Mouse-human experimental epigenetic analysis unmasks dietary targets and genetic liability for diabetic phenotypes

Abstract

Using a functional approach to investigate the epigenetics of type 2 diabetes (T2D), we combine three lines of evidence-diet-induced epigenetic dysregulation in mouse, epigenetic conservation in humans, and T2D clinical risk evidence-to identify genes implicated in T2D pathogenesis through epigenetic mechanisms related to obesity. Beginning with dietary manipulation of genetically homogeneous mice, we identify differentially DNA-methylated genomic regions. We then replicate these results in adipose samples from lean and obese patients pre- and post-Roux-en-Y gastric bypass, identifying regions where both the location and direction of methylation change are conserved. These regions overlap with 27 genetic T2D risk loci, only one of which was deemed significant by GWAS alone. Functional analysis of genes associated with these regions revealed four genes with roles in insulin resistance, demonstrating the potential general utility of this approach for complementing conventional human genetic studies by integrating cross-species epigenomics and clinical genetic risk.

Figure 1. Genome-wide significant methylation changes related to diet-induced obesity in C57BL/6 mice

(A) Two genome-wide significant DMRs are hypermethylated in adipocytes purified from mice raised on a high-fat diet. Each point represents the methylation level in adipocytes from an individual mouse at a specific probe, with smoothed lines representing group methylation averages. These points are colored blue for lean mice and red for obese mice. (B) Body weight (grams) and glucose tolerance (AUC) are associated with methylation in adipocytes at genome-wide significant levels. Each point in the top panels represents one probe, with the y-axis representing the Pearson correlation coefficients of the probes with the analyzed phenotype. Dotted lines represent the extent of the DMR as generated automatically via CHARM. The bottom panels display gene location information for the chromosomal coordinates on the x-axis.

Figure 2. Replication of mouse methylation changes in additional mice, and associated gene expression changes

(A) Methylation changes observed after CHARM analysis at two genome-wide significant DMRs are replicated using bisulfite pyrosequencing. Red boxes indicate CpGs assayed in pyrosequencing. For the lower pyrosequencing plots, the y-axis represents methylation, and individual CpGs are plotted along the x-axis. Purple dots represent control DNA artificially methylated to have 0, 25, 50, 75 and 100% methylation. (B) Gene expression changes for genes near genome-wide significant mouse adipocyte DMRs. RNA levels were normalized to same-sample 18S RNA measurements and are displayed as [C_T (high-fat samples) – C_T (low-fat samples)]². Error bars represent standard error of the C_T differences between groups. * p<0.05, ** p<0.005, *** p<0.0005. The direction of the genome-wide significant CHARM DMR closest to the gene is denoted below the gene names; + and − represent regions hyper- or hypomethylated in the high-fat samples, respectively. See also Figure S2 for whole-genome gene expression correlations, and Tables S4 and S5 for pyrosequencing and tissue purification, respectively.

Figure 3. Overlapping methylation changes in human and mouse adipose tissue

Two genome-wide significant DMRs found in mouse adipocytes over Adrbk1 (A, top) and Kcna3 (B, top) are shown along with the corresponding methylation changes in human adipose tissue in (A, bottom), and (B, bottom). For the panels denoting methylation, each point represents the methylation level from an individual mouse or human at a specific genomic location, with smoothed lines representing group methylation averages. Y-axis – methylation values. Below each methylation plot is a panel showing genomic coordinates for the respective species and any genes at those coordinates. See also Figure S3 for tissue and species overlaps, and Tables S6 and S7 for conserved adipose mouse DMRs in human and for enrichment between DIAGRAM and conserved DMRs, respectively.

Figure 4. Diagrammatic representation of the interactions between epigenetically conserved and genetically-associated genes implicated in this study

Generated using QIAGEN’s Ingenuity IPA (Ingenuity® Systems, www.ingenuity.com), these diagrams represent the connections between genes implicated in our analyses. A) Genes with genome-wide significant linkage to T2D in the DIAGRAM meta-analysis were connected to genes near directionally conserved cross-species DMRs. Genes with no connections were dropped. B) Starting with a set of 23 genes near T2D-associated directionally conserved cross-species DMRs, this network was grown by adding genes near species-conserved and mouse-only genome-wide significant DMRs in order to represent one potential regulatory network. Gene colors explained in within-figure legend. See also Figure S4 for the permutation analysis of the enrichment of interactions in Figure 4A.

Figure 5. Overexpression and shRNA-mediated knock down of selected genes in 3T3-L1 adipocytes

Selected genes from the set of 30 species conserved and T2D-SNP overlapping adipose DMRs were either stably overexpressed (A) or knocked down with shRNA (B). Glucose uptake is plotted as fold difference from normal, and significance was determined by two-way ANOVA modified by Bonferroni correction denoted as follows: * p<0.05, ** p<0.01, *** p<0.001. (C) DNA methylation and gene expression levels for high-fat-fed mice and obese human versus low-fat-fed mice and lean humans (e.g., “↓“ indicates hypomethylation / lower gene expression in high-fat-fed and obese compared to low-fat-fed and lean). Bold arrows indicate significant changes.