Abstract
Formation of the vasculature by angiogenesis is critical for proper development, but angiogenesis also contributes to the pathogenesis of various disorders, including cancer and cardiovascular diseases. Vascular endothelial zinc finger 1 (Vezf1), is a Krüppel-like zinc finger protein that plays a vital role in vascular development. However, the mechanism by which Vezf1 regulates this process is not fully understood. Here, we show that Vezf1−/− mouse embryonic stem cells (ESC) have significantly increased expression of a stem cell factor, Cbp/p300-interacting transactivator 2 (Cited2). Compared with WT ESCs, Vezf1−/− ESCs inefficiently differentiated into endothelial cells (ECs), which exhibited defects in the tube-formation assay. These defects were due to reduced activation of EC-specific genes concomitant with lower enrichment of histone 3 acetylation at Lys27 (H3K27) at their promoters. We hypothesized that overexpression of Cited2 in Vezf1−/− cells sequesters P300/CBP away from the promoters of proangiogenic genes and thereby contributes to defective angiogenesis in these cells. This idea was supported by the observation that shRNA-mediated depletion of Cited2 significantly reduces the angiogenic defects in the Vezf1−/− ECs. In contrast to previous studies that have focused on the role of Vezf1 as a transcriptional activator of proangiogenic genes, our findings have revealed a role for Vezf1 in modulating the expression of the antiangiogenic factor Cited2. Vezf1 previously has been characterized as an insulator protein, and our results now provide insights into the mechanism, indicating that Vezf1 can block inappropriate, nonspecific interactions of promoters with cis-located enhancers, preventing aberrant promoter activation.
Highlights
Formation of the vasculature by angiogenesis is critical for proper development, but angiogenesis contributes to the pathogenesis of various disorders, including cancer and cardiovascular diseases
In contrast to previous studies that have focused on the role of Vascular endothelial zinc finger 1 (Vezf1) as a transcriptional activator of proangiogenic genes, our findings have revealed a role for Vezf1 in modulating the expression of the antiangiogenic factor Cited2
We found Cited2 among the top 20 genes that were up-regulated in Vezf1Ϫ/Ϫ embryonic stem cells (ESC) by more than 5-fold
Summary
Formation of the vasculature by angiogenesis is critical for proper development, but angiogenesis contributes to the pathogenesis of various disorders, including cancer and cardiovascular diseases. To study the specific role of Vezf in endothelial development and angiogenesis, we examined the differentiation of WT and Vezf1Ϫ/Ϫ ESCs to ECs by treatment with VEGF-A165 and tested their angiogenic potential by a in vitro tube-formation assay. Our findings suggest that Vezf controls activation of angiogenesis in ECs by restricting Cited expression to basal levels, which allows Hif-1␣–mediated activation of the pro-angiogenic genes. Concomitant with this, there was reduced activation of endothelial/pro-angiogenic genes in differentiating Vezf1Ϫ/Ϫ ECs. Based on our data showing reduced levels of histone H3K27Ac at the promoters of angiogenesisspecific genes, we propose that high levels of Cited sequester the histone acetyltransferase p300 away from angiogenesisspecific gene promoters, reducing their activation and gene expression. We speculate that the insulator function of Vezf blocks inappropriate interactions of the Cited promoter with nearby enhancer/s, modulating the magnitude and spatiotemporal regulation of its expression
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