ZNF354C is a transcriptional repressor that inhibits endothelial angiogenic sprouting

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

DNA topoisomerase inhibition with the HIF inhibitor acriflavine promotes transcription of lncRNAs in endothelial cells

The transcription factor hypoxia-inducible factor 1 (HIF1) is an important driver of cancer and is therefore an attractive drug target. Acriflavine (ACF) has been suggested to inhibit HIF1, but its mechanism of action is unknown. Here we investigated the interaction of ACF with DNA and long non-coding RNAs (lncRNAs) and its function in human endothelial cells. ACF promoted apoptosis and reduced proliferation, network formation, and angiogenic capacity. It also induced changes in gene expression, as determined by RNA sequencing (RNAseq), which could not be attributed to specific inhibition of HIF1. A similar response was observed in murine lung endothelial cells. Although ACF increased and decreased a similar number of protein-coding genes, lncRNAs were preferentially upregulated under normoxic and hypoxic conditions. An assay for transposase accessibility with subsequent DNA sequencing (ATAC-seq) demonstrated that ACF induced strong changes in chromatin accessibility at lncRNA promoters. Immunofluorescence showed displacement of DNA:RNA hybrids. Such effects might be due to ACF-mediated topoisomerase inhibition, which was indeed the case, as reflected by DNA unwinding assays. Comparison with other acridine derivatives and topoisomerase inhibitors suggested that the specific function of ACF is an effect of acridinium-class compounds. This study demonstrates that ACF inhibits topoisomerases rather than HIF specifically and that it elicits a unique expression response of lncRNAs

HIF1 alpha-AS1 is a DNA:DNA:RNA triplex-forming lncRNA interacting with the HUSH complex

DNA:DNA:RNA triplexes that are formed through Hoogsteen base-pairing of the RNA in the major groove of the DNA duplex have been observed in vitro, but the extent to which these interactions occur in cells and how they impact cellular functions remains elusive. Using a combination of bioinformatic techniques, RNA/DNA pulldown and biophysical studies, we set out to identify functionally important DNA:DNA:RNA triplex-forming long non-coding RNAs (lncRNA) in human endothelial cells. The lncRNA HIF1 alpha-AS1 was retrieved as a top hit. Endogenous HIF1 alpha-AS1 reduces the expression of numerous genes, including EPH Receptor A2 and Adrenomedullin through DNA:DNA:RNA triplex formation by acting as an adapter for the repressive human silencing hub complex (HUSH). Moreover, the oxygen-sensitive HIF1 alpha-AS1 is down-regulated in pulmonary hypertension and loss-of-function approaches not only result in gene de-repression but also enhance angiogenic capacity. As exemplified here with HIF1 alpha-AS1, DNA:DNA:RNA triplex formation is a functionally important mechanism of trans-acting gene expression control. Using a composite bioinformatics approach, the DNA:DNA:RNA triplex-forming lncRNAs HIF1 alpha-AS1 was identified in human endothelial cells which recruits an epigenetic silencing complex to limit expression of triplex target genes