Loss of EZH2 causes derepression of INK4a and INK4b. (A) Neonatal human diploid fibroblasts were transduced with lentiviruses expressing either shRNAs targeting E(z)h2 mRNA (EZH2 KD) or a scrambled control. Three days following transduction, EZH2 levels were analysed by real-time quantitative polymerase chain reaction (RT-qPCR) and Western immunoblotting, as described above. The band corresponding to EZH2 is indicated with an arrowhead. In parallel, BMI1 and histone H3 levels were determined. (B) Loss of EZH2 causes transcriptional activation of INK4b and INK4a. Seventy-two hours following transduction, relative expression levels of INK4b, ARF and INK4a were determined by RT-qPCR of isolated mRNA. (C) EZH2 depletion leads to RNA POL II recruitment to INK4a and INK4b, as determined by chromatin immunoprecipitation (ChIP)-qPCR. (D-E) EZH2 depletion causes loss of PRCs from INK4a and INK4b, as revealed by ChIP-qPCR using antibodies directed against EZH2 (D) or BMI1 (E). All analyses were as described in the legend to Figures 1 and 2.