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Epigenetic and Transcriptomic Characterization of Pure Adipocyte Fractions From Obese Pigs Identifies Candidate Pathways Controlling Metabolism

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Epigenetic and Transcriptomic Characterization of Pure Adipocyte Fractions From Obese Pigs Identifies Candidate Pathways Controlling Metabolism

Mette Juul Jacobsen et al. Front Genet.

Abstract

Reprogramming of adipocyte function in obesity is implicated in metabolic disorders like type 2 diabetes. Here, we used the pig, an animal model sharing many physiological and pathophysiological similarities with humans, to perform in-depth epigenomic and transcriptomic characterization of pure adipocyte fractions. Using a combined DNA methylation capture sequencing and Reduced Representation bisulfite sequencing (RRBS) strategy in 11 lean and 12 obese pigs, we identified in 3529 differentially methylated regions (DMRs) located at close proximity to-, or within genes in the adipocytes. By sequencing of the transcriptome from the same fraction of isolated adipocytes, we identified 276 differentially expressed transcripts with at least one or more DMR. These transcripts were over-represented in gene pathways related to MAPK, metabolic and insulin signaling. Using a candidate gene approach, we further characterized 13 genes potentially regulated by DNA methylation and identified putative transcription factor binding sites that could be affected by the differential methylation in obesity. Our data constitute a valuable resource for further investigations aiming to delineate the epigenetic etiology of metabolic disorders.

Keywords: DNA methylation; RNAseq; Sus scrofa; epigenetics; metabolism; obesity.

Figures

Figure 1
Figure 1
Biplot of the Principal Component Analysis (PCA) of the anthropometric and metabolic characteristics of studied animals. The variables were scaled prior to the analysis. Lean animals are separated from obese animals according to the first component.
Figure 2
Figure 2
DMRs overlapping different genomic annotations. DMRs from mDNAcap and RRBS are shown as hypo-methylated (decrease methylation in obese animals) or hyper-methylated (increased methylation in obese animals). The overlap percentage is calculated as the number of regions of the given type overlapping an annotation compared to the total number of regions of the given type. As many of the DMRs overlap more than one annotation type, the numbers do not add up to 100% even when the “Gene” annotation is left out.
Figure 3
Figure 3
(A) Enrichment analysis of the Top5 KEGG pathways in common between the DE genes (RNAseq) and the genes with identified DMRs (RRBS and mDNAcap). (B) Pathway analysis showing the significant enrichment of the same five pathways after data was merged, accordingly the DE genes encompassed a least one DMR. Orange columns represent the expected number of genes in the pathways, and blue column display the actual number of identified differential expressed genes/methylated genes in each pathway. The specified p values are all Bonferroni corrected.
Figure 4
Figure 4
Gene expression profile of the differentially expressed genes in mature adipocytes. RT-qPCR: Orange columns, 9 lean and 10 obese. RNAseq: Blue columns, 5 lean and 5 obese. + FC; Upregulated in obese pigs, − FC; Downregulated in obese pigs. ND, Not determined. †; No significant differentially expression between the groups. *Genes encompassing a DMR. **Genes encompassing a DMR, where the gene expression level correlates with the methylation level (Pearson p < 0.05).
Figure 5
Figure 5
Representative illustrations of significant correlations between gene expression and DNA methylation levels (mDNAcap)/DNA methylation percentage (RRBS) of selected genes. The mDNAcap methylation levels of KLB correlate negatively with the gene expression, and gene expression correlates positively with the methylation of IDS. In PPARA both a positive and a negative correlation is observed between the RRBS methylation percentages and the gene expression level, whereas only negative correlation is seen for the 2 differential methylated CpGs in PNPLA2. r is Pearson's correlation coefficient. (See Supplementary Figures S4-S5) for all the 21 correlation analysis).
Figure 6
Figure 6
Representative illustrations of significant correlations between gene expression and metabolic characteristic. ISLR, LEP, and MOCOS mRNA levels correlated positively with LDL-cholesterol, retroperitoneal fat and abdomen circumference levels, respectively. r is Pearson's correlation coefficient (See Supplementary Figures 6) for the 11 correlation analysis).
Figure 7
Figure 7
Focus on the TBC1D16 gene. (A) Schematic representation of the small transcript and full-length of the porcine TBC1D16 gene. Blue boxes represent the location of the 5 DMRs identified in the gene, and yellow boxes represent the additional differentially methylated regions in intron5 and intron6. (B) Mean differences in percentage of methylation between the obese and lean animals in 3 DMRs (RRBS), where the methylation levels correlated with TBC1D16 expression level. Hypermethylation in obese animals are denoted as positive difference, and hypomethylation in obese animals is denoted as negative difference. Each peak represents a methylated CpG. (C) The correlation between the methylation of 3 CpGs and expression level of the amplicons targeting only the full-length TBC1D16 (Amplicon1) and all TBC1D16 transcripts (Amplicon2), See Supplementary Figures S7 for the correlation analysis. (D) Gene expression and protein expression of porcine TBC1D16. Lean animals; L and blue columns. Obese animals; O and orange columns. NS, Not significant. ** p < 0.0001, *** p < 0.01.
Figure 8
Figure 8
Illustration of the predicted putative binding sites for the transcriptions factors; STAT1, HIF1A, and TP53 within the differential methylated CpGs located in the TBC1D16, PNPLA2, and PPARA genes, respectively. The red bases in the sequences correspond to the positions where differential methylation is identified. For TBC1D16, this position corresponds to position 6 in the STAT1 motif, and for PNPLA2 the differential methylated CpG is localized in position 10 in the HIF1A motif. The differential methylated CpG in PNPLA2 is localized in position 8 in the TP53 motif. + FC (Fold Change); Upregulated in obesity, − FC; Downregulated in obesity. DNA methylation; + increase methylation in obesity, − decreased methylation in obesity.

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