Functional characterization of GWAS findings highlights a role for REST in endothelial plasticity and atherosclerosis [snRNA-seq]

Background: Genome-wide association studies (GWAS) have identified multiple novel loci that contribute to coronary artery disease (CAD) pathogenesis, but the mechanisms of these associations remain largely unknown. Methods: In this study we used a multi-trait colocalization approach to prioritize novel endothelial specific loci for atherosclerosis. We combined computational methods with in vitro assays and mouse models to study one of those new loci targeting gene REST. Results: A multi-trait colocalization approach across expression quantitative trait loci (eQTL) in atherosclerosis-relevant cell types followed by in vitro CRISPR interference revealed that a conserved regulatory element in a chromosome 4 genetic locus increases risk of CAD and decreases the expression of REST, a transcriptional repressor, in endothelial cells. Pcsk9-overexpressing mice with an endothelial-specific knockout of Rest exhibited increased atherosclerotic plaque formation in their aortas, with increased macrophage and lipid deposition within the plaque after 16 weeks of high-fat diet exposure compared to littermate controls. RNA-seq in human aortic endothelial cells (HAEC) after REST silencing followed by assessment of protein expression revealed that REST silencing triggers endothelial-to-mesenchymal transition (endMT). Consistently, REST silencing increased endothelial permeability and migration in vitro. Single nucleus RNA sequencing in endothelial lineage traced atherosclerotic mice with Rest knock-out revealed evidence of endothelial TGFb signalling activation and of transition smooth muscle-like cells in atherosclerotic aortas upon genetic knockout of Rest. CUT&Tag sequencing did not identify any known TGFb effector genes as direct REST transcriptional targets. Instead, joint analysis of CUT&Tag with RNA-seq, highlighted L1CAM, a known endMT activator, and its interactors as the most significant gene-set directly affected by REST in the endothelium. Simultaneous silencing of L1CAM and REST in HAEC inhibited the upregulation of mesenchymal genes and the enhanced migration induced by REST silencing and diminished the upregulation of several TGFb effectors overexpressed upon REST silencing. Conclusion: In summary, our data reveal the novel role of REST as a repressor in endothelial cells that functions to constitutively inhibit endMT and protect against atherosclerosis. Key words: GWAS, NRSF/REST, endothelial to mesenchymal transition, CRISPRi, CUT&TAG, endothelial cells, L1CAM, atherosclerosis Overall design: We assess whether endMT is observed in vivo during atherosclerosis upon endothelial Rest knock-out. To do that we crossed our endothelial Rest knock-out mice and corresponding endothelial Cre mice controls with a reporter mT/mG mouse. By treating these mice with tamoxifen after 8 weeks of age, we are able to simultaneously knock-out endothelial Rest and label endothelial cells with green fluorescence permanently, thereby permitting lineage tracing of those cells during atherosclerosis progression. We injected those mice with AAV8-m-Pcsk9 and exposed them to 16 weeks HFD as above to induce atherosclerosis. We subsequently isolated their aortas and performed single nucleus RNA-sequencing