Coronary artery disease (CAD) is the leading cause of mortality and morbidity, driven by both genetic and environmental risk factors. Meta-analyses of genome-wide association studies have identified >150 loci associated with CAD and myocardial infarction susceptibility in humans. A majority of these variants reside in non-coding regions and are co-inherited with hundreds of candidate regulatory variants, presenting a challenge to elucidate their functions. Herein, we use integrative genomic, epigenomic and transcriptomic profiling of perturbed human coronary artery smooth muscle cells and tissues to begin to identify causal regulatory variation and mechanisms responsible for CAD associations. Using these genome-wide maps, we prioritize 64 candidate variants and perform allele-specific binding and expression analyses at seven top candidate loci: 9p21.3, SMAD3, PDGFD, IL6R, BMP1, CCDC97/TGFB1 and LMOD1. We validate our findings in expression quantitative trait loci cohorts, which together reveal new links between CAD associations and regulatory function in the appropriate disease context.

Recent genome wide association studies have identified a number of genes that contribute to the risk for coronary heart disease. One such gene, TCF21, encodes a basic-helix-loop-helix transcription factor believed to serve a critical role in the development of epicardial progenitor cells that give rise to coronary artery smooth muscle cells (SMC) and cardiac fibroblasts. Using reporter gene and immunolocalization studies with mouse and human tissues we have found that vascular TCF21 expression in the adult is restricted primarily to adventitial cells associated with coronary arteries and also medial SMC in the proximal aorta of mouse. Genome wide RNA-Seq studies in human coronary artery SMC (HCASMC) with siRNA knockdown found a number of putative TCF21 downstream pathways identified by enrichment of terms related to CAD, including “vascular disease,” “disorder of artery,” and “occlusion of artery,” as well as disease-related cellular functions including “cellular movement” and “cellular growth and proliferation.” In vitro studies in HCASMC demonstrated that TCF21 expression promotes proliferation and migration and inhibits SMC lineage marker expression. Detailed in situ expression studies with reporter gene and lineage tracing revealed that vascular wall cells expressing Tcf21 before disease initiation migrate into vascular lesions of ApoE^-/- and Ldlr^-/- mice. While Tcf21 lineage traced cells are distributed throughout the early lesions, in mature lesions they contribute to the formation of a subcapsular layer of cells, and others become associated with the fibrous cap. The lineage traced fibrous cap cells activate expression of SMC markers and growth factor receptor genes. Taken together, these data suggest that TCF21 may have a role regulating the differentiation state of SMC precursor cells that migrate into vascular lesions and contribute to the fibrous cap and more broadly, in view of the association of this gene with human CAD, provide evidence that these processes may be a mechanism for CAD risk attributable to the vascular wall. Coronary artery disease (CAD) is responsible for the majority of deaths in the Western world, and is due in part to environmental and metabolic factors. However, half of the risk for developing heart disease is genetically predetermined. Genome-wide association studies in human populations have identified over 100 sites in the genome that appear to be associated with CAD, however, the mechanisms by which variation in these regions are responsible for predisposition to CAD remain largely unknown. We have begun to study a gene that contributes to CAD risk, the TCF21 gene. Through genomic studies we show that this gene is involved in processes related to alterations in vascular gene expression, and in particular those related to the smooth muscle cell biology. With cell culture models, we show that TCF21 regulates the differentiation state of this cell type, which is believed critical for vascular disease. Using mouse genetic models of atherosclerotic vascular disease we provide evidence that this gene is expressed in precursor cells that migrate into the disease lesions and contribute to the formation of the fibrous cap that is believed to stabilize these lesions and prevent heart attacks.

Both environmental factors and genetic loci have been associated with coronary artery disease (CAD), however gene-gene and gene-environment interactions that might identify molecular mechanisms of risk are not easily studied by human genetic approaches. We have previously identified the transcription factor TCF21 as the causal CAD gene at 6q23.2 and characterized its downstream transcriptional network that is enriched for CAD GWAS genes. Here we investigate the hypothesis that TCF21 interacts with a downstream target gene, the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor that mediates the cellular response to environmental contaminants, including dioxin and polycyclic aromatic hydrocarbons (e.g., tobacco smoke). Perturbation of TCF21 expression in human coronary artery smooth muscle cells (HCASMC) revealed that TCF21 promotes expression of AHR, its heterodimerization partner ARNT, and cooperates with these factors to upregulate a number of inflammatory downstream disease related genes including IL1A, MMP1, and CYP1A1. TCF21 was shown to bind in AHR, ARNT and downstream target gene loci, and co-localization was noted for AHR-ARNT and TCF21 binding sites genome-wide in regions of HCASMC open chromatin. These regions of co-localization were found to be enriched for GWAS signals associated with cardio-metabolic as well as chronic inflammatory disease phenotypes. Finally, we show that similar to TCF21, AHR gene expression is increased in atherosclerotic lesions in mice in vivo using laser capture microdissection, and AHR protein is localized in human carotid atherosclerotic lesions where it is associated with protein kinases with a critical role in innate immune response. These data suggest that TCF21 can cooperate with AHR to activate an inflammatory gene expression program that is exacerbated by environmental stimuli, and may contribute to the overall risk for CAD. Coronary heart disease is the leading cause of death in the world. Both genes and the environment are important risk factors for the progression of disease, however, how genes may modulate the harmful response to the disease promoting environment is unknown and difficult to study. Here, we show that a common heritable variation in the gene TCF21 may regulate coronary heart disease risk by regulating the response of downstream gene activation by the disease environment. We find that a well-known environmental sensor, aryl-hydrocarbon receptor (AHR), is regulated by TCF21 and also interacts with TCF21, resulting in regulation of pro-inflammatory gene expression in coronary artery smooth muscle cells. We further show that oxidized LDL, a well-known driver of atherosclerosis in the plaque can activate the AHR pathway. This work describes a heritable form of gene-environment interaction identified through genome wide association studies in coronary artery disease, and presents an opportunity to define causal gene-gene and gene-environment interactions.

Multiplex experimental assays coupled to computational predictions are being increasingly employed for the simultaneous analysis of many specimens at the genome scale, which quickly generates very large amounts of data. However, inferring valuable biological information from the comparisons of very large genomic datasets still represents an enormous challenge.

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. Coronary artery disease (CAD) is the worldwide leading cause of death. The majority of risk for CAD is genetic in nature, i.e., a feature of the genetic information that is transmitted to each individual from both parents, and primarily affects the disease processes in the blood vessel wall that regulate the disease molecular pathways. Modern genetic approaches have allowed mapping of the regions of the human genome that encode information that mediates this risk. The SMAD3 gene has been identified through these studies, a known master regulatory of other genes and molecular pathways, and we have investigated the functions of this gene that are important for disease risk. SMAD3 affects basic functions of a cellular component of the vessel wall, the smooth muscle cell (SMC), that is responsible for responding to vascular stresses to heal the lesions that are produced in conjunction with elevated lipids and other classic risk factors. Studies reported here show that SMAD3 actually inhibits the cellular processes that allow SMC to repair the vascular lesions, and its expression is promoted by the disease related variable sequences in the disease associated regions of the genome. SMAD3 is opposed by another CAD gene, TCF21, that functions to block the effects of SMAD3 expression, and these studies identify genetic mechanisms by which this is done. Thus, these studies identify an interactive pathway that directly contributes to disease risk, and the ability to block SMAD3 or promote TCF21 function could be exploited to inhibit vascular events such as myocardial infarction.

To functionally link coronary artery disease (CAD) causal genes identified by genome wide association studies (GWAS), and to investigate the cellular and molecular mechanisms of atherosclerosis, we have used chromatin immunoprecipitation sequencing (ChIP-Seq) with the CAD associated transcription factor TCF21 in human coronary artery smooth muscle cells (HCASMC). Analysis of identified TCF21 target genes for enrichment of molecular and cellular annotation terms identified processes relevant to CAD pathophysiology, including “growth factor binding,” “matrix interaction,” and “smooth muscle contraction.” We characterized the canonical binding sequence for TCF21 as CAGCTG, identified AP-1 binding sites in TCF21 peaks, and by conducting ChIP-Seq for JUN and JUND in HCASMC confirmed that there is significant overlap between TCF21 and AP-1 binding loci in this cell type. Expression quantitative trait variation mapped to target genes of TCF21 was significantly enriched among variants with low P-values in the GWAS analyses, suggesting a possible functional interaction between TCF21 binding and causal variants in other CAD disease loci. Separate enrichment analyses found over-representation of TCF21 target genes among CAD associated genes, and linkage disequilibrium between TCF21 peak variation and that found in GWAS loci, consistent with the hypothesis that TCF21 may affect disease risk through interaction with other disease associated loci. Interestingly, enrichment for TCF21 target genes was also found among other genome wide association phenotypes, including height and inflammatory bowel disease, suggesting a functional profile important for basic cellular processes in non-vascular tissues. Thus, data and analyses presented here suggest that study of GWAS transcription factors may be a highly useful approach to identifying disease gene interactions and thus pathways that may be relevant to complex disease etiology. While coronary artery disease (CAD) is due in part to environmental and metabolic factors, about half of the risk is genetically predetermined. Genome-wide association studies in human populations have identified approximately 150 sites in the genome that appear to be associated with CAD. The mechanisms by which mutations in these regions are responsible for predisposition to CAD remain largely unknown. To begin to explore how disease-specific gene sequences and disease gene function promotes pathology, we have mapped the loci and genes that are downstream of the transcription factor TCF21, which is strongly associated with CAD. By identifying genes that are regulated by TCF21 we have been able to link together multiple other CAD associated genes and begin to identify the critical molecular processes that mediate atherosclerosis in the blood vessel wall and contribute to the genesis of ischemic cardiovascular events.