Integrative genomic analyses in adipocytes implicate DNA methylation in human obesity and diabetes

Abstract

DNA methylation variations are prevalent in human obesity but evidence of a causative role in disease pathogenesis is limited. Here, we combine epigenome-wide association and integrative genomics to investigate the impact of adipocyte DNA methylation variations in human obesity. We discover extensive DNA methylation changes that are robustly associated with obesity (N = 190 samples, 691 loci in subcutaneous and 173 loci in visceral adipocytes, P < 1 × 10-7). We connect obesity-associated methylation variations to transcriptomic changes at >500 target genes, and identify putative methylation-transcription factor interactions. Through Mendelian Randomisation, we infer causal effects of methylation on obesity and obesity-induced metabolic disturbances at 59 independent loci. Targeted methylation sequencing, CRISPR-activation and gene silencing in adipocytes, further identifies regional methylation variations, underlying regulatory elements and novel cellular metabolic effects. Our results indicate DNA methylation is an important determinant of human obesity and its metabolic complications, and reveal mechanisms through which altered methylation may impact adipocyte functions.

Fig. 1. DNA methylation sites associated with extreme human obesity in subcutaneous and visceral adipocytes.

a Genome-wide associations between 5mC and extreme obesity in subcutaneous and visceral adipocytes (N = 401,595 sites); -log10 pvalue in combined discovery and replication samples ordered by autosomal chromosome; threshold line epigenome-wide significance (EWS, P < 1 × 10^–7). b 5mC differences between obese cases and controls relative to mean 5mC levels at N = 691 subcutaneous and N = 173 visceral sentinel methylation sites (%-methylation). c Comparisons of association models without and with adjustment for potential confounding variables to evaluate the effects of genetic variations and potential contaminating cell genes on 5mC-obesity relationships. Top panels: adjustment for cis-SNPs associated with each sentinel 5mC site (FDR < 0.01); effect size (beta) and -log10 pvalue in the combined discovery and replication cohorts; solid threshold EWS; dashed threshold Bonferroni corrected pvalue (0.05/N sentinels); G genetic effects; E non-genetic effects. Bottom panels: adjustment for principal components (PC1-5) derived from expression of 12 potential contaminating cell genes; effect size (beta) and -log10 pvalue in the replication cohort. d Cross tissue effects. Methylation-obesity association pvalue in subcutaneous and visceral adipocytes (combined discovery and replication samples); solid threshold EWS; dashed threshold Bonferroni corrected pvalue (0.05/N sentinels). All genome-wide association analyses were carried out separately in the discovery and replication cohorts using linear regression, and combined by inverse variance weighted meta-analysis. e Genomic annotation of obesity-associated DNA methylation sites. Numbered by fold change (observed compared to mean expected) and coloured by enrichment (red) or depletion (blue) -log10 pvalue (Fishers Exact Test, two-sided).

Fig. 2. DNA methylation-target gene associations in human adipocytes.

a–c Locus plots of sentinel 5mC sites (diamond) and their predicted target effector genes (dark grey). a Methylation at cg01558212 in the SATB2 promoter was associated with SATB2 and SATB2-AS1 gene transcription (subcutaneous). b Methylation at two sites, cg11307296 and cg13390388, within distinct functional loops in human adipocyte promoter capture HiC connectivity maps, was associated with transcription of the adipocyte browning/beigeing gene EBF2 (subcutaneous). c Methylation at cg03779326 was associated with transcription of RPN1 but not other putative target genes within a shared human adipocyte TAD (visceral). Presented as %-difference in methylation between obese cases and controls, annotated by UCSC CpG island (CGI) and Roadmap adipose (E063) and adipocyte (E025) chromatin states. d Frequency of sentinel methylation-expression associations at FDR < 0.01 according to target gene assignment method. Genic: sentinel in promoter, 5/3ʹUTR or exon. Functional: intronic/intergenic sentinel sharing functional interaction with distal target gene. TAD: intronic/intergenic sentinel and distal target gene(s) within shared human adipocyte topologically associated domain. Adi C-HiC: human adipocyte promoter capture Hi-C interaction. Other C-HiC: promoter capture Hi-C interaction in another human tissue. eRNA coexprN: co-expression of distal eRNA and proximal promoter RNA. eQTLs: Association of distal SNP with proximal promoter expression. TF coexprN: TF binding in distal site (ChIP-seq) and TF-target gene co-expression. 1 to >5 assocN: Number of sentinel-target gene associations in shared TAD. e Subcutaneous sentinel-target gene associations at FDR < 0.01 grouped by target gene annotation method, coloured by adipocyte chromatin state (Roadmap E025). Left panel: distance to TSS and -log10 pvalue according to direction of effect, and sentinel density distribution. Right panel: frequencies of observed associations compared to the null background (sentinel-gene associations at FDR > 0.01). Fold change: log2 fold change in gene expression for each unit change in methylation. f Enriched pathways and genesets at P < 0.001 (Empirical, one-sided) based on the nearest cis−gene to each 5mC sentinel in subcutaneous and visceral adipocytes. Bar represents fold change of observed compared to mean expected frequency, number is the observed gene counts, in each pathway/geneset. All methylation-expression analyses were carried out using mixed-effects linear regression in combined adipocyte samples.

Fig. 3. Interactions between DNA methylation and transcription factors in human subcutaneous adipocytes.

a Enrichment of extreme obesity-associated DNA methylation sentinels in 7 transcription factor binding motifs (subcutaneous sentinels). Left panel: the predicted DNA sequence corresponding to each enriched motif, based on observed nucleotide frequencies (Homer). Motifs 2 and 4 both contained CG sites within their predicted DNA binding sequence. Centre panel: heatmap of -log10 pvalue for enrichment of: i. hypo- (lower in obesity); and ii. hyper-methylated subcutaneous sentinels (relative to permuted background, hypergeometric test, one-sided). Right panel: bar plot of the number of subcutaneous sentinels mapping to each motif. b Human adipocyte Roadmap chromatin state annotation of Motifs 1, 4 and 5; bar plots of observed over expected ratio in selected roadmap states, coloured by observed counts (number of sentinel-motif pairs). Motif 1 was over-represented in enhancers, Motif 4 in active TSS, and Motif 5 in enhancers and active TSS. c Density/ridge plots of pairwise correlation between TF expression and methylation level at each of its corresponding sentinels in subcutaneous adipocytes, split by Motif and ranked by mean correlation. d Distribution of genomic CG sites in the ±150-bp regions flanking Motifs 1, 2 and 4, centred on the motif (coloured in orange). Genomic CG sites were enriched at Motif 4 (peak), and depleted at Motif 1 (trough), relative to the flanking DNA sequences. A peak of genomic CGs was also observed immediately upstream of Motif 2 though other genomic CG peaks were present in its flanking DNA sequences. e Relationships between expression of 4 TFs predicted to bind at Motif 4 (ELK1, ELK3, ELF1, ELF4) and expression of the assigned target genes of each methylation sentinel corresponding to Motif 4. Presented as association beta without (Effect) and with (Effect + Sentinel) adjustment for sentinel DNA methylation level (combined adipocyte samples), with regression line and 95% confidence intervals. Adjustment for sentinel DNA methylation levels systematically influenced the associations between the ELK1, ELK3 and ELF4 TFs and their target genes, but not the ELF1 TF.

Fig. 4. Causal inference analyses.

a Mendelian Randomisation analysis using genetic variants as instrumental variables to evaluate cause-consequence relationships between DNA methylation sentinels and human obesity phenotypes. Required evidence: robust association between: i. the instrumental genetic variant and the methylation exposure; and ii. the instrumental genetic variant and the outcome phenotype. Assumptions: i. the instrument only influences the outcome through the exposure not through any other pathway (horizontal pleiotropy); and ii. the instrument is not associated with confounders. b Forest plots of the effect sizes of subcutaneous and visceral adipocyte sentinels causally associated with human obesity phenotypes through two sample MR in adipocytes (FDR < 0.01 in both MR causal and Steiger directionality tests). Centre values mark effect size estimates (MR beta) and error bars show the 95% confidence intervals. Sentinels causally associated with both adiposity and its metabolic consequences are annotated (connected lines). MR causal tests: Wald Ratio for single SNP IV (WR); Inverse Variance Weighted for >1 SNP IV (IVW). BMI: body mass index as a measure of obesity (GIANT, N ≤ 795,640). WHRadjBMI: Waist-hip ratio adjusted for BMI as a measure of central adiposity (GIANT, N ≤ 694,649). T2D and T2D adjusted for BMI as measures of T2D risk (DIAGRAM, N ≤ 231,422). Fasting glucose and insulin (MAGIC, N ≤ 138,589) and HbA1c (MAGIC, N ≤ 159,940) as measures of glycaemic traits linked to T2D.

Fig. 5. Adipocyte genomic and functional studies.

a Oil Red O (ORO, red/brown) lipid staining in day 6 differentiated 3T3-L1 adipocytes reverse transfected with non-silencing (NS), Prrc2a or Limd2 siRNA at day 2 of differentiation. b Equivalent spectrophotometric measurements of eluted ORO, normalised for cell number using crystal violet (CV, day 6, N = 4 independent samples, Prrc2a P = 3.5 × 10^–5, Limd2 P = 0.0047). Presented as mean ± SEM relative to NS control, compared by Student’s t test (two-sided). c Expression of adipogenesis, insulin signalling and lipid metabolism genes at day 6 of differentiation in 3T3-L1 adipocytes transfected with siRNA against Prrc2a, Limd2 or NS control at day 2 of differentiation (N = 6 independent samples). Real-time qPCR values were normalised to housekeeping genes (Nono, Ywhaz). Presented as mean ± SEM relative to NS control, compared by Student’s t test (two-sided). d Targeted methylation sequencing at the PRRC2A (left panel, subcutaneous adipocytes, N = 43) and LIMD2 (right panel, visceral adipocytes, N = 46) loci. CGI: UCSC CpG islands. E063 and E025: Roadmap adipose and adipocyte chromatin states. Sentinel: Sentinel methylation site in combined discovery and replication data. Δ-Array and Δ-TMS: Difference in methylation (range −10 to 10%) in obesity in combined discovery and replication array data, and targeted methylation sequencing data (red higher, blue lower). %-TMS: Mean methylation level (0 to 100%) in targeted methylation sequencing data. ATAC1 and ATAC2: ATAC sequencing of human undifferentiated preadipocytes and mature differentiated adipocytes. Hi-C: Human adipocyte Hi-C functional connectivity maps at day 3 of differentiation. e Targeted activation at the PRRC2A locus in human adipocytes (using two pairs of guides, F1/R2 N = 6 and F2/R3 N = 6 independent samples) had no effect on PRRC2A expression. Targeted activation at the LIMD2 locus increased LIMD2 expression in two (F1/R2 N = 7 and F1/R3 N = 6 independent samples) but not a third cell line (F2/R3 N = 6 independent samples). Presented as mean ± SEM relative to AAVS1 control cells, standardised to housekeeping genes (ACTB, GAPDH). AAVS1 represents the combined results for the AAVS1 F1/R2 and F1/R3 guide pairs (N = 13 independent samples, One-Way ANOVA test, two-sided, Dunnett’s test for multiple comparisons). **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are provided in the Source Data file.