DNA methylation at differentially methylated regions of imprinted genes is resistant to developmental programming by maternal nutrition

Abstract

The nutritional environment in which the mammalian fetus or infant develop is recognized as influencing the risk of chronic diseases, such as type 2 diabetes and hypertension, in a phenomenon that has become known as developmental programming. The late onset of such diseases in response to earlier transient experiences has led to the suggestion that developmental programming may have an epigenetic component, because epigenetic marks such as DNA methylation or histone tail modifications could provide a persistent memory of earlier nutritional states. One class of genes that has been considered a potential target or mediator of programming events is imprinted genes, because these genes critically depend upon epigenetic modifications for correct expression and because many imprinted genes have roles in controlling fetal growth as well as neonatal and adult metabolism. In this study, we have used an established model of developmental programming-isocaloric protein restriction to female mice during gestation or lactation-to examine whether there are effects on expression and DNA methylation of imprinted genes in the offspring. We find that although expression of some imprinted genes in liver of offspring is robustly and sustainably changed, methylation of the differentially methylated regions (DMRs) that control their monoallelic expression remains largely unaltered. We conclude that deregulation of imprinting through a general effect on DMR methylation is unlikely to be a common factor in developmental programming.

Figure 1. Expression analysis of imprinted and other genes in offspring from dams experiencing dietary protein restriction during gestation (GLP) or lactation (PLP). (A) RT-qPCR analysis for the indicated genes at week 3 after birth. (B) RT-qPCR analysis for the indicated genes at week 12 after birth. mRNA levels are expressed as the percentage of the means of the control groups (set at 100%) following normalization for each gene against three housekeeping genes. Bars represent standard errors; *p < 0.05, **p < 0.01 compared with control.

Figure 2. Representation of the location of the amplicons tested in relation to the DMRs within each imprinted domain. Each vertical line represents an individual CpG site. Thick bars above the CpG track represent exons of the indicated transcripts, with transcription start sites and direction of transcription indicated by arrows. The locations of the amplicons used for methylation analysis is given by the short bars under each DMR. In addition, the locations of the amplicons to assay methylation at the Pparα promoter and upstream enhancer are shown similarly.

Figure 3. Methylation analysis of the Gnas exon 1A DMR. (A) Methylation analysis by EpiTYPER of the DMR in control, GLP and PLP liver samples at week 3 (above) and week 12 (below) after birth. Methylation values on a scale 0.0 to 1.0 for each CpG unit that can be resolved by EpiTYPER are given. Values expressed are means and bars represent standard errors; n = 7–9. Where individual CpG units exhibited a significant difference in methylation level in treated group compared with control, * p < 0.05. (B) Bisulphite sequencing analysis of two representative samples from each of the control, GLP and PLP groups (week 3). Each row of circles represents a single, non-redundant bisulphite sequence clone. Individual CpGs are represented by circles, with open circles indicating unmethylated and filled circles methylated CpGs. The numbering at the top refers to the CpG units identified by EpITYPER analysis; note that some CpG units contain multiple CpGs (bracketed), while other CpGs are not scored by EpiTYPER analysis.

Figure 4. Methylation analysis of the Grb10 DMR. EpiTYPER analysis of the DMR in control, GLP and PLP liver samples at week 3 (above) and week 12 (below) after birth. Methylation values for each CpG unit that can be resolved by EpiTYPER are given. Values expressed are means and bars represent standard errors. n = 7–9; *p ≤ 0.055 compared with control.