Gestational choline supplementation normalized fetal alcohol-induced alterations in histone modifications, DNA methylation, and proopiomelanocortin (POMC) gene expression in β-endorphin-producing POMC neurons of the hypothalamus

Abstract

Background: Prenatal exposure to ethanol (EtOH) reduces the expression of hypothalamic proopiomelanocortin (POMC) gene, known to control various physiological functions including the organismal stress response. In this study, we determined whether the changes in POMC neuronal functions are associated with altered expressions of histone-modifying and DNA-methylating enzymes in POMC-producing neurons, because these enzymes are known to be involved in regulation of gene expression. In addition, we tested whether gestational choline supplementation prevents the adverse effects of EtOH on these neurons.

Methods: Pregnant rat dams were fed with alcohol-containing liquid diet or control diet during gestational days 7 and 21 with or without choline, and their male offspring rats were used during the adult period. Using double-immunohistochemistry, real-time reverse transcription polymerase chain reaction (RT-PCR) and methylation-specific RT-PCR, we determined protein and mRNA levels of histone-modifying and DNA-methylating enzymes and the changes in POMC gene methylation and expression in the hypothalamus of adult male offspring rats. Additionally, we measured the basal- and lipopolysaccharide (LPS)-induced corticosterone levels in plasma by enzyme-linked immunosorbent assay.

Results: Prenatal EtOH treatment suppressed hypothalamic levels of protein and mRNA of histone activation marks (H3K4me3, Set7/9, acetylated H3K9, phosphorylated H3S10), and increased the repressive marks (H3K9me2, G9a, Setdb1), DNA-methylating enzyme (Dnmt1), and the methyl-CpG-binding protein (MeCP2). The treatment also elevated the level of POMC gene methylation, while it reduced levels of POMC mRNA and β-EP and elevated corticosterone response to LPS. Gestational choline normalized the EtOH-altered protein and the mRNA levels of H3K4me3, Set7/9, H3K9me2, G9a, Setdb1, Dnmt1, and MeCP2. It also normalizes the changes in POMC gene methylation and gene expression, β-EP production, and the corticosterone response to LPS.

Conclusions: These data suggest that prenatal EtOH modulates histone and DNA methylation in POMC neurons that may be resulting in hypermethylation of POMC gene and reduction in POMC gene expression. Gestational choline supplementation prevents the adverse effects of EtOH on these neurons.

Keywords: Choline; DNA Methylation; Fetal Alcohol; Histone Modification; Proopiomelanocortin; Stress Axis.

Figure 1

Effects of gestational choline with or without ethanol on histone modifying enzyme proteins levels in β-endorphin (β-EP) neurons in the arcuate nucleus of the hypothalamus of male PND60 offspring. Pregnant rats were fed ad lib rat chow (AD), alcohol-containing liquid diet (AF) or alcohol and choline-containing liquid fed (CAF), or pair-fed isocaloric liquid diet without (PF) or with choline (CPF). Histone modifying enzyme proteins in β-EP neurons are measured using double-labeled Immunohistochemistry. Changes in the number of β-EP cell-positive to H3K4m2,3 (A, B), H3K9me2 (C, D), AcetylH3K9 (E,F) and pH3S10 (G,H) in the hypothalamus (histone proteins shown in green and β-EP shown in red). Representative photographs show the double-labeled cells (A, C, E & G; red and green combined), in each treatment group, and histograms (B, D, F & H) show the mean ± SEM values of the percentage of β-EP cells that were double-labeled. Magnification 200×. N = 5. H3K4me2,3 (*p < 0.05, compared to the rest). H3K9me2 (*p<0.05, compared to the rest). AceH3K9 (*p<0.05, compared to rest except CAF). pH3S10 (*p<0.05, compared to the rest except AF or CAF). Data were analyzed using one-way analysis of variance (ANOVA) followed by Student Neuman-Keuls post-hoc test.

Figure 2

Effects of gestational choline with or without ethanol on the protein level of DNA methyltransferases, MeCP2 and β-EP in cells containing β-EP in the arcuate nucleus of the hypothalamus of male PND60 offspring treated similarly as in Fig. 1. Changes in the number of β-EP neurons positive to Dnmt1 (A, B), Dnmt3a (C, D), MeCP2 (E,F) and the number of β-EP neurons (G,H) in the hypothalamus (Dnmts and MeCP2 shown in green and β-EP shown in red). Representative photographs show the double-labeled cells (A, C, E & G), indicated by arrows, in each treatment group, and histograms (B, D, F & H) show the mean ± SEM values of the percentage of β-EP cells that were double-labeled. N = 5. Dnmt1 (*p <0.05, compared to the rest). Dnmt3a, (*p<0.05, AF compared to AD and PF;^a, p<0.05, PF compared to CPF). MeCP2 (*p<0.05, compared to the rest). β-EP (*p <0.05, compared to the rest;^a, p<0.05, compared to AD). Data were analyzed using one-way ANOVA followed by Student Neuman-Keuls post hoc test.

Figure 3

Effects of gestational choline with or without ethanol on the mRNA level of histone-modifying enzymes, DNA methylating enzymes and methylbinding protein MeCP2 gene in the mediobasal hypothalamus of male PND60 offspring treated similarly as in Fig. 1. Changes in mRNA levels of Set7/9 (A), G9a (B), Setdb1 (C), Dnmt1 (D), Dnmt3a (E) and MeCP2 (F). Data presented are mean ± SEM. N=5–9. Set7/9 (*p <0.05, compared to the rest). G9a (*p<0.05, compared to the rest;^a, p<0.05, compared to the rest except CAF or CPF). Setdb1 (*p<0.05, compared to the rest). Dnmt1 (*p<0.05, compared to the rest). Dnmt3a (**p<0.01, compared to the rest except CPF or CAF). MeCP2 (*p<0.05, compared to the rest). Data were analyzed using one-way ANOVA followed by Student Neuman-Keuls post hoc test.

Figure 4

Effects of gestational choline on methylation of POMC gene promoter (A) and POMC mRNA levels (B) in the mediobasal hypothalamus of male PND60 offspring treated similarly as in Fig. 1. Rats were injected intraperitoneally with LPS (100 μg/kg body) in saline or saline alone 2 hrs prior to blood sampling for corticosterone measurements. Data presented are mean ± SEM. N = 7–8. *p<0.05, compared to the rest.^a, p<0.05, compared to the rest except CPF.^b, p<0.05, compared to the rest AF. Data were analyzed using one-way ANOVA followed by Student Neuman-Keuls post hoc test.

Figure 5

Effects of gestational choline on basal (saline) and lipopolysaccharide (LPS)-stimulated corticosterone levels in plasma of male PND60 offspring treated similarly as in Fig. 1. Data presented are mean ± SEM. N = 7–8. *p<0.05, compared to the rest.^a, p<0.05, compared with similarly treated control. Data were analyzed using one-way analysis of variance (ANOVA) with Student Neuman-Keuls post hoc test.