Functional genomics and assays of regulatory activity detect mechanisms at loci for lipid traits and coronary artery disease

Abstract

Many genome-wide association studies (GWAS) have identified signals located in non-coding regions, and an increasing number of functional genomics annotations of regulatory elements and assays of regulatory activity have been used to investigate mechanisms. Genome-wide datasets that characterize chromatin structure help detect potential regulatory elements. Assays to experimentally assess candidate variants include transcriptional reporter assays, and recently, massively parallel reporter assays (MPRAs). Additionally, the effect of candidate regulatory elements and variants on gene expression and function can be evaluated using genomic editing with the CRISPR-Cas9 technology. We highlight some recent studies that employed these strategies to identify variant effects and elucidate molecular and/or biological mechanisms at GWAS loci for lipid traits and coronary artery disease.

Figure 1. Recent strategies for identifying regulatory variants and elements using functional genomics assays

DNA segments can be cloned into a vector containing a reporter gene, one at a time or in a library. Reporter activity is then measured and compared to the reporter activity of a control vector or input DNA. These assays can be used to identify variants that show allelic differences in transcriptional activity. Candidate regulatory variants and their effect on gene expression and function can be further investigated by genomic editing methods. The DNA segment containing the candidate regulatory variant(s) can be targeted using CRISPR-Cas9 methods and a guide RNA to generate double-stranded breaks. Insertions or deletions can be generated by non-homologous end joining, or allelic substitutions can be generated by homology-directed repair with the addition of a donor DNA template. The effects of these edits can be tested by evaluating gene expression levels, protein levels, and biological function.

Figure 2. Chromatin regulatory annotations predict rs10872142 is located within an enhancer

, rs10872142 is strongly linked with LDL-C-associated lead GWAS variant rs11153594 and was reported as a regulatory variant associated with FRK gene expression in liver [37]. H3K4me1 ChIP-seq peaks are shown for adipose-derived mesenchymal stem cells and primary adipose nuclei (top green tracks) from the NIH Epigenomics Roadmap Consortium's Human Epigenome Atlas [10]. H3K4me1, H3K4me2, or H3K4me3 represent mono-, di- or tri-methylation of lysine 4 of histone H3, and are modifications commonly observed in regulatory enhancer or promoter regions [18,19]. ENCODE H3K4me1,2,3 ChIP-seq, DNase, and FAIRE peaks are shown for HepG2 hepatocellular carcinoma cells (purple tracks), and H3K4me1 ChIP-seq and DNase peaks are shown for human adult liver and hepatocytes (black tracks) [9]. The DNase and FAIRE peaks represent open, accessible chromatin regions devoid of histones, and the peaks corresponding to H3K4me1, H3K4me2 and H3K4me3 marks on the adjacent histones are commonly observed at regulatory enhancer and/or promoter regions. The multiple tracks provide greater evidence that the variant is located within a regulatory region.