Gestational choline supply regulates methylation of histone H3, expression of histone methyltransferases G9a (Kmt1c) and Suv39h1 (Kmt1a), and DNA methylation of their genes in rat fetal liver and brain

Abstract

Choline is an essential nutrient that, via its metabolite betaine, serves as a donor of methyl groups used in fetal development to establish the epigenetic DNA and histone methylation patterns. Supplementation with choline during embryonic days (E) 11-17 in rats improves memory performance in adulthood and protects against age-related memory decline, whereas choline deficiency impairs certain cognitive functions. We previously reported that global and gene-specific DNA methylation increased in choline-deficient fetal brain and liver, and these changes in DNA methylation correlated with an apparently compensatory up-regulation of the expression of DNA methyltransferase Dnmt1. In the current study, pregnant rats were fed a diet containing varying amounts of choline (mmol/kg: 0 (deficient), 8 (control), or 36 (supplemented)) during E11-17, and indices of histone methylation were assessed in liver and frontal cortex on E17. The mRNA and protein expression of histone methyltransferases G9a and Suv39h1 were directly related to the availability of choline. DNA methylation of the G9a and Suv39h1 genes was up-regulated by choline deficiency, suggesting that the expression of these enzymes is under negative control by methylation of their genes. The levels of H3K9Me2 and H3K27Me3, tags of transcriptionally repressed chromatin, were up-regulated by choline supplementation, whereas the levels of H3K4Me2, associated with active promoters, were highest in choline-deficient rats. These data show that maternal choline supply during pregnancy modifies fetal histone and DNA methylation, suggesting that a concerted epigenomic mechanism contributes to the long term developmental effects of varied choline intake in utero.

FIGURE 1.

H3K9Me2 in the liver (A) and cortex (B). H3K9Me2 protein levels on E17 were analyzed by immunoblot and normalized to total histone 3 (see example in blot insets). See “Results” for statistics. Bar and blot labels: S, choline-supplemented; C, controls; D, choline-deficient.

FIGURE 2.

H3K27Me3 in the liver (A) and cortex (B). H3K27Me3 protein levels were analyzed on E17 by immunoblot and normalized to total histone 3 (see example in blot insets). See “Results” for statistics. Bar and blot labels: S, choline-supplemented; C, controls; D, choline-deficient.

FIGURE 3.

H3K4Me2 in the liver. H3K4Me2 protein levels in E17 liver were analyzed by immunoblot and normalized to total histone 3 (see example in blot insets). See “Results” for statistics. Bar and blot labels: S, choline-supplemented; C, controls; D, choline-deficient.

FIGURE 4.

G9a in the liver and cortex. A, RNA was isolated from the liver of E17 embryos and used for RT-PCR of G9a and normalized to β-actin (see example in gel insets). B, G9A protein levels in E17 liver were analyzed by immunoblot and normalized to β-ACTIN (see example in blot insets). C, RNA was isolated from the cortex of E17 embryos and used for RT-PCR of G9a and normalized to β-actin (see example in gel insets). D, G9A protein levels in E17 cortex were analyzed by immunoblot and normalized to the levels ofβ-ACTIN (see example in blot insets). See “Results” for statistics. Bar labels: S, choline-supplemented; C, controls; D, choline-deficient.

FIGURE 5.

Suv39h1 in the liver and cortex. A, RNA was isolated from the liver of E17 embryos and used for RT-PCR of Suv39h1 and normalized to β-actin (see example in gel insets). B, SUV39H1 protein levels in E17 liver were analyzed by immunoblot and normalized to β-ACTIN (see example in blot insets). C, RNA was isolated from the cortex of E17 embryos and used for RT-PCR of Suv39h1 and normalized to β-actin (see example in gel insets). D, SUV39H1 protein levels in E17 cortex were analyzed by immunoblot and normalized to the levels of β-ACTIN (see example in blot insets). See “Results” for statistics. Bar labels: S, choline-supplemented; C, controls; D, choline-deficient.

FIGURE 6.

Methylation-specific PCR (MSP) analysis of G9a and Suv39h1 promoter CpG islands. A–C, genomic DNA isolated from E17 liver (A and B) or cortex (C) was treated with sodium bisulfite and analyzed by methylation-specific PCR. The inset shows examples of the PCR products obtained using the primers designed to amplify the methylated (m) and unmethylated (u) DNA templates. See “Results” for statistics. Bar and PCR product labels: S, choline-supplemented; C, controls; D, choline-deficient.