<i>TCF21</i> and the environmental sensor aryl-hydrocarbon receptor cooperate to activate a pro-inflammatory gene expression program in coronary artery smooth muscle cells

<div><p>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 <i>TCF21</i> 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 <i>TCF21</i> expression in human coronary artery smooth muscle cells (HCASMC) revealed that TCF21 promotes expression of <i>AHR</i>, its heterodimerization partner <i>ARNT</i>, and cooperates with these factors to upregulate a number of inflammatory downstream disease related genes including <i>IL1A</i>, <i>MMP1</i>, and <i>CYP1A1</i>. TCF21 was shown to bind in <i>AHR</i>, <i>ARNT</i> 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.</p></div

Schematic representation of AHR-TCF21 interactions that may modulate the effect of environmental stimuli on the progression of inflammation and atherosclerotic plaque formation.

<p>Environmental toxins, including dioxin and tobacco, as well as endogenous activators such as ox-LDL activate the AHR pathway, leading to increased inflammatory burden in the plaque. TCF21 can further increase the burden by increasing AHR expression as well as interacting with AHR at its downstream genes.</p

TCF21 binds in the <i>CYP1A1</i> locus, and interacts with AHR to promote transcription.

<p>(a) UCSC Genome Browser view of TCF21 binding upstream of the <i>CYP1A1</i> gene in a region of open chromatin as defined by ATAC-seq with a high predicted affinity for AHR-ARNT binding. (b) TCF21 binding to <i>CYP1A1</i> upstream region is demonstrated with ChIP-qPCR. (c) Transfection studies with a composite TCF21-AHR binding site reporter plasmid shows additive effects on transcription with combined <i>TCF21</i> expression plasmid and treatment of the cells with dioxin. <i>TCF21</i>-DRE-pLuc is a luciferase construct with alternating TCF21 and AHR binding motifs subcloned proximal to the promoter. DRE, dioxin response element. (d) Deletion of the TCF21 binding motif in the reporter plasmid abrogates the effects of <i>TCF21</i> over-expression, verifying that TCF21 has a direct transcriptional effect in these experiments. (*p<0.05; **p<0.005).</p

Endogenous regulation of the AHR pathway in SMC, and in vivo expression of <i>AHR</i> in atherosclerotic vascular disease.

<p>(a) <i>IL1A</i> mRNA levels are increased by dioxin and ox-LDL, and co-treatment with αNF, an AHR pathway antagonist, reverses these effects. (b) <i>AHR</i> mRNA levels are elevated in ligated carotid arteries with ruptured plaques compared to stable plaques. (c) Laser capture microdissection shows increased <i>AHR</i> mRNA levels to be located in the plaque region of the atherosclerotic aortic root. (d) <i>AHR</i> mRNA levels are elevated in atherosclerotic human carotid arteries compared to normal arteries. (e) In vivo protein expression patterns in human carotid artery plaque tissues. AHR-TCF21 unique interactors from the BIOGRID protein-protein interaction database display clustering patterns in mass-spectrometry and high resolution isoelectric focusing proteomic data from human carotid artery plaques. Proteomic datasets were constructed from patients with asymptomatic (10) and symptomatic (10) carotid stenoses. Mass-spec and high resolution isoelectric focusing yielded 8–9000 recovered proteins. Hierarchical clustering revealed AHR to be located in a cluster of genes that include immune related genes, transcription factors, and cell cycle regulated genes, such as IRAK4, SP1, and XPO1, respectively.</p

AHR expression in HCASMC is mediated by an eQTL that modulates <i>TCF21</i> binding.

<p>(a) The downstream region of the <i>AHR</i> gene on chr7 (located on >20kb distance from the transcription end site) contains a strong open chromatin region and enhancer in HCASMC marked by ATAC-Seq, H3K27ac tracks and binding of AP1 transcription factors, JUN and JUND, as well as TCF21. Enhanced view shows that SNP rs10265174 directly overlaps the ATAC-Seq open chromatin, H3K27ac enhancer mark, JUN and JUND ChIP-Seq peaks, and is within a broad TCF21 ChIP-Seq peak. (b) The LocusZoom plot shows <i>AHR</i> gene eQTL distribution in HCASMC across 1Mb (16.9–17.9Mb) on chr7 encompassing <i>AHR</i> gene, with SNP rs10265174 being the top eQTL in the locus. (c) rs10265174 alters the position weight matrix scores for AP1 and TCF4 transcription factors (data from Haploreg). (d) TCF21 ChIP-qPCR shows enrichment at the <i>AHR</i> upstream genomic region compared to IgG control (p<0.05).</p

<i>TCF21</i> overexpression perturbs expression of chronic inflammatory genes and genes linked to vascular disease pathophysiology.

<p><b>(</b>a). Representation of GO categories that are significantly altered by the <i>TCF21</i> overexpression. Red text indicates immune related GO terms, black text indicates other categories. CTRL, control cell growth conditions; <i>TCF21</i>, <i>TCF21</i> over-expression. (b) Selected DAVID GO ontology analysis restricted to genes that show significant up- or down- regulation with <i>TCF21</i> overexpression in HCASMC. Upregulated GO clusters colored in blue, downregulated in red. (c) Co-expression modules of <i>TCF21</i> and <i>AHR-ARNT</i> show a high degree of connectivity. <i>TCF21</i> and <i>AHR</i> co-expression modules were defined using 4164 human microarray datasets through GeneFriends and visualized with Cytoscape. A few representative genes are identified by gene symbol. (d) Co-expression network of 77 CAD GWAS related genes was built using GeneFriends:Microarray, and visualized with Cytoscape. Transcription modules identify three primary clusters of CAD GWAS genes, with <i>TCF21</i> and <i>AHR</i> appearing in the same cluster. Genes <i>PDGFD</i>, <i>SMAD3</i> and <i>COL4A1</i> also appear in this cluster. A second cluster is composed primarily of lipid-related genes, such as APOA1, APOA5, APOB, LPA, etc.</p

Rotational phasing of PWM predicts steric interaction of TCF21 and AHR differentially regulates transcriptional programs.

<p>(a) Schematic representation of rotationally phased TCF21 and AHR spaced at n(10bp) distances along the DNA indicating direct interaction between proteins. (b) GO terms enriched in genes proximal to rotationally phased TCF12, employed here as surrogate for TCF21, and AHR-ARNT JASPAR matrices. (c) Schematic of rotationally unphased TCF21 and AHR proteins spaced at 5bp+n(10bp) distances indicating indirect interaction through an intermediary protein or a protein complex. (d) GO terms enriched in genes proximal to rotationally unphased TCF12 and AHR-ARNT JASPAR matrices.</p

ChIP-seq data show co-localization of TCF21, AHR and ARNT binding in loci containing genes related to inflammation and programmed cell death.

<p>(a) Venn diagram showing intersection of the TCF21, AHR and ARNT ChIP-seq peaks. Note that the shown number of overlapping AHR-ARNT-TCF21 sites is colored in light gray to indicate that it is a subgroup of the shown intersections of individual pairs. (b) GO terms related to the overlap of TCF21, AHR and ARNT ChIP-seq sites. GO terms were defined using 119 overlapping sites from a, and by identification of related genes with GREAT. Calcium signaling GO terms are noted to represent a broad array of cytokines and chemokines in addition to other types of molecules. (c) Overlap of AHR ChIP-seq binding sites and GWAS SNPs in a window of +/-2000bp. GWAS SNPs were obtained from the GWAS Catalog, parsed into categories and the binomial enrichment of overlap with the AHR ChIP-seq sites was calculated for each parsed category. Plotted are binomial fold change vs.–log binomial p-value and terms were colored according to the four major categories: brain, cancer, cardiovascular, chronic inflammatory. (d) Overlap of ARNT ChIP-seq binding sites and GWAS SNPs in a window of +/-2000bp. (e) Overlap of TCF21 ChIP-seq binding sites and GWAS SNPs in a window of +/-2000bp. (f) Overlap of AHR-ARNT-TCF21 ChIP-seq binding sites and GWAS. SNPs in a window of +/-2000bp.</p