Background & Purpose:
Atherosclerosis is associated with complex molecular pathophysiological processes, such as chronic inflammation and endothelial-to-mesenchymal transition (EndMT). Initiation and resolution of the inflammation processes need to be tightly regulated. On the level of RNA elongation, inflammation can be induced by a rapid response mechanism in which paused RNA Polymerase II (RNAPII) at promoter-proximal sites is activated upon stimulation. This rapid response mechanism is controlled by the Super Elongation Complex (SEC) built around the scaffold proteins AFF1 and AFF4. The SEC controls the RNA elongation rate through pausing and release of RNAPII. We hypothesized, that EndMT is regulated by rapid responses on the level of RNA elongation. Therefore, we analyzed the contribution of the RNA elongation proteins AFF1 and AFF4 in the process of EndMT. 

Methods & Results:
We found the SEC scaffold proteins AFF1 and AFF4 enriched in primary human endothelial cells (EC) and upregulated upon induction of EndMT (AFF1: 1.9-fold and AFF4: 2.7-fold, P<0.05). The SEC can be inhibited using two small molecules leading to degradation of AFF1 and AFF4 on protein level. Treatment with these inhibitors resulted in a reduced induction of EndMT as measured by the expression of several mesenchymal markers, such as SM22 (-87.6%, P<0.05) and Calponin (-93.8%, P<0.05). Consistently, overexpression of AFF4 led to enhanced expression of mesenchymal markers. Using chromatin immunoprecipitation (ChIP) in EC, we found AFF1 and AFF4 as well as paused RNAPII located at the proximal promotor site of EndMT marker genes, such as Calponin. A subsequent ChIP-sequencing revealed, that the treatment with the small molecule inhibitors targeting AFF1/AFF4 in EC led to an overall higher occupation of paused RNAPII at promoter sites, indicating a regulation on level of RNA elongation. Sequencing of newly transcribed RNA after treatment with small molecule inhibitors targeting the AFF1/AFF4 SEC revealed a lower transcript density downstream of promoter sites, indicating a reduced rate of RNA elongation.  Upon induction of EndMT, we observed an upregulation in a variety of newly synthesized RNA transcripts. After treatment with small molecule inhibitors, 63.6% of EndMT-induced transcripts were decreased. Among those, we identified a transcript of the SPARC gene induced during EndMT. This induction was diminished after the inhibition of the SEC. We validated this regulation using qRT-PCR. Less occupancy of paused RNAPII at promoter-proximal sites upon induction of EndMT further indicated a SEC-mediated regulation of SPARC during EndMT. 

Conclusion:
AFF1 and AFF4, scaffold proteins of the SEC are upregulated in EndMT. They are co-enriched with paused RNAPII at promoters of EndMT genes. Inhibition of the SEC using two small molecules resulted in reduced induction of EndMT and higher levels of paused RNAPII in proximity of promoter sites of target genes such as Calponin or SPARC. Our data suggest a potential role of RNA elongation in the rapid gene response to stress stimuli contributing to EndMT.