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 . 85G9 . 64 . 096). However, epigenetic modifications on fetal hepatic H3 associated with gene repression were absent or subtle (PO0 . 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 . 61G0 . 25; PZ0 . 011). Consistent with our mRNA expression profile, fetal nuclear extracts from offspring of highfat diet animals were observed to be significantly relatively deplete of HDAC1 protein (36 . 07G6 . 73 vs 83 . 18G7 . 51; PZ0 . 006) and in vitro HDAC functional activity (0 . 252G0 . 03 vs 0 . 698G0 . 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 . 59G0 . 23-fold; PZ0 . 08), DNAJA2 (1 . 36G0 . 21; PZ0 . 09), and Rdh12 (1 . 88G0 . 15; PZ0 . 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 . 66G0 . 08; PZ0 . 03). In this study, we show that a current significant in utero exposure (caloric-dense hi...

Intrauterine growth retardation (IUGR) increases the risk of neuroendocrine reprogramming. In the rat, IUGR leads to persistent changes in cerebral mRNA levels. This suggests lasting alterations in IUGR cerebral transcriptional regulation, which may result from changes in chromatin structure. Candidate nutritional triggers for these changes include altered cerebral zinc and one-carbon metabolite levels. We hypothesized that IUGR affects cerebral chromatin structure in neonatal and postnatal rat brains. Rats were rendered IUGR by bilateral uterine artery ligation; controls (Con) underwent sham surgery. At day of life 0 (d0), we measured cerebral DNA methylation, histone acetylation, expression of chromatin-affecting enzymes, and cerebral levels of one-carbon metabolites and zinc. At day of life 21 (d21), we measured cerebral DNA methylation and histone acetylation, as well as the caloric content of Con and IUGR rat breast milk. At d0, IUGR significantly decreased genome-wide and CpG island methylation, as well as increased histone 3 lysine 9 (H3/K9) and histone 3 lysine 14 (H3/K14) acetylation in the hippocampus and periventricular white matter, respectively. IUGR also decreased expression of the chromatin-affecting enzymes DNA methyltransferase 1 (DNMT1), methyl-CpG binding protein 2 (MeCP2), and histone deacetylase (HDAC)1 in association with increased cerebral levels of zinc. In d21 female IUGR rats, cerebral CpG DNA methylation remained lower, whereas H3/K9 and H3/K14 hyperacetylation persisted in hippocampus and white matter, respectively. In d21 male rats, IUGR decreased acetylation of H3/K9 and H3/K14 in these respective regions compared with controls. Despite these differences, caloric, fat, and protein content were similar in breast milk from Con and IUGR dams. We conclude that IUGR results in postnatal changes in cerebral chromatin structure and that these changes are sex specific.

Studies in humans and rats suggest that intrauterine growth retardation (IUGR) permanently resets the hypothalamic-pituitary-adrenal (HPA) axis. HPA axis reprogramming may involve persistently altered expression of the hippocampal glucocorticoid receptor (hpGR), an important regulator of HPA axis reactivity. Persistent alteration of gene expression, long after the inciting event, is thought to be mediated by epigenetic mechanisms that affect mRNA and mRNA variant expression. GR mRNA variants in both humans and rats include eleven 5'-end variants and GRalpha, the predominant 3'-end variant. The 3'-end variants associated with glucocorticoid resistance in humans (GRbeta, GRgamma, GRA, and GRP) have not been reported in rats. We hypothesized that in the rat hippocampus IUGR would decrease total GR mRNA, increase GRbeta, GRgamma, GRA, and GRP, and affect epigenetics of the GR gene at birth (D0) and at 21 days of life (D21). IUGR increased hpGR and exon 1.7 hpGR mRNA in males at D0 and D21, associated with increased trimethyl H3/K4 at exon 1.7 at both time points. IUGR also increased hpGRgamma in males at D0 and D21, associated with increased acetyl H3/K9 at exon 3 at both time points. hpGRA increased in female IUGR rats at D0 and D21. In addition, our data support the existence of hpGRbeta and hpGRP in the rat. IUGR has sex-specific, persistent effects on GR expression and its histone code. We speculate that postnatal changes in hippocampal GR variant and total mRNA expression may underlie IUGR-associated HPA axis reprogramming.

INTRODUCTION Uteroplacental insufficiency (UPI) produces significant neurodevelopmental deficits affecting the hippocampus of intrauterine growth restricted (IUGR) offspring. IUGR males have worse deficits compared to IUGR females. The exact mechanisms underlying these deficits are unclear. Alterations in hippocampal cellular composition along with altered expression of neural stem cell (NSC) differentiation molecules may underlie these deficits. HYPOTHESIS We hypothesized that IUGR hippocampi would be endowed with altered neuronal, astrocytic, and immature oligodendrocytic proportions at birth, with males showing greater cellular deficits. We further hypothesized that UPI would perturb rat hippocampal expression of ErbB receptors (ErbB-Rs) and Neuregulin 1 (NRG1) at birth and at weaning to account for the short- and long-term IUGR neurological sequelae. METHODS A well established rat model of bilateral uterine artery ligation at embryonic day (E) 19.5 was used to induce IUGR. RESULTS Compared to gender-matched controls, IUGR offspring have altered hippocampal neuronal, astrocytic, and immature oligodendrocytic composition in a subregion- and gender-specific manner at birth. In addition, IUGR hippocampi have altered receptor-type- and gender-specific ErbB-R expression at birth and at weaning. CONCLUSIONS These cellular and molecular alterations may account for the neurodevelopmental complications of IUGR and for the male susceptibility to worse neurologic outcomes.

A caspase-3-activated DNase produces internucleosomal DNA cleavage (DNA laddering). We determined whether caspase-3 is activated by lithium-pilocarpine-induced status epilepticus in six brain regions with necrosis-induced DNA laddering. The thymuses of adult rats given methamphetamine or normal saline were used as controls for apoptosis. Some 6-8 h after methamphetamine treatment, thymocytes showed apoptosis by electron-microscopic examination, positive terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), DNA laddering, cleavage of caspase-3 into its active p17 subunit, active caspase-3 immunoreactivity, and a 25-fold increase in caspase-3-like activity. Six hours after SE, necrotic neurons by electron-microscopic examination in hippocampus, amygdala and piriform, entorhinal and frontal cortices showed no TUNEL and no DNA laddering. Twentyfour hours after seizures, most necrotic neurons were negative for TUNEL, some were positive, but all regions showed DNA laddering. However, 6 and 24 h after seizures, active caspase-3 immunoreactivity was negative, caspase-3-like activity did not increase, and western blot analysis failed to show the p17 subunit. In addition, 24 h after seizures, microdialytic perfusion of carbobenzoxy-valyl-alanyl-aspartyl (O-methylester) fluoromethylketone was not neuroprotective. Thus, caspase-3 is not activated in brain regions with seizureinduced neuronal necrosis with DNA laddering. Either caspase-activated DNase is activated by another enzyme, or a caspase-independent DNase is responsible for the DNA cleavage.