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Lipid-induced epigenomic changes in human macrophages identify a coronary artery disease-associated variant that regulates PPAP2B Expression through Altered C/EBP-beta binding.

Reschen ME, Gaulton KJ, Lin D, Soilleux EJ, Morris AJ, Smyth SS, O'Callaghan CA - PLoS Genet. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

An intronic SNP at the PPAP2B locus regulates enhancer activity and oxLDL-induced expression of PPAP2B.(A) Chromatin profile at the PPAP2B locus where rs72664324 is in a dynamic chromatin, enhancer and C/EBP beta site (red box). The region is part of an oxLDL-induced super enhancer. (B) Comparison of the human rs72664324 alleles and the corresponding mouse sequence with the CEBP motif aligned above. (C) EMSA demonstrating that only the A allele at rs72664324 binds nuclear protein, which is shown to be C/EBP beta by a super-shift in the presence of anti-C/EBP beta antibody. Also, only a cold probe with the A allele competes off binding to a C/EBP consensus probe. (D) Luciferase reporter assays in primary human macrophages and foam cells with a reporter element containing rs72664324 with either allele (* p < 0.05, ** p < 0.005). (E) Effect of C/EBP beta overexpression on luciferase reporter activity with either the A or G allele relative to empty vector (* p < 0.05). (F) Induction of PPAP2B expression by oxLDL in primary human macrophages from individuals with allele G or at least one copy of the A allele (long lines indicate mean, short lines indicate median, Mann-Whitney U test, p = 0.0013, GG n = 10, GA n = 2, AA n = 6).
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pgen.1005061.g008: An intronic SNP at the PPAP2B locus regulates enhancer activity and oxLDL-induced expression of PPAP2B.(A) Chromatin profile at the PPAP2B locus where rs72664324 is in a dynamic chromatin, enhancer and C/EBP beta site (red box). The region is part of an oxLDL-induced super enhancer. (B) Comparison of the human rs72664324 alleles and the corresponding mouse sequence with the CEBP motif aligned above. (C) EMSA demonstrating that only the A allele at rs72664324 binds nuclear protein, which is shown to be C/EBP beta by a super-shift in the presence of anti-C/EBP beta antibody. Also, only a cold probe with the A allele competes off binding to a C/EBP consensus probe. (D) Luciferase reporter assays in primary human macrophages and foam cells with a reporter element containing rs72664324 with either allele (* p < 0.05, ** p < 0.005). (E) Effect of C/EBP beta overexpression on luciferase reporter activity with either the A or G allele relative to empty vector (* p < 0.05). (F) Induction of PPAP2B expression by oxLDL in primary human macrophages from individuals with allele G or at least one copy of the A allele (long lines indicate mean, short lines indicate median, Mann-Whitney U test, p = 0.0013, GG n = 10, GA n = 2, AA n = 6).

Mentions: We sought to identify and characterize candidate causal CAD-associated variants at the PPAP2B locus. A single CAD-associated variant rs72664324 overlapped a dynamic chromatin and enhancer site, which in turn lies in an oxLDL-induced ‘super-enhancer’ cluster (Fig. 8A).


Lipid-induced epigenomic changes in human macrophages identify a coronary artery disease-associated variant that regulates PPAP2B Expression through Altered C/EBP-beta binding.

Reschen ME, Gaulton KJ, Lin D, Soilleux EJ, Morris AJ, Smyth SS, O'Callaghan CA - PLoS Genet. (2015)

An intronic SNP at the PPAP2B locus regulates enhancer activity and oxLDL-induced expression of PPAP2B.(A) Chromatin profile at the PPAP2B locus where rs72664324 is in a dynamic chromatin, enhancer and C/EBP beta site (red box). The region is part of an oxLDL-induced super enhancer. (B) Comparison of the human rs72664324 alleles and the corresponding mouse sequence with the CEBP motif aligned above. (C) EMSA demonstrating that only the A allele at rs72664324 binds nuclear protein, which is shown to be C/EBP beta by a super-shift in the presence of anti-C/EBP beta antibody. Also, only a cold probe with the A allele competes off binding to a C/EBP consensus probe. (D) Luciferase reporter assays in primary human macrophages and foam cells with a reporter element containing rs72664324 with either allele (* p < 0.05, ** p < 0.005). (E) Effect of C/EBP beta overexpression on luciferase reporter activity with either the A or G allele relative to empty vector (* p < 0.05). (F) Induction of PPAP2B expression by oxLDL in primary human macrophages from individuals with allele G or at least one copy of the A allele (long lines indicate mean, short lines indicate median, Mann-Whitney U test, p = 0.0013, GG n = 10, GA n = 2, AA n = 6).
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Related In: Results  -  Collection

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pgen.1005061.g008: An intronic SNP at the PPAP2B locus regulates enhancer activity and oxLDL-induced expression of PPAP2B.(A) Chromatin profile at the PPAP2B locus where rs72664324 is in a dynamic chromatin, enhancer and C/EBP beta site (red box). The region is part of an oxLDL-induced super enhancer. (B) Comparison of the human rs72664324 alleles and the corresponding mouse sequence with the CEBP motif aligned above. (C) EMSA demonstrating that only the A allele at rs72664324 binds nuclear protein, which is shown to be C/EBP beta by a super-shift in the presence of anti-C/EBP beta antibody. Also, only a cold probe with the A allele competes off binding to a C/EBP consensus probe. (D) Luciferase reporter assays in primary human macrophages and foam cells with a reporter element containing rs72664324 with either allele (* p < 0.05, ** p < 0.005). (E) Effect of C/EBP beta overexpression on luciferase reporter activity with either the A or G allele relative to empty vector (* p < 0.05). (F) Induction of PPAP2B expression by oxLDL in primary human macrophages from individuals with allele G or at least one copy of the A allele (long lines indicate mean, short lines indicate median, Mann-Whitney U test, p = 0.0013, GG n = 10, GA n = 2, AA n = 6).
Mentions: We sought to identify and characterize candidate causal CAD-associated variants at the PPAP2B locus. A single CAD-associated variant rs72664324 overlapped a dynamic chromatin and enhancer site, which in turn lies in an oxLDL-induced ‘super-enhancer’ cluster (Fig. 8A).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus