Genome-wide methylation and gene expression changes in newborn rats following maternal protein restriction and reversal by folic acid

Abstract

A large body of evidence from human and animal studies demonstrates that the maternal diet during pregnancy can programme physiological and metabolic functions in the developing fetus, effectively determining susceptibility to later disease. The mechanistic basis of such programming is unclear but may involve resetting of epigenetic marks and fetal gene expression. The aim of this study was to evaluate genome-wide DNA methylation and gene expression in the livers of newborn rats exposed to maternal protein restriction. On day one postnatally, there were 618 differentially expressed genes and 1183 differentially methylated regions (FDR 5%). The functional analysis of differentially expressed genes indicated a significant effect on DNA repair/cycle/maintenance functions and of lipid, amino acid metabolism and circadian functions. Enrichment for known biological functions was found to be associated with differentially methylated regions. Moreover, these epigenetically altered regions overlapped genetic loci associated with metabolic and cardiovascular diseases. Both expression changes and DNA methylation changes were largely reversed by supplementing the protein restricted diet with folic acid. Although the epigenetic and gene expression signatures appeared to underpin largely different biological processes, the gene expression profile of DNA methyl transferases was altered, providing a potential link between the two molecular signatures. The data showed that maternal protein restriction is associated with widespread differential gene expression and DNA methylation across the genome, and that folic acid is able to reset both molecular signatures.

Figure 1. Differentially expressed genes in maternal undernutrition model.

(A) Venn's diagrams showing the number of differentially expressed gene probes in comparisons MLP vs. C (P-C), MLP-F vs. C (F-C) and MLP vs. MLP-F (P-F) at FDR 5% (top) and FDR 1% (bottom). Number of gene probes whose expression was unaltered is indicated in black (right). Numbers of altered gene probes in MLP-C contrast at different FDR are indicated in green (left). (B) qRT-PCR mRNA analysis for 8 genes: Dnmt1, Dnmt3a, Dnmt3b, Aurkb, Mcm6, Hat1, Gnmt and Gmnn. Results are given in relative units, as ratio of number of copies in assessed gene and housekeeping gene GAPDH. ^× P<0.05; ^×× P<0.01; ^××× P<0.001. ns P>0.05.

Figure 2. Figure panel representing five genomic loci (Agtrap, Fgf5, Dnmt1, Mcm6, Sfmbt1) containing differentially methylated regions (DMRs) between livers obtained from newborns whose mothers were fed a control diet (18% protein) and livers obtained from newborns whose mothers were fed a low protein (9% protein) diet.

For each locus, Panel A shows the validation data obtained from bisulfite conversion followed by cloning and sequencing, which provides methylation status at single CpG site resolution for different livers and different clones (black circles represent methylated CpG, white circles unmethylated CpG). All loci except for Sfmbt1 validated significant difference in DNA Methylation (Agtrap P<0.01, Fgf5 P<0.01; Dnmt1 P<0.05; Mcm6 P<0.01, Mann-Whitney U test), in line with that indicated by genome-wide MBD-Seq data. Panel B provides an overview of the genomic locus, the gene structure, the location of the DMR validated, and the level of methylation across the locus for each of the animal groups analyzed (Control = “cmbd”, Low Protein = “pmbd”, and Low Protein with Folic Acid Supplementation = “fmbd”). Panel C summarises the percent methylation in control and low protein for each locus assessed by bisulphite sequencing.

Figure 3. Intersection of differentially expressed (GEX), separated in up-regulated (UP) and down-regulated (DW), and DMRs-neighboring (MBD) genes.

At FDR 5% only 15 genes (hypergeometric probability = 0.69) are found in both lists, of which 6 were down-regulated (Dym, Glrx2, Mlph, Myh10, Pftk1, And Prep) and 9 up-regulated (Abcc8, Abhd6, Cep350, Ctsa, Eif2b3, Pdk1, Serpina11, Tpcn2, And Tyw3). The total number of mapped genes is 555 in MBD and 577 in GEX.