Lipid-Induced Epigenomic Changes in Human Macrophages Identify a Coronary Artery Disease-Associated Variant that Regulates PPAP2B Expression through Altered C/EBP-Beta Binding

摘要

Genome-wide association studies (GWAS) have identified over 40 loci that affect risk of coronary artery disease (CAD) and the causal mechanisms at the majority of loci are unknown. Recent studies have suggested that many causal GWAS variants influence disease through altered transcriptional regulation in disease-relevant cell types. We explored changes in transcriptional regulation during a key pathophysiological event in CAD, the environmental lipid-induced transformation of macrophages to lipid-laden foam cells. We used a combination of open chromatin mapping with formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) and enhancer and transcription factor mapping using chromatin immuno-precipitation (ChIP-seq) in primary human macrophages before and after exposure to atherogenic oxidized low-density lipoprotein (oxLDL), with resultant foam cell formation. OxLDL-induced foam cell formation was associated with changes in a subset of open chromatin and active enhancer sites that strongly correlated with expression changes of nearby genes. OxLDL-regulated enhancers were enriched for several transcription factors including C/EBP-beta, which has no previously documented role in foam cell formation. OxLDL exposure up-regulated C/EBP-beta expression and increased genomic binding events, most prominently around genes involved in inflammatory response pathways. Variants at CAD-associated loci were significantly and specifically enriched in the subset of chromatin sites altered by oxLDL exposure, including rs72664324 in an oxLDL-induced enhancer at the PPAP2B locus. OxLDL increased C/EBP beta binding to this site and C/EBP beta binding and enhancer activity were stronger with the protective A allele of rs72664324. In addition, expression of the PPAP2B protein product LPP3 was present in foam cells in human atherosclerotic plaques and oxLDL exposure up-regulated LPP3 in macrophages resulting in increased degradation of pro-inflammatory mediators. Our results demonstrate a genetic mechanism contributing to CAD risk at the PPAP2B locus and highlight the value of studying epigenetic changes in disease processes involving pathogenic environmental stimuli. Author Summary Coronary artery disease is a complex disease where over 40 genomic loci contributing to genetic risk have been identified. However, identifying the precise variants, genomic elements and genes that mediate this risk at each locus has proved challenging. We hypothesized that some genetic risk variants may influence a key step in development of coronary artery disease, which occurs when macrophages encounter environmentally-derived lipid. These cells take up lipid and accumulate in atherosclerotic plaques in the walls of blood vessels where they contribute to the inflammatory atherosclerotic disease process. Therefore, we studied the effects of this lipid exposure on the genomic activity of these cells. Environmental lipid exposure triggered changes in transcriptional regulation and gene expression. Variants at coronary artery disease risk loci were enriched for genomic regions altered by lipid exposure. We studied one such risk variant rs72664324 in detail and found that it altered binding of the C/EBP-beta transcription factor and altered expression of the PPAP2B gene. PPAP2B encodes an enzyme that degrades pro-inflammatory substances. Our study demonstrates a hitherto unknown genetic mechanism underlying atherosclerotic heart disease and demonstrates the value of studying changes in transcriptional regulation in key disease processes involving environmental influences.