Abstract
Zinc finger proteins (ZNF) are a large group of transcription factors with diverse functions. We recently discovered that endothelial cells harbour a specific mechanism to limit the action of ZNF354C, whose function in endothelial cells is unknown. Given that ZNF354C has so far only been studied in bone and tumour, its function was determined in endothelial cells. ZNF354C is expressed in vascular cells and localises to the nucleus and cytoplasm. Overexpression of ZNF354C in human endothelial cells results in a marked inhibition of endothelial sprouting. RNA-sequencing of human microvascular endothelial cells with and without overexpression of ZNF354C revealed that the protein is a potent transcriptional repressor. ZNF354C contains an active KRAB domain which mediates this suppression as shown by mutagenesis analysis. ZNF354C interacts with dsDNA, TRIM28 and histones, as observed by proximity ligation and immunoprecipitation. Moreover, chromatin immunoprecipitation revealed that the ZNF binds to specific endothelial-relevant target-gene promoters. ZNF354C suppresses these genes as shown by CRISPR/Cas knockout and RNAi. Inhibition of endothelial sprouting by ZNF354C is dependent on the amino acids DV and MLE of the KRAB domain. These results demonstrate that ZNF354C is a repressive transcription factor which acts through a KRAB domain to inhibit endothelial angiogenic sprouting.
Highlights
Zinc finger proteins (ZNF) are a large group of transcription factors with diverse functions
We previously demonstrated that ZNF354C inhibits transcription of sphingosine-1-phosphate receptor-1 (S1PR1) in human umbilical vein endothelial cells (HUVECs) and limits S1P signaling
RNA and protein expression and Encode Cap Analysis of Gene Expression (CAGE) promoter analysis revealed that ZNF354C is expressed in all tissues tested but, most importantly, it is strongly expressed in vascular cells such as human umbilical vein endothelial cells (HUVECs), human microvascular endothelial cells-1 (HMEC-1) and human aortic smooth muscle cells (HAoSMCs)
Summary
Zinc finger proteins (ZNF) are a large group of transcription factors with diverse functions. We previously reported that ZNF354C is active in human umbilical vein endothelial cells: it inhibits transcription of sphingosine-1-phosphate receptor-1 (S1PR1) and endothelial cells express the long non-coding RNA “long intergenic noncoding RNA antisense to S1PR1” (LISPR1) to prevent the interaction of ZNF354C with the endothelial S1PR1 promoter[14]. Despite this observation, ZNF354C is poorly characterised in the cardiovascular system. We hypothesise that ZNF354C is an important protein in the cardiovascular system contributing to vascular transcriptional regulation
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