Experimental intrauterine growth restriction induces alterations in DNA methylation and gene expression in pancreatic islets of rats

Abstract

Intrauterine growth restriction (IUGR) increases susceptibility to age-related diseases, including type 2 diabetes (T2DM), and is associated with permanent and progressive changes in gene expression. Our study was designed to test whether epigenomic dysregulation mediates the cellular memory of this intrauterine event. To test this hypothesis, we isolated pancreatic islets from control and IUGR (induced by bilateral uterine artery ligation at day 18 of fetal life) animals at 7 weeks of age. Using the HELP (HpaII tiny fragment enrichment by ligation-mediated PCR) assay, we generated the first DNA methylation map at almost 1 million unique sites throughout the rat genome in normal pancreatic islet cells, allowing us to identify the changes that occur as a consequence of IUGR. We validated candidate dysregulated loci with quantitative assays of cytosine methylation and gene expression. IUGR changes cytosine methylation at approximately 1,400 loci (false discovery rate of 4.2%) in male rats at 7 weeks of age, preceding the development of diabetes and thus representing candidate loci for mediating the pathogenesis of metabolic disease that occurs later in life. Epigenetic dysregulation occurred preferentially at conserved intergenic sequences, frequently near genes regulating processes known to be abnormal in IUGR islets, such as vascularization, beta-cell proliferation, insulin secretion, and cell death, associated with concordant changes in mRNA expression. These results demonstrate that epigenetic dysregulation is a strong candidate for propagating the cellular memory of intrauterine events, causing changes in expression of nearby genes and long term susceptibility to type 2 diabetes.

FIGURE 1.

Significant differences in cytosine methylation between IUGR and control rats. A, histogram distribution of p values calculated from an unpaired t test of IUGR (n = 4) in one group and controls (n = 4) in another, with p values along the x axis and frequency along the y axis. B, the analogous comparison with data obtained from a representative random grouping (n = 4 per group). C, heat map of the top 1000 loci that distinguish IUGR from control rats. Each row in this heat map corresponds to data from a single locus, whereas columns correspond to individual samples, IUGR (≫≪) and controls. The branching dendrogram corresponds to the relationships among samples, as determined by clustering using these 1000 sites. IUGR and controls show similar numbers of loci becoming relatively hyper- and hypomethylated (red to yellow, respectively).

FIGURE 2.

Genomic feature distributions of HELP data. Frequencies were calculated for the overlap between four sets of loci (all loci, constitutively hypomethylated, constitutively hypermethylated, and top 1000 differentially methylated) with 7 genomic features (gene body, 10 kb upstream of promoter, intergenic region, conserved element, repetitive element, CpG island, and CG cluster). Associated probabilities that observed differences are non-random (tailed hypergeometric distribution) are shown in supplemental Table 2. Most differential methylation occurs at intergenic and conserved sequences.

FIGURE 3.

Bisulfite MassArray validation confirms IUGR-specific hypermethylation of the Gch1 locus. MassArray was performed for two genomic regions as described by the PCR conditions in supplemental Table 1. Three replicate assays were performed for each of three IUGR offspring and controls. A, MassArray validation results are shown as group-median percent methylation values with standard error bars (y axis) for each individual CG site (in sequential order along the x axis). IUGR is depicted with red lines and diamonds, whereas control data are shown in black lines and filled circles. HpaII sites are highlighted by yellow rectangles. One single nucleotide polymorphism (SNP) was identified in the underlying genomic sequence and was removed from affected samples in the dataset (blue, starred symbol). B, MassArray data are similarly shown for a second genomic region at the Pdx1 gene. No differences in DNA methylation were observed at this age, with relative hypomethylation in all samples.

FIGURE 4.

Quantitative RT-PCR shows changes in mRNA expression concordant with the cytosine methylation changes. Primers were designed to target mRNA transcripts for eight loci, four of which were genes associated with differential methylation (Fgfr1, Vgf, Gch1, and Pcsk5), and four with no changes in methylation, including Hint3 and three genes regulating cytosine methylation (Gadd45a, Dnmt3a, and Dnmt3b). Average log₂ ratio (± S.D.) of IUGR expression data relative to controls was calculated for each gene. The color scheme indicates corresponding methylation data (green for hypomethylation, red for hypermethylation) in IUGR samples compared with controls. All methylation values displayed are from HELP data except Hint3, for which MassArray data proved more reliable because of a local sequence polymorphism.