Targeting RNA elongation in endothelial-to-mesenchymal transition

Abstract

Abstract Background and purpose EndMT describes a process in which endothelial cells lose their endothelial features such as the maintenance of the vascular barrier function and trans-differentiate into mesenchymal like cells. ECs which have underwent EndMT are enriched in vulnerable plaques in humans. Several stress stimuli such as hypoxia or inflammation demand immediate induction of adaptive gene expression to improve cell survival. In contrast to the usual induction of gene expression which requires several hours, release of paused RNA Polymerase II (RNAPII) from promoter allows rapid gene induction via RNA elongation. This rapid response mechanism is mediated by the super elongation complex (SEC), which is built around the scaffold proteins AFF1 and AFF4. Here, we hypothesized that RNA elongation regulates EndMT and therefore investigated on the contribution of the SEC scaffold proteins during the process of EndMT. Methods and results After induction of EndMT in human primary endothelial cells (EC) the elongation factors AFF1 and AFF4 were induced (AFF1: 1.9-fold and AFF4: 2.7-fold, P<0.05). Overexpression of AFF4 resulted in increased expression of the mesenchymal marker genes SM22 (1.8-fold, P<0.05) and VCAN (2.0-fold, P<0.05). Accordingly, treatment of EC with two small molecules inhibiting the SEC reduced the expression of several mesenchymal markers SM22 (−87.6%, P<0.05) and Calponin (−93.8%, P<0.05) during EndMT. After induction of EndMT, a large number of newly synthesized RNA transcripts were upregulated, indicating regulation of gene expression on the level of RNA elongation during EndMT. Sequencing of newly transcribed RNA (4sU-sequencing) after treatment with the SEC inhibitors revealed a lower transcript density downstream of promoter sites, suggesting a decreased RNA elongation rate in ECs. SEC inhibitors decreased the expression of the majority of EndMT-induced transcripts (63.6%). Among these rapid induced genes we found the mRNA of RE1 (+10,8-fold). We validated the regulation of RE1 by qPCR (2.8-fold, P<0.05) and single-cell sequencing. Chromatin immunoprecipitation (ChIP) sequencing revealed a reduced occupation of RNAPII in proximity of promotor sites of RE1 upon induction of EndMT. These data suggest a SEC-mediated regulation on the level of RNA elongation of RE1 during EndMT. Conclusion SEC scaffold proteins AFF1 and AFF4 are upregulated during EndMT. Targeting the SEC during EndMT reduces the expression of common mesenchymal markers, consistent with an overall higher occupancy of paused RNAPII at promoter-proximal sites. We identify RE1 as a rapidly induced gene during EndMT that is mediated by the SEC scaffold proteins AFF1 and AFF4. Our findings suggest that RNA elongation is an inducer of EndMT. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)