From the Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (Y.O., N.F., T.T., Y.N., K.S., Y.K., H.N., A.S., M.S., J.Z., K.I., N.H., M.K., Y.M., S.N., T.D.); Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA (L.Y., W.C.A.); Department of Pathology, Center for Excellence in Vascular Biology, Harvard Medical School, Boston, MA (A.S.T., G.G.-C.); Department of Material Sciences, Massachusetts Institute of Technology, Boston (A.S.T.); and Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (K.K.).
Arterioscler Thromb Vasc Biol. 2014 Jul;34(7):1531-8. doi: 10.1161/ATVBAHA.114.303818. Epub 2014 May 22.
The molecular basis of endothelial cell (EC)-specific gene expression is poorly understood. Roundabout 4 (Robo4) is expressed exclusively in ECs. We previously reported that the 3-kb 5'-flanking region of the human Robo4 gene contains information for lineage-specific expression in the ECs. Our studies implicated a critical role for GA-binding protein and specificity protein 1 (SP1) in mediating overall expression levels. However, these transcription factors are also expressed in non-ECs. In this study, we tested the hypothesis that epigenetic mechanisms contribute to EC-specific Robo4 gene expression.
Bisulfite sequencing analysis indicated that the proximal promoter of Robo4 is methylated in non-ECs but not in ECs. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine increased Robo4 gene expression in non-ECs but not in ECs. Proximal promoter methylation significantly decreased the promoter activity in ECs. Electrophoretic mobility shift assays showed that DNA methylation of the proximal promoter inhibited SP1 binding to the -42 SP1 site. In DNase hypersensitivity assays, chromatin condensation of the Robo4 promoter was observed in some but not all nonexpressing cell types. In Hprt (hypoxanthine phosphoribosyltransferase)-targeted mice, a 0.3-kb proximal promoter directed cell-type-specific expression in the endothelium. Bisulfite sequencing analysis using embryonic stem cell-derived mesodermal cells and ECs indicated that the EC-specific methylation pattern of the promoter is determined by demethylation during differentiation and that binding of GA-binding protein and SP1 to the proximal promoter is not essential for demethylation.
The EC-specific DNA methylation pattern of the Robo4 proximal promoter is determined during cell differentiation and contributes to regulation of EC-specific Robo4 gene expression.
内皮细胞（EC）特异性基因表达的分子基础尚不清楚。Roundabout 4（Robo4）仅在 EC 中表达。我们之前报道过，人 Robo4 基因的 3kb 5'-侧翼区包含在 EC 中特异性表达的信息。我们的研究表明 GA 结合蛋白和特异性蛋白 1（SP1）在介导整体表达水平方面起着关键作用。然而，这些转录因子也在非 EC 中表达。在这项研究中，我们检验了这样一个假设，即表观遗传机制有助于 EC 特异性 Robo4 基因表达。
亚硫酸氢盐测序分析表明，Robo4 的近端启动子在非 EC 中被甲基化，但在 EC 中没有。用 DNA 甲基转移酶抑制剂 5-氮杂-2'-脱氧胞苷处理可增加非 EC 中 Robo4 基因的表达，但不能增加 EC 中的表达。近端启动子甲基化显著降低了 EC 中的启动子活性。电泳迁移率变动分析显示，近端启动子的 DNA 甲基化抑制了 SP1 与-42 SP1 结合位点的结合。在 DNase 超敏性分析中，在一些而非所有非表达细胞类型中观察到 Robo4 启动子的染色质浓缩。在 Hprt（次黄嘌呤磷酸核糖基转移酶）靶向小鼠中，0.3kb 的近端启动子在血管内皮细胞中具有细胞类型特异性表达。使用胚胎干细胞衍生的中胚层细胞和 EC 进行的亚硫酸氢盐测序分析表明，启动子的 EC 特异性甲基化模式是通过分化过程中的去甲基化决定的，并且 GA 结合蛋白和 SP1 与近端启动子的结合对于去甲基化不是必需的。
Robo4 近端启动子的 EC 特异性 DNA 甲基化模式是在细胞分化过程中确定的，有助于调节 EC 特异性 Robo4 基因表达。
