Developmental origins of disease and determinants of chromatin structure: maternal diet modifies the primate fetal epigenome

Abstract

Chromatin structure is epigenetically altered via covalent modifications of histones to allow for heritable gene regulation without altering the nucleotide sequence. Multiple lines of evidence from rodents have established a role for epigenetic remodeling in regulating gene transcription in response to an altered gestational milieu. However, to date, it is unknown whether variations in the intrauterine environment in primates similarly induce changes in key determinants of hepatic chromatin structure. We hypothesized that a maternal high-fat diet would alter the epigenomic profile of the developing offspring, which would result in alterations in fetal gene expression. Age- and weight-matched adult female Japanese macaques were placed on control (13% fat) or high-fat (35% fat) breeder diets and mated annually over a 4-year interval. Fetuses in successive years were delivered near term (e130 of 167 days) and underwent necropsy with tissue harvest. Fetal histones were acid extracted for characterization of H3 modification and chromatin immunoprecipitation (ChIP) with differential display PCR; fetal RNA, DNA, and cytoplasmic and nuclear protein extracts were similarly extracted for comparison. Chronic consumption of a maternal high-fat diet results in a threefold increase in fetal liver triglycerides and histologic correlates of non-alcoholic fatty liver disease. These gross changes in the fetal liver are accompanied by a statistically significant hyperacetylation of fetal hepatic tissue at H3K14 (199.85+/-9.64 vs 88.8+/-45.4; P=0.038) with a trend towards the increased acetylation at H3K9 (140.9+/-38.7 vs 46.6+/-6.53; P=0.097) and at H3K18 (69.0+/-3.54 vs 58.0+/-4.04; P=0.096). However, epigenetic modifications on fetal hepatic H3 associated with gene repression were absent or subtle (P>0.05). Subsequent characterization of key epigenetic determinants associated with H3 acetylation marks revealed similar significant alterations in association with a high-fat maternal diet (e.g., relative fetal histone deacetylase 1 (HDAC1) gene expression 0.61+/-0.25; P=0.011). Consistent with our mRNA expression profile, fetal nuclear extracts from offspring of high-fat diet animals were observed to be significantly relatively deplete of HDAC1 protein (36.07+/-6.73 vs 83.18+/-7.51; P=0.006) and in vitro HDAC functional activity (0.252+/-0.03 vs 0.698+/-0.02; P<0.001). We employ these observations in ChIP differential display PCR to attempt to identify potential fetal genes whose expression is reprogramed under conditions of a high-fat maternal diet. We quantitatively confirm a minimum of a 40% alteration in the expression of several genes of interest: glutamic pyruvate transaminase (alanine aminotransferase) 2 (GPT2) (1.59+/-0.23-fold; P=0.08), DNAJA2 (1.36+/-0.21; P=0.09), and Rdh12 (1.88+/-0.15; P=0.01) are appreciably increased in fetal hepatic tissue from maternal caloric-dense diet animals when compared with control while Npas2, a peripheral circadian regulator, was significantly downmodulated in the offspring of high-fat diet animals (0.66+/-0.08; P=0.03). In this study, we show that a current significant in utero exposure (caloric-dense high-fat maternal diet) induces site-specific alterations in fetal hepatic H3 acetylation. Employing ChIP, we extend these observations to link modifications of H3 acetylation with alterations in gene-specific expression. These results suggest that a caloric-dense maternal diet leading to obesity epigenetically alters fetal chromatin structure in primates via covalent modifications of histones and hence lends a molecular basis to the fetal origins of adult disease hypothesis.

Conflict of interest statement

Conflict of interest

The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

Figures

Figure 1

Fetal histone H3 undergoes modification in response to a maternal high-fat diet. (a) Western blotting with antibodies specific to H3AcK9, H3AcK14, and H3AcK18 and (b) H3K4me2, K9me2, K9me3, and K27me2me3 were employed to determine the relative degree of lysine-specific acetylation (a) and methylation (b) after normalization to total histone H3. Histones were acid extracted from fetal hepatic tissue from control (n=3) and high-fat diet (n=4)-exposed animals in year 2, and resolved on SDS-PAGE gels. Relative densitometry was used in quantification (mean±S.E.M). Shown are representative bands of a minimum of three separate experiments. (c) Acid-extracted histones were resolved on an acidic urea gel under conditions of reverse polarity to enable resolution of post-translationally modified histone variants by virtue of their charge difference. Thus, retardation reflects increasing modified acetylation states following western blotting (e.g., anti-H3AcK14, left panel). Relative densitometry was used in quantification for the determination of the ratio of the arbitrarily designated ‘A’ versus ‘B’ variants AcK14A/B (mean± S.E.M). Shown are the representative bands of a minimum of three separate experiments.

Figure 2

A maternal caloric-dense diet modifies expression of fetal hepatic epigenetic determinants. Given our observed site-specific modification in H3 acetylation, we quantitated alterations in the expression and activity of key epigenetic determinants in the offspring of high-fat-fed animals. (a) Real-time RT-PCR estimate of relative expression of MeCP2, Dnmt1, and HDAC1 of high-fat (n=9) relative to control (n=10) maternal diet following normalization to GAPDH (ΔΔCT). Data are plotted as the mean ΔΔCT of high-fat diet-exposed animals in separate experiments (circles) relative to control diet offspring (dotted line); bars are thus the mean derived from a minimum of three separate experiments. (b) Quantification of fetal hepatic MeCP2, Dnmt1, and HDAC1 protein expression (left panel), and HDAC nuclear in vitro enzymatic activity (right panel). One hundred micrograms of cytosolic (Dnmt1) or nuclear (MeCP2 and HDAC1) protein were resolved on 2–12% SDS-PAGE gels, and quantification of protein was performed by western blotting. Relative densitometry following normalization to GAPDH was performed and protein expression values are plotted as mean±S.E.M. For the determination of HDAC enzymatic activity, 80 μg nuclear extracts were assayed for HDAC enzymatic activity using previously described methods. Data are plotted as mean±S.E.M. absorbance at 405 nm for control (n=3) and high-fat diet (n=4) offspring.

Figure 3

Quantification of fetal hepatic mRNA expression of ‘reprogramed’ primate genes under conditions of a maternal high-fat diet. Real-time RT-PCR primers and probes were designed from cloned Japanese macaque sequence for GPT2, DNAJA2, Rdh12, and Npas2. Quantification of fetal hepatic mRNA expression is shown as fold mRNA (mean±S.E.M.) expression among high-fat (n=9) relative to control maternal diet (n=10)-exposed offspring following normalization to GAPDH ΔΔCT). Data are plotted as the mean ΔΔCT of high-fat diet-exposed animals in separate experiments (circles) relative to control diet offspring (dotted line); bars are thus the mean derived from a minimum of three separate experiments.

Figure 4

Quantification of fetal hepatic (a) mRNA expression of Hsp27 and Hsp70 and (b) Hsp 40 and Hsp90 protein expression in the primate under conditions of a maternal high-fat diet. (A) Real-time RT-PCR estimate of relative expression of Hsp27 and Hsp70 (ΔCT RNA was isolated from fetal hepatic liver under conditions of control (n=9) and high-fat (n=17) maternal diet in years 1 and 2, and quantification of fetal hepatic mRNA expression is shown as fold mRNA (mean±S.E.M.) expression relative following normalization ΔΔCT). Data are representative of a minimum of three separate experiments. (B) Translational expression of fetal hepatic Hsp40 and Hsp90. Quantification of 20 μg cytosolic protein was performed by western blotting following normalization. Data are representative of a minimum of three separate experiments.