<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

Characterization of TCF21 Downstream Target Regions Identifies a Transcriptional Network Linking Multiple Independent Coronary Artery Disease Loci

<div><p>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 <i>P</i>-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.</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

Two TCF21 antibodies show overlapping patterns of TCF21 chromosomal binding.

<p>A) Two replicate experiments with Antibody 2 (Ab2) identified 4828 binding sites. All but 72 of these peaks were also identified by similar replicate experiments with Ab1, which recognized an additional 5695 peaks. B) In addition to analyzing data for each of the two antibody ChIP-Seq datasets, we have intersected those identified with both Ab1 and Ab2 (Ab_Shared), with the smaller of the peaks being employed if there was complete overlap of one versus the other, and the region of overlap used if the two peaks shared incomplete overlap. C) High throughput next-generation sequencing reads were aligned to the genome, peaks present in both biological replicates of each of the two antibody precipitations were identified by IDR, and visualized on the UCSC browser [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005202#pgen.1005202.ref008" target="_blank">8</a>]. In addition to the TCF21 ChIP-Seq data, also shown are ATAC-Seq data for HCASMC and DNse I hypersensitivity data obtained with human aortic smooth muscle cells (HAoSMC DHS) indicating that TCF21 peaks localize to regions of active chromatin conformation.</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

Analysis of peak sequences identifies TCF21 binding motifs as well as motifs for JUN related and other transcription factors that likely cooperate with TCF21.

<p>A) HOMER analysis of known TF motif enrichment within TCF21 peaks in the Ab_Shared data revealed several distinct motif families. The bHLH motif CAGCTG is identical to that attributed to TCF12, a known heterodimer partner of TCF21 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005202#pgen.1005202.ref026" target="_blank">26</a>], and a second highly enriched bHLH motif CATCTG is attributed to nervous system TF OLIG2, suggesting that TCF21 can bind either of these two motifs. The bZIP motif TGA(G/C)TCA most closely resembles the binding sequence for TFs within the AP-1/ATF super family. Other motifs found to be enriched in the TCF21 peaks include those mediating binding of TEAD, CEBP, and ATF transcription factor family members, and an unknown element identified by ChIP-Seq with NANOG in human embryonic stem cells (ESC). B) Distribution (density) plots for top 7 motifs from panel A: TCF12, OLIG2, AP-1, unknown-NANOG (left), and CEBP, TEAD4, ATF1 (right). C) TCF21 binds in close proximity to JUN and JUND in a number of loci, including developmentally important <i>WT1</i> and <i>PDGFRB</i> loci.</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

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

Analysis of linkage disequilibrium between SNPs in GWAS loci of select phenotypes and TCF21 peak regions for chosen phenotypes.

<p>*GWAS catalog genes were supplemented with additional CAD genes identified from CARDIoGRAM+C4D, which are not currently included in the GWAS catalog. Traits with bold text are those significant at P<0.05 in both the GWAS gene enrichment analysis shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005202#pgen.1005202.t004" target="_blank">Table 4</a> and the current analysis.</p><p>Analysis of linkage disequilibrium between SNPs in GWAS loci of select phenotypes and TCF21 peak regions for chosen phenotypes.</p

GREAT pathway terms from analysis of TCF21 Ab_Shared peaks.

<p>GREAT pathway terms from analysis of TCF21 Ab_Shared peaks.</p