Many obesity-associated SNPs strongly associate with DNA methylation changes at proximal promoters and enhancers

Abstract

Background: The mechanisms by which genetic variants, such as single nucleotide polymorphisms (SNPs), identified in genome-wide association studies act to influence body mass remain unknown for most of these SNPs, which continue to puzzle the scientific community. Recent evidence points to the epigenetic and chromatin states of the genome as having important roles.

Methods: We genotyped 355 healthy young individuals for 52 known obesity-associated SNPs and obtained DNA methylation levels in their blood using the Illumina 450 K BeadChip. Associations between alleles and methylation at proximal cytosine residues were tested using a linear model adjusted for age, sex, weight category, and a proxy for blood cell type counts. For replication in other tissues, we used two open-access datasets (skin fibroblasts, n = 62; four brain regions, n = 121-133) and an additional dataset in subcutaneous and visceral fat (n = 149).

Results: We found that alleles at 28 of these obesity-associated SNPs associate with methylation levels at 107 proximal CpG sites. Out of 107 CpG sites, 38 are located in gene promoters, including genes strongly implicated in obesity (MIR148A, BDNF, PTPMT1, NR1H3, MGAT1, SCGB3A1, HOXC12, PMAIP1, PSIP1, RPS10-NUDT3, RPS10, SKOR1, MAP2K5, SIX5, AGRN, IMMP1L, ELP4, ITIH4, SEMA3G, POMC, ADCY3, SSPN, LGR4, TUFM, MIR4721, SULT1A1, SULT1A2, APOBR, CLN3, SPNS1, SH2B1, ATXN2L, and IL27). Interestingly, the associated SNPs are in known eQTLs for some of these genes. We also found that the 107 CpGs are enriched in enhancers in peripheral blood mononuclear cells. Finally, our results indicate that some of these associations are not blood-specific as we successfully replicated four associations in skin fibroblasts.

Conclusions: Our results strongly suggest that many obesity-associated SNPs are associated with proximal gene regulation, which was reflected by association of obesity risk allele genotypes with differential DNA methylation. This study highlights the importance of DNA methylation and other chromatin marks as a way to understand the molecular basis of genetic variants associated with human diseases and traits.

Fig. 1

Raw p value as a function of distance between SNP and CpG. Each point represents an associated SNP–CpG pair (107 pairs). Most associated SNP–CpG pairs are close to each other, as illustrated by the box plot of the distance between SNP and CpG (bottom of the plot). The closer the associated SNP–CpG pairs are, the lower the p value

Fig. 2

Associations between genotypes at rs713586 and methylation at cg01884057. Distribution of methylation levels at cg01884057 is displayed for individuals carrying zero (C/C), one (T/C), or two (T/T) risk alleles at rs713586

Fig. 3

Distribution of associated versus all tested CpGs in promoters, gene bodies, and intergenic regions. A CpG was classified as “promoter” if located within 1500 bp of the TSS of a gene, and as “ambiguous” if it was both in a promoter and within a gene body. Associated CpGs (top) were underrepresented in promoters, and overrepresented in intergenic regions (Fisher’s exact test). *q value < 0.05

Fig. 4

Distribution of associated versus all tested CpGs in seven chromatin states in 11 tissues. Associated CpGs were overrepresented in enhancers in PBMCs (Fisher’s exact test). **q value < 0.01

Fig. 5

Genomic context of the CpGs associated with rs2444217. Genomic positions of RefSeq genes and rs2444217 are displayed in the top panel. Within the two vertical red dotted lines, the LD r² > 0.8. The positions of the tested CpGs are displayed. Long-range interactions as defined by ChIA-PET libraries from five cell lines using chromatin immunoprecipitation with antibodies targeting three transcription factors (Additional file 5) are displayed as arcs. For clarity of visualization, we chose to display only the long-range interactions of genomic regions containing associated CpGs. Two interacting genomic regions are represented by an arc that links them, and the thickness of the arc line is proportional to the strength of this interaction. The color of the arc corresponds to the target transcription factor and the shade of the color corresponds to the cell line: red for RNA polymerase II, blue for ERα, and green for CTCF. In the bottom panel, chromatin states in 11 tissues are displayed. Chromatin states were obtained using chromHMM prediction using data on seven histone marks (see “Methods”). The color of each band corresponds to a particular state. AN adipose nuclei, BrainAC brain anterior caudate, BrainAG brain angular gyrus, BrainCG brain cingulate gyrus, BrainHIPPO brain hippocampus, BrainITL brain inferior temporal lobe, BrainSN brain substantia nigra, PBMC peripheral blood mononuclear primary cells, PI pancreatic islets, SM skeletal muscle

Fig. 6

Genomic context of the CpGs associated with rs3934834. a The entire investigated region. b Zoom on the region surrounding rs3934834. Genomic positions of RefSeq genes and rs3934834 are displayed in the top panel. Within the two vertical red dotted lines, the LD r² > 0.8. The positions of the tested CpGs are displayed. Long-range interactions as defined by ChIA-PET libraries from five cell lines using chromatin immunoprecipitation with antibodies targeting three transcription factors (Additional file 5) are displayed as arcs. For clarity of visualization, we chose to display only the long-range interactions of genomic regions containing associated CpGs. Two interacting genomic regions are represented by an arc that links them, and the thickness of the arc line is proportional to the strength of this interaction. The color of the arc corresponds to the target transcription factor and the shade of the color corresponds to the cell line: red for RNA polymerase II, blue for ERα, and green for CTCF. In the bottom panel, chromatin states in 11 tissues are displayed. Chromatin states were obtained using chromHMM prediction using data on seven histone marks (see “Methods”). The color of each band corresponds to a particular state. AN adipose nuclei, BrainAC brain anterior caudate, BrainAG brain angular gyrus, BrainCG brain cingulate gyrus, BrainHIPPO brain hippocampus, BrainITL brain inferior temporal lobe, BrainSN brain substantia nigra, PBMC peripheral blood mononuclear primary cells, PI pancreatic islets, SM skeletal muscle

Fig. 7

Distribution of the number of long-range interactions for associated versus all tested CpGs. For each associated and tested CpG, we counted the number of genomic regions containing the CpG that interacted with another genomic region. For clarity and because the number of interactions was skewed, we chose to display the log₁₀(number of interactions)