EZH2 attenuation during progenitor cell differentiation. (A-D) PRC2 subunit EZH2, but not the PRC1 subunit BMI1, is down-regulated during cellular ageing and differentiation. The expression of EZH2 was analysed by RT-qPCR and Western immunoblotting in: (A) neonatal and adult human diploid fibroblasts (HDFs); (B) proliferating and differentiating erythroblasts, (C) immature CD34+ progenitors and mature CD34- cells isolated directly from umbilical cord blood and CD34+ cells that differentiated and lost CD34 expression following 6 weeks of culture; (D) malignant rhabdoid tumour cells that either lack or express hSNF5. For characterization of these cells see Figure 1. In parallel, BMI1 and histone H3 levels were determined. (E) Selective RNA POL II recruitment to INK4a and INK4b, but not ARF, in ageing HDFs. Chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR) analysis of RNA POL II binding to the INK4b-ARF-INK4a locus in neonatal (light blue) and adult (dark blue) HDFs. The following primer sets were used: H (INK4b), I (ARF) and L (INK4a). All ChIP data presented in this study are the result of at least three biological replicates. Background levels were determined using antibodies directed against GST. The abundance of specific DNA sequences in the immunoprecipitates was analysed by qPCR and corrected for the independently determined amplification curves for each primer set. ChIP signal levels for each region are presented as percentage of input chromatin. Bar graphs represent the mean of three independent experiments, each analysed in triplicate by qPCR. Error bars represent standard error of the mean.(F) PRCs bind INK4a and INK4b in neonatal but not in adult HDFs. ChIP-qPCR analysis revealed highly localized binding of EZH2 to the INK4a promoter region (primer sets K and L) and an area ~3 kb upstream of the INK4b promoter (primer sets C and D) in neonatal HDFs. In adult HDFs, EZH2 protein binding is strongly reduced. Analysis was as described above. (G) Following waning of EZH2, BMI1 binding to INK4a and INK4b is reduced significantly. ChIP-qPCR analysis of BMI1 binding to the INK4b-ARF-INK4a locus in neonatal and adult HDFs. The positions of the amplified regions (A-M) of the INK4b-ARF-INK4a locus are indicated at the bottom. (H) ChIPs using antibodies directed against histone H3K37 me3 revealed increased H3K27 methylation at- and around the PRC binding sequences upstream of INK4b and at the INK4a promoter. H3K27 me3 ChIPs were normalized against histone H3.