College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China.
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin 300457, China.
Acta Biochim Biophys Sin (Shanghai). 2020 Dec 11;52(11):1236-1246. doi: 10.1093/abbs/gmaa109.
Arterial marker genes EphrinB2 and HEY2 are essential for cardiovascular development and postnatal 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 augmented 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 interaction 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 immunoprecipitation. 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.
动脉标记基因 EphrinB2 和 HEY2 对于心血管发育和出生后新生血管形成是必不可少的。我们之前的研究证实，E2F1 可以在人骨髓间充质干细胞中激活 EphrinB2 和 HEY2 的转录；然而，其详细机制尚未得到解决。在这项研究中，我们专注于 E2F1 与从头 DNA 甲基转移酶 DNMT3A 之间的相互作用，以调节 EphrinB2 和 HEY2 的表达，并探索潜在的机制。E2F1 的获得和缺失功能实验表明其对 EphrinB2 和 HEY2 的表达以及人脐动脉内皮细胞管形成有正向作用。DNMT3A 的积累降低了 EphrinB2 和 HEY2 的水平，并损害了 E2F1 诱导的管形成，而通过 RNA 干扰抑制 DNMT3A 则增强了 EphrinB2 和 HEY2 在 E2F1 转染细胞中的表达和血管生成。然后，我们询问了 DNMT3A 诱导的 EphrinB2 和 HEY2 低表达是否与它们启动子的甲基化状态有关。令人惊讶的是，外源性 DNMT3A 过表达对启动子区 CpG 岛的甲基化状态没有显著影响。此外，E2F1 和 DNMT3A 之间的相互作用通过共免疫沉淀得到了证实。DNMT3A 可以通过影响 E2F1 与其识别序列的结合来抑制 EphrinB2 和 HEY2 启动子的转录，这一点通过荧光素酶报告基因检测和染色质免疫沉淀得到了证实。这些结果确定了 DNMT3A 与 E2F1 合作调节靶基因表达的新机制，并揭示了它们在血管生成过程中的作用。
