Figure 8.

A, ChIP-qPCR of H3K27Ace showed an increase in the fold-enrichment of H3K27Ace at VEGF-A and Flk1 promoters in WT ECs that were reduced in Vezf1^−/− ECs. At the Oct4 promoter, post-differentiation, deacetylation of H3K27 is accompanied by the loss of gene expression. Therefore, it serves as a negative control. B, DNA methylation was analyzed at Cited2 CpGi using bisulfite sequencing in undifferentiated ESCs. The average percent methylation of 27 CpG sites in the CpGi was plotted. C, gene expression analysis of Cited2 in WT and Dnmt3b^−/− ESCs. There was no significant difference in Cited2 expression levels. D, ChIP-qPCR using anti-Vezf1 antibody showed an increase in the fold-enrichment at Cited2 promoter in WT ESCs. Fold-enrichment below 1 in Vezf1^−/− ESCs indicates absence of binding, and was used as negative control. The data in each bar graph represents average and S.D. of 3 to 4 replicates. E, model showing the effect of Cited2 on Hif-1α-mediated regulation of pro-angiogenic genes. Vezf1 regulates the expression of Cited2 at basal levels in ESCs before the induction of endothelial differentiation. This allows the interaction of Hif-1α and P300 that activate the pro-angiogenic genes. In the absence of Vezf1, high expression of Cited2 sequesters P300 away from Hif-1α, thus inhibiting the activation of pro-angiogenic genes. WT, wildtype; Vezf1^−/−, Vezf1 knockout; Dnmt3b^−/−, Dnmt3b knockout; UD, undifferentiated; D7, days post-differentiation; P1 and P2, two primer pairs in Cited2 promoter used in ChIP-qPCR.