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Background: To investigate the epigenetic and transcriptional mechanisms of coronary artery disease (CAD) risk, as well as the functional regulation of chromatin structure and function, we create a catalog of genetic variants associated with three stages of transcriptional cis-regulation in primary human coronary artery vascular smooth muscle cells (HCASMCs)., Results: We use a pooling approach with HCASMC lines to map regulatory variants that mediate binding of the CAD-associated transcription factor TCF21 with ChIPseq studies (bQTLs), variants that regulate chromatin accessibility with ATACseq studies (caQTLs), and chromosomal looping with Hi-C methods (clQTLs). We examine the overlap of these QTLs and their relationship to smooth muscle-specific genes and transcription factors. Further, we use multiple analyses to show that these QTLs are highly associated with CAD GWAS loci and correlate to lead SNPs where they show allelic effects. By utilizing genome editing, we verify that identified functional variants can regulate both chromatin accessibility and chromosomal looping, providing new insights into functional mechanisms regulating chromatin state and chromosomal structure. Finally, we directly link the disease-associated TGFB1-SMAD3 pathway to the CAD-associated FN1 gene through a response QTL that modulates both chromatin accessibility and chromosomal looping., Conclusions: Together, these studies represent the most thorough mapping of multiple QTL types in a highly disease-relevant primary cultured cell type and provide novel insights into their functional overlap and mechanisms that underlie these genomic features and their relationship to disease risk.

Background: Genome-wide association studies have identified over 160 loci that are associated with coronary artery disease. As with other complex human diseases, risk in coronary disease loci is determined primarily by altered expression of the causal gene, due to variation in binding of transcription factors and chromatin-modifying proteins that directly regulate the transcriptional apparatus. We have previously identified a coronary disease network downstream of the disease-associated transcription factor TCF21, and in work reported here extends these studies to investigate the mechanisms by which it interacts with the AP-1 transcription complex to regulate local epigenetic effects in these downstream coronary disease loci., Methods: Genomic studies, including chromatin immunoprecipitation sequencing, RNA sequencing, and protein-protein interaction studies, were performed in human coronary artery smooth muscle cells., Results: We show here that TCF21 and JUN regulate expression of two presumptive causal coronary disease genes, SMAD3 and CDKN2B-AS1, in part by interactions with histone deacetylases and acetyltransferases. Genome-wide TCF21 and JUN binding is jointly localized and particularly enriched in coronary disease loci where they broadly modulate H3K27Ac and chromatin state changes linked to disease-related processes in vascular cells. Heterozygosity at coronary disease causal variation, or genome editing of these variants, is associated with decreased binding of both JUN and TCF21 and loss of expression in cis, supporting a transcriptional mechanism for disease risk., Conclusions: These data show that the known chromatin remodeling and pioneer functions of AP-1 are a pervasive aspect of epigenetic control of transcription, and thus, the risk in coronary disease-associated loci, and that interaction of AP-1 with TCF21 to control epigenetic features, contributes to the genetic risk in loci where they co-localize.

Although numerous genetic loci have been associated with coronary artery disease (CAD) with genome wide association studies, efforts are needed to identify the causal genes in these loci and link them into fundamental signaling pathways. Recent studies have investigated the disease mechanism of CAD associated gene SMAD3, a central transcription factor (TF) in the TGFβ pathway, investigating its role in smooth muscle biology. In vitro studies in human coronary artery smooth muscle cells (HCASMC) revealed that SMAD3 modulates cellular phenotype, promoting expression of differentiation marker genes while inhibiting proliferation. RNA sequencing and chromatin immunoprecipitation sequencing studies in HCASMC identified downstream genes that reside in pathways which mediate vascular development and atherosclerosis processes in this cell type. HCASMC phenotype, and gene expression patterns promoted by SMAD3 were noted to have opposing direction of effect compared to another CAD associated TF, TCF21. At sites of SMAD3 and TCF21 colocalization on DNA, SMAD3 binding was inversely correlated with TCF21 binding, due in part to TCF21 locally blocking chromatin accessibility at the SMAD3 binding site. Further, TCF21 was able to directly inhibit SMAD3 activation of gene expression in transfection reporter gene studies. In contrast to TCF21 which is protective toward CAD, SMAD3 expression in HCASMC was shown to be directly correlated with disease risk. We propose that the pro-differentiation action of SMAD3 inhibits dedifferentiation that is required for HCASMC to expand and stabilize disease plaque as they respond to vascular stresses, counteracting the protective dedifferentiating activity of TCF21 and promoting disease risk., Competing Interests: The authors have declared that no competing interests exist.