NCoR1 limits angiogenic capacity by altering Notch signaling [RNA]
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. Overall design: RNA sequencing (n = 3) of human umbilical vein endothelial cells (HUVEC) treated with either negative control siRNA #12935300 or siRNA #HSS114352, which knocks down NCoR1  expression.