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.

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Notes/Citation Information

Published in PLOS Genetics, v. 11, no. 4, article e1005061, p. 1-33.

© 2015 Reschen et al.

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

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Funding Information

This work was supported by the Wellcome Trust (097089/Z/11/Z), the Medical Research Council (G116/165) and the National Institute for Health Research Oxford Comprehensive Biomedical Research Centre Program. We thank the High Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics (Wellcome Trust grant reference 090532/Z/09/Z and MRC Hub grant G0900747 91070) for the generation of the sequencing data. This study was supported by grants from the NIH, American Heart Association and Department of Veterans Affairs and conducted in part using resources provided by the Lexington Veterans Affairs Medical Center. The recruitment of participants from the Oxford Biobank was supported by the NIHR Oxford Biomedical Research Centre and the National NIHR Bioresource. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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S1 Fig. Foam cell formation in human primary macrophages.

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S2 Fig. Genomic distribution of open chromatin sites in macrophages and foam cells.

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S3 Fig. Width of dynamic FAIRE sites.

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S4 Fig. Up-regulation of IL6R and PPAP2B in foam cells compared to macrophages as assessed by quantitative RT-PCR.

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S5 Fig. EMSA with cold probe competition assays.

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S6 Fig. EMSA with probes for the rs72664324 alleles.

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S1 Table. (A) List of differentially expressed genes (probe centric) between macrophages and foam cells using paired data (FDR adjusted p < 0.05). For some genes several different probes are reported. (B) List of differentially expressed genes (probe centric) without using pairing information (FDR adjusted p < 0.05). For some genes several different probes are reported.

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S2 Table. CAD loci and SNPs in high linkage disequilibrium.

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S3 Table. Genes within 50kb of CAD SNPs.

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S4 Table. Gene set enrichment analysis for genes within 50kb of CAD SNPs.

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S5 Table. Macrophage open chromatin sites identified by FAIRE-seq.

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S6 Table. Foam cell open chromatin sites identified by FAIRE-seq.

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S7 Table. Dynamic FAIRE-seq sites.

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S8 Table. Top 10 enriched transcription factor binding site motifs in dynamic open chromatin sites (top 3000 open dynamic chromatin sites, see also S9 Table).

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S9 Table. Complete output of transcription factor motif enrichment analysis in dynamic chromatin sites (top 3000 open chromatin sites, see also S8 Table).

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S10 Table. Dynamic H3K27ac sites.

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S11 Table. Macrophage super enhancer clusters.

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S12 Table. Foam cell super enhancer clusters.

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S13 Table. Complete output of transcription factor motif enrichment analysis in the subset of 1743 dynamic chromatin sites with dynamic enhancer status.

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S14 Table. Dynamic C/EBP beta binding sites.

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S15 Table. Transcription factor motif enrichment in dynamic C/EBP beta sites to confirm C/EBP beta motif enrichment (on top 3000 sites).

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S16 Table. Count of overlaps of CAD SNPs with chromatin and enhancer maps.

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S17 Table. Transcription factor motif affinities for rs72664324 alleles.

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