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

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

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

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

Enrichment of TCF21 target genes from Ab_Shared among GWAS candidate trait genes using all GWAS genes as background and a permutation strategy to correct for the differences in the numbers of GWAS genes between traits.

<p>*GWAS catalog genes were supplemented with additional CAD genes identified from CARDIoGRAM+C4D, these genes are not currently included in the GWAS catalog.</p><p>Enrichment of TCF21 target genes from Ab_Shared among GWAS candidate trait genes using all GWAS genes as background and a permutation strategy to correct for the differences in the numbers of GWAS genes between traits.</p

A CAD gene network identified among TCF21 target genes.

<p>Genes in CAD-associated loci that have TCF21 peaks identified by ChIP-Seq were assembled into a network, with incorporation of additional non-associated genes to promote the connectivity shown here. This transcriptional network was created by STRING [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005202#pgen.1005202.ref035" target="_blank">35</a>]. Many additional CAD genes downstream of TCF21 were not incorporated into the network, due to an overall lack of functional annotation for these loci.</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

Integrative functional genomics identifies regulatory mechanisms at coronary artery disease loci

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 4150 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

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