Maps of Open Chromatin Guide the Functional Follow-Up of Genome-Wide Association Signals: Application to Hematological Traits

Abstract

Turning genetic discoveries identified in genome-wide association (GWA) studies into biological mechanisms is an important challenge in human genetics. Many GWA signals map outside exons, suggesting that the associated variants may lie within regulatory regions. We applied the formaldehyde-assisted isolation of regulatory elements (FAIRE) method in a megakaryocytic and an erythroblastoid cell line to map active regulatory elements at known loci associated with hematological quantitative traits, coronary artery disease, and myocardial infarction. We showed that the two cell types exhibit distinct patterns of open chromatin and that cell-specific open chromatin can guide the finding of functional variants. We identified an open chromatin region at chromosome 7q22.3 in megakaryocytes but not erythroblasts, which harbors the common non-coding sequence variant rs342293 known to be associated with platelet volume and function. Resequencing of this open chromatin region in 643 individuals provided strong evidence that rs342293 is the only putative causative variant in this region. We demonstrated that the C- and G-alleles differentially bind the transcription factor EVI1 affecting PIK3CG gene expression in platelets and macrophages. A protein–protein interaction network including up- and down-regulated genes in Pik3cg knockout mice indicated that PIK3CG is associated with gene pathways with an established role in platelet membrane biogenesis and thrombus formation. Thus, rs342293 is the functional common variant at this locus; to the best of our knowledge this is the first such variant to be elucidated among the known platelet quantitative trait loci (QTLs). Our data suggested a molecular mechanism by which a non-coding GWA index SNP modulates platelet phenotype.

Maps of open chromatin highlight cell type–restricted patterns of regulatory sequence variation at hematological trait loci

“…Further experiments have to be carried out to investigate whether such SNPs affect the platelet phenotype through alternative molecular mechanisms. Several examples of such suitable experimental strategies have recently been described in the literature (Pomerantz et al 2009;Tuupanen et al 2009;Gaulton et al 2010;Musunuru et al 2010;Harismendy et al 2011;Paul et al 2011).…”

“…We recently demonstrated that the formaldehyde-assisted isolation of regulatory elements (FAIRE) technique is a valuable tool in mapping nucleosome-depleted regions (NDRs) at selected genetic loci associated with hematological traits, and in prioritizing candidate variants for experimental validation (Paul et al 2011). Hematological traits, such as the count and volume of cells in peripheral blood and the hemoglobin content of erythrocytes, are under genetic control and vary extensively between individuals (Evans et al 1999;Garner et al 2000).…”

“…All samples were analyzed on the CyAn ADP 9-Color flow cytometer using the software Summit v4.3.02 (Beckman Coulter). Formaldehyde-assisted isolation of regulatory elements CHRF-288-11 cells were cultured according to the method previously described (Paul et al 2011). We used approximately 10 3 10 6 CHRF-288-11 cells for each FAIRE experiment.…”

“…Each FAIRE assay in primary human MKs, EBs, and MOs was performed with approximately 15 3 10 6 cells from two independent extractions. FAIRE was performed according to the method previously described (Paul et al 2011), except that cross-linked washed cell pellets were resuspended in 2 mL of lysis buffer (10 mM Tris [Thermo Fisher Scientific] …”

“…EMSAs using nuclear extracts from CHRF-288-11 cells were performed according to the method previously described (Paul et al 2011). Oligonucleotide probes were designed based on the genomic sequence surrounding the candidate SNPs.…”