Abstract
BackgroundTo 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).ResultsWe 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.ConclusionsTogether, 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.
Highlights
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)
We have identified QTLs for binding of the CAD-associated transcription factor TCF21, chromatin accessibility, and chromosomal looping and investigated their relationship to one another and to eQTLs mapped in the same cell type, as well as their overlap with CAD-associated genetic variation
We obtained more than 400 M reads each for pooled TCF21 ChIPseq and ATACseq libraries and 800 M reads for the pooled Hi-C library
Summary
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). While there has been considerable success identifying loci in the human genome that are associated with a broad range of human diseases, including coronary artery disease (CAD) [1,2,3], most identified regions represent non-exonic regulatory sequence and are difficult to associate to a particular gene or functional annotation of the genome Toward this end, numerous studies have explored the genetics of gene expression and mapped expression quantitative trait locus variants to inform on which SNPs and which genes are causally related to disease in the associated loci [4,5,6,7]. QTLs for histone modification and chromatin accessibility and more recently chromosomal looping have been mapped [9,10,11,12] Such efforts have provided important information regarding the regulatory structure of the non-coding sequence in the genome and identified how such variation may regulate the risk for complex human disease. This pooling minimizes experimental variability between samples (both within and between experimental “batches”), considerably reducing the cost and effort of QTL mapping when compared to standard individual-level approaches [13]
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