FIG. 4.
Restoration of SWI/SNF causes eviction of PcG silencers and loss of H3-K27 methylation. ChIPs using antibodies directed against BMI1 (A), EZH2 (B), SUZ12 (C), and H3-K27me3 (D) were performed. Cross-linked chromatin was isolated from MRT cells that either lack (yellow bars) or express (dark green bars) hSNF5. ChIPs were analyzed by qPCR using primer sets specific for the regions indicated by A to I along the INK4b-ARF-INK4a locus, revealing that PcG silencer binding peaks at the p16INK4a promoter. After hSNF5 induction both PRC1 (BMI1) and PRC2 (EZH2 and SUZ12) were removed, and H3-K27me3 was strongly reduced. H3-K27me3 ChIPs were normalized to H3 ChIP. Procedures were as described in the legend to Fig. 1. (E) hSNF5 expression does not affect BMI1, SUZ12, and EZH2 levels. Western immunoblotting analysis of BMI1, SUZ12, and EZH2 expression in MON cells transduced by either GFP- or SNF5-expressing lentiviruses. Cell lysates were resolved by SDS-PAGE and analyzed by Western immunoblotting with antibodies to BMI1, SUZ12, and EZH2, respectively. Histone H3 serves as a loading control.