Abstract 16925: KLF and AP1 Having Opposing Roles in Regulating Chromatin Accessibility and the Endothelial Enhancer Landscape Under Laminar vs. Disturbed Shear Stress

Abstract

Background: Laminar shear stress (LSS) is associated with a vasculoprotective endothelial phenotype, while exposure to disturbed shear stress (DSS) is linked to endothelial dysfunction and vascular disease. Epigenetic changes in chromatin accessibility and enhancer dynamics are important regulators of gene expression. We hypothesized that LSS and DSS induce distinct transcription factors that recruit chromatin remodelers to change the chromatin landscape, making it permissive for either protective or disease-related gene expression profiles. Methods & Results: Human pulmonary arterial endothelial cells were exposed to LSS (15 dyne/cm2) or DSS (±3 dyne/cm2, 0.8 Hz; and static) for 24h and analyzed by ATAC-Seq and RNA-Seq. Exposure to LSS resulted in increased chromatin accessibility, predominantly in regions within 100 kb of transcription start sites. These changes correlated with induction of protective, and suppression of disease-related genes. Many differentially accessible regions were also differentially marked by H3K27ac and H3K4me1 enhancer marks, as identified by ChIP-Seq. Motif analyses found enrichment for KLF binding sites in enhancers that are open under LSS vs. enrichment for AP1 at enhancers that are open under DSS. ChIP-Seq for KLF and AP1 confirmed differential binding at those sites. Gain-and-loss of function studies using a constitutive active mutant of MEK5, which induces KLF akin to LSS, and RNAi for KLF and AP1 confirmed that these factors were required to differentially mediate accessibility changes under LSS and DSS. We then used IP-MS to identify the chromatin remodelers recruited by KLF, and uncovered several components of the SWI/SNF nucleosome remodeling complex. Co-IP assays confirmed interaction of SWI/SNF with KLF under LSS. This was further documented by in situ proximity ligation assays. Under conditions of DSS we observed nuclear translocation of AP1 member ATF2 that interacts with c-Jun, which is enriched at enhancers that are open under DSS. Conclusions: KLF and AP1 differentially regulate the endothelial enhancer landscape under laminar vs. disturbed shear stress, which culminates in protective or disease-related gene expression profiles.