRCs simultaneously bind the INK4a promoter and the INK4b upstream promoter region. This creates a looped chromatin structure that links the silenced INK4b and INK4a, but excludes ARF. EZH2 is critical for PRC binding and silencing. During progenitor cell differentiation and ageing, EZH2 attenuates, causing reduced locus occupancy of PRCs and release of the repressive loop. Concomitantly, RNA POL II is recruited selectively to INK4b and INK4a, but not to ARF, leading to gene transcription. MRT cells contain a crippled SWI/SNF complex, due to loss of the hSNF5 tumor suppressor subunit. hSNF5 re-expression in these cells restores SWI/SNF targeting to INK4a and INK4b, causing PRC eviction and release of the repressive loop. For details see discussion.