RNA elongation contributes to gene expression regulation during early endothelial-to-mesenchymal transition

Abstract

Abstract Background Endothelial dysfunction is characterized by endothelial-to-mesenchymal transition (EndMT), a process in which endothelial cells (EC) transform into mesenchymal cells and lose part of their endothelial identity. EndMT-positive cells are present in vulnerable atherosclerotic plaques. EndMT is triggered by stress stimuli such as inflammation or hypoxia that require an adaptive change in gene expression mediated at the level of RNA elongation. RNA elongation is controlled by the super elongation complex (SEC), which is built around scaffolding proteins of the AF family (AFF1-AFF4). The SEC controls the pausing of RNA Polymerase II (RNAPII). We hypothesized that RNA elongation plays a regulatory role in EndMT. Methods and Results Single cell sequencing data revealed that the SEC scaffold proteins AFF1 and AFF4 are ubiquitously expressed in human carotid artery plaques. AFF1 and AFF4 are higher expressed in EC compared to AFF2 and AFF3. In an in vitro EndMT model, AFF1 and AFF4 were upregulated compared to controls (AFF1: 1.9-fold; AFF4: 2.6-fold). SEC inhibition using chemical compounds reduced the protein levels of AFF1 (-67.4%) and AFF4 (-46.5%). EndMT induction was reduced by SEC inhibition, as measured by mesenchymal marker gene expression (SM22: -88.8%; CNN1: -94.0%). The rapid induction of gene expression depends on the release of paused RNAPII present at the promoter site. ChIP-sequencing revealed a reduced occupancy of RNAPII at promoter sites in EndMT as early as 2 h compared to controls, suggesting a rapid pause release of RNAPII during EndMT. Indeed, transcriptome-wide RNA elongation rates were increased by 20.2% as early as 10 min after induction of EndMT, as determined by analysis of newly synthesized 4sU-labelled RNA. SEC inhibition reduced transcriptome-wide RNA elongation rates (-26.8%). These data suggest a regulatory contribution of RNA elongation in early EndMT. Among the genes that were immediately and prominently upregulated after the induction of EndMT was the SPARC mRNA. RNA initiation and elongation of SPARC mRNA was increased 10 min after EndMT induction, which was reduced by SEC inhibition. 72 hours after EndMT induction, inhibition of SEC resulted in a reduction in SPARC mRNA (-72.2%), protein concentration (-97.2%) and reduced SPARC delivery to the supernatant (-43.9%). Knockdown of SPARC enhanced EndMT induction (SM22: 1.6-fold; CNN1: 3.6-fold), which was attenuated by treatment with recombinant SPARC. Conclusions We observed increased RNA elongation rates during EndMT associated with decreased pausing of RNA Polymerase II. The RNA elongation mediated specific gene expression such as the upregulation of SPARC. Taken together, our results indicate that RNA elongation plays a regulatory role in EndMT.