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Arterial marker genes EphrinB2 and HEY2 are essential for cardiovascular development and post-natal neovascularization.Our previous study confirmed that E2F1 could activate the transcription of EphrinB2 and HEY2 in human mesenchymal stem cells;however,the detailed mechanism has not been resolved yet.In this study,we focused on the interaction between E2F1 and DNMT3A,a de novo DNA methyltransferase,on regulating the expression of EphrinB2 and HEY2,and explored the potential mechanisms.Gain-and loss-of-function experiments implicated the positive effect of E2F1 on the expression of EphrinB2 and HEY2 and tube formation in human umbilical artery endothelial cells.Accumulation of DNMT3A decreased the levels of EphrinB2 and HEY2,and impaired tube formation induced by E2F1,while inhibiting DNMT3A by RNA interference aug-mented their expression and angiogenesis in E2F1-trasfected cells.We then asked whether the low expressions of EphrinB2 and HEY2 induced by DNMT3A are related to the methylation status of their promoters.Surprisingly,the methylation status of the CpG islands in the promoter region was not significantly affected by overexpression of exogenous DNMT3A.Furthermore,the inter-action between E2F1 and DNMT3A was confirmed by co-immunoprecipitation.DNMT3A could inhibit the transcription of EphrinB2 and HEY2 promoters by affecting the binding of E2F1 to its recognition sequences as revealed by luciferase reporter assay and chromatin immunoprecipita-tion.These results identified a novel mechanism underlying the cooperation of DNMT3A with E2F1 on regulating target gene expression,and revealed their roles in the angiogenic process.