Abstract
Ets transcription factors function as important developmental regulators and are known to be modulators of cell fate. Previously, in conjunction with co-factors, we have described Ets variant 2 (Etv2) as an essential regulator of the hematopoietic and endothelial lineages. But the mechanism and the Etv2 interacting partners involved in achieving this critical function remains poorly understood. Through Yeast two-hybrid analysis we identified Vascular Endothelial Zinc Finger 1 (Vezf1) as an interacting factor with Etv2. Vezf1 is a conserved C2H2 zinc finger transcription factor known to regulate the formation, proliferation, and migration of endothelial cells through several gene targets. We verified Vezf1 as a binding partner of Etv2 through co-immunoprecipitation and GST-pull down studies. Bioinformatic analysis of ChIP-seq and Etv2-expressing single cell RNA sequencing was conducted to identify candidate genes containing both Etv2 and Vezf1 binding motifs in their regulatory regions. Histone deacetylase 7 (Hdac7) and angiomotin like protein 2 (Amotl2) were identified as genes of interest involved in endothelial development. Hdac7 is a conserved class II deacetylase that functions in endothelial cell adhesion and vascular integrity. Amotl2 is a Motin family protein that functions in angiogenesis, endothelial cell polarity and migration. RT-qPCR analysis showed upregulation of Hdac7 and Amotl2 in response to doxycycline inducible Etv2 and Vezf1; whereas significant reduction of expression of these two genes was observed in the Etv2 and Vezf1 knockout cells. Chromatin immunoprecipitation (ChIP) and electrophoresis mobility shift assays (EMSA) confirmed Etv2-Vezf1 adjacent binding sites in the promoters of Hdac7 and Amotl2. Histone Acetyl transferase (HAT) assays was performed to investigate Etv2-Vezf1 on global histone acetylation conditions in doxycycline inducible embryoid bodies. Vezf1 overexpression results in a significant reduction of acetylated histone. In summary, this study identifies Vezf1 as a novel binding partner of Etv2 and their combined role in regulating downstream target genes Amotl2 and Hdac7 in hematoendothelial development.
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