Figure 3. Active enhancers located in intergenic DMRs Figure 3. Active enhancers located in intergenic DMRs. RefSeq annotation of common regions between active enhancers (H3K4me¹) and 3360 DMRs. The relative enrichment ratios of the DMRs are compared with elements of each category on the mouse methylation array. (a) Number of DMRs that overlap with H3K4me¹ regions as shown. (b) Relative enrichment ratio compared with the representation of the different elements on the methylation array. TSS, transcription start site; TTS, transcription termination site; UTR, untranscribed region. From: In Utero Exposure to a High-Fat Diet Programs Hepatic Hypermethylation and Gene Dysregulation and Development of Metabolic Syndrome in Male Mice Endocrinology. 2017;158(9):2860-2872. doi:10.1210/en.2017-00334 Endocrinology | Copyright © 2017 Endocrine Society

Figure 1. Flowcharts of study design and data processing Figure 1. Flowcharts of study design and data processing. (a) Study design: female mice were maintained on a control or HF diet for 2 weeks before mating, throughout pregnancy, and throughout lactation. Offspring were weaned onto a LF diet at postnatal day 21. Wild-type offspring were analyzed at embrionic day 18.5 (e18.5), 17 days, 5 weeks, and 26 weeks of age. Ln, number of litters represented in each assay and time point. (b) Data processing scheme: genome-wide methylation status and gene expression were measured on 5-week-old liver using the HELP assay and microarray, respectively. Hot spot analysis and methylation and gene expression validation analyses were performed based on data obtained from the array experiments using MassARRAY and qRT-PCR, respectively. From: In Utero Exposure to a High-Fat Diet Programs Hepatic Hypermethylation and Gene Dysregulation and Development of Metabolic Syndrome in Male Mice Endocrinology. 2017;158(9):2860-2872. doi:10.1210/en.2017-00334 Endocrinology | Copyright © 2017 Endocrine Society

Figure 2. Exposure to HF diet in utero induced hepatic hypermethylation. (a) Technical validation studies using bisulphite MassARRAY confirmed the data obtained from the HELP assay. Three loci were identified, each representing constitutively hypomethylated and hypermethylated sites. HELP data as log (Hpall/Mspl) ratio are shown along the x-axis, with hypermethylation toward the left and hypomethylation toward the right. MassARRAY data for the same loci are plotted along the y-axis, from 0% (hypomethylation) to 100% (hypermethylation). (b) Global correlation using the entire dataset demonstrates the difference between control and HF. A tree-pair plot was generated using R package (The R Foundation for Statistical Computing) (15). Pairwise correlations were calculated from 801,224 independent loci. R value indicates the Pearson correlation for each pair of samples (n = 3 per group). (c) Volcano plot of HF diet–induced changes in global DNA methylation of control (n = 3) vs HF diet (n = 3). Each dot corresponds to every locus showing average differences between control and HF [x-axis, below zero is hypomethylation; above zero is hypermethylation with negative log-transformed significance (P values) along the y-axis]. Larger values are more significant. (d) Analysis of 3360 DMRs were assigned by genomic context. (e) Top seven significant IPA canonical pathways of genes with DMRs. (f) Cell signaling and small molecule biochemistry and lipid metabolism interaction network. The top IPA network of genes displaying significant differences in both methylation and gene expression. An asterisk (*) indicates that a given gene is represented in the array set with multiple probes. Cont, control; GB, gene body; Hyper, hypermethylation; Hypo, hypomethylation; IL, interleukin. From: In Utero Exposure to a High-Fat Diet Programs Hepatic Hypermethylation and Gene Dysregulation and Development of Metabolic Syndrome in Male Mice Endocrinology. 2017;158(9):2860-2872. doi:10.1210/en.2017-00334 Endocrinology | Copyright © 2017 Endocrine Society

Figure 4. Increased DNMT mRNA and enzymatic activity in e18 Figure 4. Increased DNMT mRNA and enzymatic activity in e18.5 liver from offspring of HF. (a) DNMT mRNA expression (target normalized to 36B4 in arbitrary units; n = 4 to 5 per group) and (b) enzymatic activity (n = 3 per group) were measured in e18.5 and 5-week-old liver. *P < 0.05 using an unpaired two-tailed Student t test. From: In Utero Exposure to a High-Fat Diet Programs Hepatic Hypermethylation and Gene Dysregulation and Development of Metabolic Syndrome in Male Mice Endocrinology. 2017;158(9):2860-2872. doi:10.1210/en.2017-00334 Endocrinology | Copyright © 2017 Endocrine Society

Figure 5. Chromosomal distribution of DMRs in HF compared with (a) control, hypermethylation clusters on Chrs 4 and 18 and qRT-PCR of these hotspot hypermethylated genes in (b) 26-week- and (c) 5-week-old liver. (a) There were 3360 DMRs assigned by Chr. Exposure to HF diet induced the most hypermethylation on Chr 4. Hypermethylation DMRs are indicated by black lollipops. One lollipop represents five hypermethylated DMRs. There were 211 DMRs found on Chr 4, with 120 DMRs located in the atherosclerosis QTL 1 (black underline). There were 84 DMRs found on Chr 18, with 58 DMRs located in the insulin-dependent diabetes susceptibility 21a QTL (black underline). mRNA expression in (b) 26-week and (c) 5-week liver (target normalized to 36B4 in arbitrary units; n = 4 to 5 per group). *P < 0.05 using an unpaired two-tailed Student t test. From: In Utero Exposure to a High-Fat Diet Programs Hepatic Hypermethylation and Gene Dysregulation and Development of Metabolic Syndrome in Male Mice Endocrinology. 2017;158(9):2860-2872. doi:10.1210/en.2017-00334 Endocrinology | Copyright © 2017 Endocrine Society

Figure 6. Bisulphite MassARRAY validation and mRNA expression of Mmp9 in 5-week liver. (a) Data were uploaded into browser extensible data–formatted tracks for visualization with the USCS Genome Brower. Hypomethylated (upward peaks) and hypermethylated (downward peaks) sites are shown. (b, d–f) The degree of difference in cytosine methylation between liver from offspring of control and HF as measured by Bisulphite MassARRAY at (b) 5 weeks, (d) e18.5, (e) 17 days, and (f) 26 weeks. Gray bars in the rectangle indicate unanalyzable CpG sites (n = 3 to 6 per group). *P < 0.05 for control vs HF. (c) Mmp9 mRNA expression at different postnatal time points (target normalized to 36B4 in arbitrary units; n = 4 to 5 per group). *P < 0.05 using an unpaired two-tailed Student t test. From: In Utero Exposure to a High-Fat Diet Programs Hepatic Hypermethylation and Gene Dysregulation and Development of Metabolic Syndrome in Male Mice Endocrinology. 2017;158(9):2860-2872. doi:10.1210/en.2017-00334 Endocrinology | Copyright © 2017 Endocrine Society