DNA Methylation of Genes Participating in Hepatic Metabolisms and Function in Fetal Calf Liver Is Altered by Maternal Undernutrition during Gestation

Abstract

This study aimed to elucidate the effects of maternal undernutrition (MUN) on epigenetic modification of hepatic genes in Japanese Black fetal calves during gestation. Using a previously established experimental design feeding the dams with 60% (LN) or 120% (HN) of their global nutritional requirements during the 8.5-month gestational period, DNA methylation in the fetal liver was analyzed with reduced representation bisulfite sequencing (RRBS). The promoters and gene bodies in the LN fetuses were hypomethylated compared to HN fetuses. Pathway analysis showed that the genes with DMR in the exon/intron in the LN group were associated with pathways involved in Cushing syndrome, gastric acid secretion, and aldosterone synthesis and secretion. Promoter hypomethylation in the LN group was frequently observed in genes participating in various signaling pathways (thyroid hormone, Ras/Rap1, PIK3-Akt, cAMP), fatty acid metabolism, and cholesterol metabolism. The promoter hypomethylated genes ALPL and GNAS were upregulated in the LN group, whereas the promoter hypermethylated genes GRB10 and POR were downregulated. The intron/exon hypomethylated genes IGF2, IGF2R, ACAD8, TAT, RARB, PINK1, and SOAT2 were downregulated, whereas the hypermethylated genes IGF2BP2, NOS3, and NR2F1 were upregulated. Collectively, MUN alters the promoter and gene body methylation of genes associated with hepatic metabolisms (energy, cholesterol, mitochondria) and function, suggesting an impact of altered gene methylation on the dysregulation of gene expression in the fetal liver.

Keywords: DNA methylation; RRBS; epigenetics; fetal growth restriction; liver; maternal undernutrition; mitophagy.

Figure 1

Genomic locus-dependent distribution of CG methylation level in the context of functional genetic elements (A) and upstream/downstream 2kb-regions of gene body (B). After combining the samples with biological repeats, each region is divided into 20 bins (A), or 50 bins (B), and the methylation level is calculated in each bin. X-axis: functional genetic elements or regions, y-axis: methylation level. LN: red, HN: blue.

Figure 2

DML distribution in CG context in different functional regions. The x-axis is functional regions; the y-axis is the number of hyper/hypo DMR in each region. Hyper and hypo DMLs in LN fetuses are indicated as red and blue, respectively.

Figure 3

Circos plot for DMR condition in CG context. The circos plot represents (from outside to inside): (1) Hyper DMR statistical value: log5 (|areaStat|). The higher and bigger the point, the larger differences between the two groups. (2) TE, the heatmap of the percentage of repeat elements (if repeats are provided). (3) Heatmap of gene density. (4) Hypo DMR statistical value: log5 (|areaStat|). The higher and bigger the point, the larger differences between the two groups. The number shown in the outside indicates the chromosome number.

Figure 4

The scattered plot of DMR-related pathways analyzed by KEGG enrichment. (A) Genes of all hyper DMR regions, (B) genes of all hypo DMR regions. The x-axis represents the Rich factor, and the y-axis represents the pathway name. The size of points stands for DMR-related gene counts, and the colors stand for different q-values ranges.

Figure 5

The scattered plot of DMR-related pathways analyzed by KEGG enrichment. (A) Genes of hyper DMR in promotor, (B) genes of hypo DMR in promotor. The x-axis represents the Rich factor, and the y-axis represents the pathway name. The size of points stands for DMR-related gene counts, and the colors stand for different q-values ranges.

Figure 6

Hypothetic scheme of DM gene-associated molecular regulatory networks and metabolisms in MUN fetal liver. Up- and downward arrows indicate hyper- and hypomethylation in promotor (red) or gene-body (blue), respectively. The pathway/metabolism boxes filled in blue are significantly extracted as relevant to DM genes. Grayed genes are not determined in this study. Solid and chain lines indicate direct and indirect regulations and indirect relationships, respectively.