NCoR1 limits angiogenic capacity by altering Notch signaling

Corepressors negatively regulate gene expression by chromatin compaction. Targeted regulation of gene expression could provide a means to control endothelial cell phenotype. We hypothesize that by targeting corepressor proteins, endothelial angiogenic function can be improved. To study this, the expression and function of nuclear corepressors in human umbilical vein endothelial cells (HUVEC) and in murine organ culture was studied. RNA-seq revealed that nuclear receptor corepressor 1 (NCoR1), Silencing Mediator of Retinoid and Thyroid hormone receptors (SMRT) and repressor element-1 silencing transcription factor (REST) are the highest expressed corepressors in HUVECs. Knockout and knockdown strategies demonstrated that the depletion of NCoR1 increased the angiogenic capacity of endothelial cells, whereas depletion of SMRT or REST did not. Interestingly, the effect was VEGF signaling independent. NCoR1 depletion significantly upregulated angiogenesis-associated genes, especially tip cell genes, including ESM1, DLL4 and NOTCH4, as observed by RNA- and ATAC-seq. Confrontation assays comparing cells with and without NCoR1-deficiency revealed that loss of NCoR1 promotes a tip-cell position during spheroid sprouting. Moreover, a proximity ligation assay identified NCoR1 as a direct binding partner of the Notch-signaling-related transcription factor RBPJk. Luciferase assays showed that siRNA-mediated knockdown of NCoR1 promotes RBPJk activity. Furthermore, NCoR1 downregulation prompts upregulation of several elements in the Notch signaling cascade. Downregulation of NOTCH4, but not NOTCH1, prevented the positive effect of NCoR1 knockdown on spheroid outgrowth. Collectively, these data indicate that decreasing NCOR1 expression is an attractive approach to promote angiogenic function. This SuperSeries is composed of the SubSeries listed below. RNA sequencing (n=3) and ATAC sequencing (n=3) of of human umbilical vein endothelial cells (HUVEC) treated with either negative control siRNA or siRNA HSS114352, which knocks down NCoR1 expression. Please refer to individual Series

共抑制因子（corepressors）通过染色质压缩负向调控基因表达。靶向调控基因表达可为控制内皮细胞表型提供可行手段。本研究假设，靶向共抑制因子蛋白可改善内皮细胞的血管生成功能。为验证该假设，本研究针对人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVEC）及小鼠器官培养模型中的核共抑制因子的表达与功能展开了探究。RNA测序（RNA-seq）结果显示，核受体共抑制因子1（nuclear receptor corepressor 1, NCoR1）、维甲酸与甲状腺激素受体沉默介导因子（Silencing Mediator of Retinoid and Thyroid hormone receptors, SMRT）以及阻遏元件1沉默转录因子（repressor element-1 silencing transcription factor, REST）是HUVEC中表达量最高的共抑制因子。采用基因敲除与敲低策略的实验结果表明，敲除NCoR1可增强内皮细胞的血管生成能力，而敲低SMRT或REST则无此效果。值得注意的是，该效应不依赖于血管内皮生长因子（VEGF）信号通路。通过RNA测序与转座酶可及性测序（ATAC-seq）分析发现，敲低NCoR1可显著上调血管生成相关基因的表达，其中尤以尖端细胞相关基因如ESM1、DLL4及NOTCH4最为显著。通过对比NCoR1缺陷与正常细胞的对峙实验发现，NCoR1缺失可促进球体出芽过程中的尖端细胞定位。此外，邻近连接实验（proximity ligation assay）证实，NCoR1是Notch信号通路相关转录因子RBPJk的直接结合伴侣。萤光素酶报告实验结果显示，通过小干扰RNA（siRNA）敲低NCoR1可增强RBPJk的转录活性。此外，下调NCoR1的表达可促使Notch信号级联反应中多个元件的表达上调。敲低NOTCH4（而非NOTCH1）可阻断NCoR1敲低对球体出芽的促进作用。综上，上述实验数据表明，下调NCOR1的表达是提升内皮细胞血管生成功能的极具潜力的策略。

本超系列由以下子系列构成：
分别用阴性对照siRNA及靶向敲低NCoR1表达的siRNA HSS114352处理的人脐静脉内皮细胞（HUVEC）的RNA测序（n=3）与ATAC测序（n=3）数据。详情请参阅各独立系列数据集。