Figure 5: A subset of p53 target genes is subject to epigenetic silencing in hESCs.
(A) H3K4me3 and H3K27me3 levels at the TSSs of putative p53 target genes in H1 hESCs were plotted (left). The same set of genes were also plotted for H3K4me3 and H3K27me3 levels in H1-derived hMSCs (right). The p21 gene is marked in black open circle and shows high levels of H3K27me3 in hESCs, indicating that it is epigenetically repressed. The color corresponds to the mRNA expression level (FPKM) for each gene in the respective cell type. (B) Heatmap showing relative mRNA expression of p53 target gene candidates in H1 hESCs and hMSCs. Genes were sorted by the ratio of H3K27me3 levels in hESCs:hMSCs. FPKM values were normalized per gene to obtain relative expression values. The p21 gene and the genes selected for further analysis in Fig. 5C are indicated in red. (C) A subset of p53 target gene candidates is suppressed in H9 hESCs and requires p53 for expression in hMSCs. RNA was extracted from H9 hESCs and hMSCs and the indicated genes were analyzed by qRT-PCR (n = 3, means ± SD) (left plot). The mean value of mRNA expression in H9 hESCs is set at 1, and relative expression is shown in the left plot. H9 hMSCs were transfected with control and p53 siRNAs, and mRNA levels of each gene were analyzed similarly (right plot). The mean value of mRNA expression in control siRNA transfected H9 MSCs is set at 1, and relative expression is shown in the right plot. The passage number of H9 hMSCs is P8. (D) A subset of known p53 target genes is expressed in a p53-dependent manner in mouse ESCs. RNA was extracted from mouse p53+/− ESCs and littermate p53−/− ESCs, and the indicated genes was analyzed as in (C). The mean value of mRNA expression in p53+/− ESCs is set at 1, and relative expression is shown. (E) A conclusion model: A subset of p53 target genes such as p21 is poised but silenced by bivalent promoters in hESCs, and activated by releasing H3K27me3 marks upon differentiation.
