Figure 3: p21 expression in hESCs is repressed by histone H3K27me3.

(A) p21 promoter DNA is not significantly methylated in H9 hESCs and hMSCs. The CpG rich promoter regions of p21 (−131− + 97 bp and +73 − + 370 bp in relation to the transcriptional start site, TSS) were subjected to bisulfite sequencing. Each line shows independent sequencing results (n = 8–11). Filled circles indicate methylated CpGs and open circles represent unmethylated CpGs (X denotes un-identified nucleotide sequence). Clones with at least 90% cytosine conversion were considered as unmethylated sites. (B–E) ChIP analysis on the p21 gene locus was performed using protein lysates of H9 hESC and hMSCs with the indicated antibodies. ChIP-enriched DNA was quantified by qPCR (n = 3, mean ± SD). Values are shown as a percentage of input DNA. The amplicon’s name indicates the position of the central base pair of the amplicon relative to the TSS of p21 gene. Note that p21 gene locus is silenced by H3K27me3. (F) p53 localizes in the nucleus. Immunofluorescence staining of p53 and LMNB1 (nuclear envelope marker) in hESC line H9 and HES3. Scale bar, 20 μm. (G) p53 protein binds equally well to the p21 promoter in H9 hESC and hMSC. ChIP analysis with p53 antibody was performed as in (B). Mouse IgG was used as the negative control for immunoprecipitation.