Background and Purpose— Genome-wide association studies have identified the HDAC9 (histone deacetylase 9) gene region as a major risk locus for atherosclerotic stroke and coronary artery disease in humans. Previous results suggest a role of altered HDAC9 expression levels as the underlying disease mechanism. rs2107595, the lead single nucleotide polymorphism for stroke and coronary artery disease resides in noncoding DNA and colocalizes with histone modification marks suggestive of enhancer elements. Methods— To determine the mechanisms by which genetic variation at rs2107595 regulates HDAC9 expression and thus vascular risk we employed targeted resequencing, proteome-wide search for allele-specific nuclear binding partners, chromatin immunoprecipitation, genome-editing, reporter assays, circularized chromosome conformation capture, and gain- and loss-of-function experiments in cultured human cell lines and primary immune cells. Results— Targeted resequencing of the HDAC9 locus in patients with atherosclerotic stroke and controls supported candidacy of rs2107595 as the causative single nucleotide polymorphism. A proteomic search for nuclear binding partners revealed preferential binding of the E2F3/TFDP1/Rb1 complex (E2F transcription factor 3/transcription factor Dp-1/Retinoblastoma 1) to the rs2107595 common allele, consistent with the disruption of an E2F3 consensus site by the risk allele. Gain- and loss-of-function studies showed a regulatory effect of E2F/Rb proteins on HDAC9 expression. Compared with the common allele, the rs2107595 risk allele exhibited higher transcriptional capacity in luciferase assays and was associated with higher HDAC9 mRNA levels in primary macrophages and genome-edited Jurkat cells. Circularized chromosome conformation capture revealed a genomic interaction of the rs2107595 region with the HDAC9 promoter, which was stronger for the common allele as was the in vivo interaction with E2F3 and Rb1 determined by chromatin immunoprecipitation. Gain-of-function experiments in isogenic Jurkat cells demonstrated a key role of E2F3 in mediating rs2107595-dependent transcriptional regulation of HDAC9 . Conclusions— Collectively, our findings imply allele-specific transcriptional regulation of HDAC9 via E2F3 and Rb1 as a major mechanism mediating vascular risk at rs2107595.

Objective- A large number of genetic loci have been associated with risk of coronary artery disease (CAD) through genome-wide association studies, however, for most loci the underlying biological mechanism is unknown. Determining the molecular pathways and cellular processes affected by these loci will provide new insights into CAD pathophysiology and may lead to new therapies. The CAD-associated variants at 10p11.23 fall in JCAD, which encodes an endothelial junction protein, however, its molecular function in endothelial cells is not known. In this study, we characterize the molecular role of JCAD (junctional cadherin 5 associated) in endothelial cells. Approach and Results- We show that JCAD knockdown in endothelial cells affects key phenotypes related to atherosclerosis including proliferation, migration, apoptosis, tube formation, and monocyte binding. We demonstrate that JCAD interacts with LATS2 (large tumor suppressor kinase 2) and negatively regulates Hippo signaling leading to increased activity of YAP (yes-associated protein), the transcriptional effector of the pathway. We also show by double siRNA knockdown that the phenotypes caused by JCAD knockdown require LATS2 and that JCAD is involved in transmission of RhoA-mediated signals into the Hippo pathway. In human tissues, we find that the CAD-associated lead variant, rs2487928, is associated with expression of JCAD in arteries, including atherosclerotic arteries. Gene co-expression analyses across disease-relevant tissues corroborate our phenotypic findings and support the link between JCAD and Hippo signaling. Conclusions- Our results show that JCAD negatively regulates Hippo signaling in endothelial cells and we suggest that JCAD contributes to atherosclerosis by mediating YAP activity and contributing to endothelial dysfunction.