Ian C. G. Weaver

Reversal of maternal programming of stress responses in adult offspring through methyl supplementation: altering epigenetic marking later in life.

Stress responses in the adult rat are programmed early in life by maternal care and associated with epigenomic marking of the hippocampal exon 17 glucocorticoid receptor (GR) promoter. To examine whether such epigenetic programming is reversible in adult life, we centrally infused the adult offspring with the essential amino acid l-methionine, a precursor to S-adenosyl-methionine that serves as the donor of methyl groups for DNA methylation. Here we report that methionine infusion reverses the effect of maternal behavior on DNA methylation, nerve growth factor-inducible protein-A binding to the exon 17 promoter, GR expression, and hypothalamic-pituitary-adrenal and behavioral responses to stress, suggesting a causal relationship among epigenomic state, GR expression, and stress responses in the adult offspring. These results demonstrate that, despite the inherent stability of the epigenomic marks established early in life through behavioral programming, they are potentially reversible in the adult brain.

The Transcription Factor Nerve Growth Factor-Inducible Protein A Mediates Epigenetic Programming: Altering Epigenetic Marks by Immediate-Early Genes

Maternal care alters epigenetic programming of glucocorticoid receptor (GR) gene expression in the hippocampus, and increased postnatal maternal licking/grooming (LG) behavior enhances nerve growth factor-inducible protein A (NGFI-A) transcription factor binding to the exon 17 GR promoter within the hippocampus of the offspring. We tested the hypothesis that NGFI-A binding to the exon 17 GR promoter sequence marks this sequence for histone acetylation and DNA demethylation and that such epigenetic alterations subsequently influence NGFI-A binding and GR transcription. We report that (1) NGFI-A binding to its consensus sequence is inhibited by DNA methylation, (2) NGFI-A induces the activity of exon 17 GR promoter in a transient reporter assay, (3) DNA methylation inhibits exon 17 GR promoter activity, and (4) whereas NGFI-A interaction with the methylated exon 17 GR promoter is reduced, NGFI-A overexpression induces histone acetylation, DNA demethylation, and activation of the exon 17 GR promoter in transient transfection assays. Site-directed mutagenesis assays demonstrate that NGFI-A binding to the exon 17 GR promoter is required for such epigenetic reprogramming. In vivo, enhanced maternal LG is associated with increased NGFI-A binding to the exon 17 GR promoter in the hippocampus of pups, and NGFI-A-bound exon 17 GR promoter is unmethylated compared with unbound exon 17 GR promoter. Knockdown experiments of NGFI-A in hippocampal primary cell culture show that NGFI-A is required for serotonin-induced DNA demethylation and increased exon 17 GR promoter expression. The data are consistent with the hypothesis that NGFI-A participates in epigenetic programming of GR expression.

Early Environmental Regulation of Hippocampal Glucocorticoid Receptor Gene Expression: Characterization of Intracellular Mediators and Potential Genomic Target Sites

Abstract: Environmental conditions in early life permanently alter the development of glucocorticoid receptor gene expression in the hippocampus and hypothalamic‐pituitary‐adrenal responses to acute or chronic stress. In part, these effects can involve an activation of ascending serotonergic pathways and subsequent changes in the expression of transcription factors that might drive glucocorticoid receptor expression in the hippocampus. This paper summarizes the evidence in favor of these pathways as well as recent studies describing regulatory targets within the chromatin structure of the promoter region of the rat hippocampal glucocorticoid receptor gene.

Maternal programming of steroid receptor expression and phenotype through DNA methylation in the rat

Increased levels of pup licking/grooming and arched-back nursing by rat mothers over the first week of life alter the epigenome at a glucocorticoid receptor gene promoter in the hippocampus of the offspring. Differences in the DNA methylation pattern between the offspring of High and Low licking/grooming--arched-back mothers emerge over the first week of life, are reversed with cross-fostering, persist into adulthood and are associated with altered histone acetylation and transcription factor (NGFI-A) binding to the glucocorticoid receptor promoter. Central infusion of the adult offspring with the histone deacetylase inhibitor trichostatin A removes the previously defined epigenomic group differences in histone acetylation, DNA methylation, NGFI-A binding, glucocorticoid receptor expression, and hypothalamic-pituitary-adrenal responses to stress, thus suggesting a causal relation between the epigenomic state, glucocorticoid receptor expression and the effects of maternal care on stress responses in the offspring. These findings demonstrate that an epigenomic state of a gene can be established through a behavioral mode of programming and that in spite of the inherent stability of this epigenomic mark, it is dynamic and potentially reversible.

From maternal care to gene expression: DNA methylation and the maternal programming of stress responses.

Maternal care of pups influences the development of hypothalamic‐pituitary‐ adrenal (HPA) responses to stress in the rat. Thus, the adult offspring of mothers which naturally show an increased frequency of pup licking=grooming and arched back nursing (i.e., high LG-ABN mothers) show more modest HPA responses to stress. These effects are, in part, mediated by changes in hippocampal glucocorticoid receptor (GR) expression which mediates glucocorticoid negative feedback regulation of CRF expression. The effects of maternal care on GR expression in the neonate are associated with increased expression of the transcription factor NGFI-A and enhanced activation of GR gene expression through a neuron-specific promoter on exon 1 (exon 1Z) of the GR gene. In adulthood, the offspring of the high LG-ABN mothers show decreased methylation of the exon 1Z sequence. Using NaBis mapping of individual CpG sites we found hypomethylation of the NGFI-A consensus sequence on exon 1Z. The results of gel-shift assay with differentially methylated oligo nucleotide sequences containing the NGFI-A consensus site show that the pattern of hypomethylation apparent in the offspring of high LG-ABN mothers is associated with increased NGFI-A binding. These findings suggest that maternal behavior increases NGFI-A expression in infancy resulting in differential methylation of selected DNA sites and thus stable, long-term differences in gene expression over the lifespan.

Early environmental regulation of hippocampal glucocorticoid receptor gene expression: characterization of intracellular mediators and potential genomic target sites.

Environmental conditions in early life permanently alter the development of glucocorticoid receptor gene expression in the hippocampus and hypothalamic-pituitary-adrenal responses to acute or chronic stress. In part, these effects can involve an activation of ascending serotonergic pathways and subsequent changes in the expression of transcription factors that might drive glucocorticoid receptor expression in the hippocampus. This paper summarizes the evidence in favor of these pathways as well as recent studies describing regulatory targets on the promoter region of the rat hippocampal glucocorticoid receptor gene.

Maternal Programming of Individual Differences in Defensive Responses in the Rat

Abstract: This paper describes the results of a series of studies showing that variations in mother‐pup interactions program the development of individual differences in behavioral and endocrine stress responses in the rat. These effects are associated with altered expression of genes in brain regions, such as the amygdala, hippocampus, and hypothalamus, that regulate the expression of stress responses. Studies from evolutionary biology suggest that such “maternal effects” are common and often associated with variations in the quality of the maternal environment. Together these findings suggest an epigenetic process whereby the experience of the mother alters the nature of the parent‐offspring interactions and thus the phenotype of the offspring.

Regional-specific global cytosine methylation and DNA methyltransferase expression in the adult rat hippocampus.

Recent observations suggest that DNA methylation plays an important role in memory and long-term potentiation (LTP) in the hippocampus and is involved in programming the offspring epigenome in response to maternal care. Global DNA methylation is believed to be stable postnatally and to be similar across tissues in the adult mammal. It has also been a long held belief that DNA methyltransferases (DNMTs) play a very limited role in postmitotic tissues. Recent data suggests a more dynamic role for DNA methylation in the brain postnatally, therefore we examined the global state of methylation and the expression of the known DNMTs in the different regions of the hippocampus. We observed strikingly different levels of global methylation in the adult rat dentate gyrus (DG) and CA1 region in comparison with the CA2 and CA3 regions. mRNA levels of DNA methyltransferases exhibited similar regional specificity and were correlated with global DNA methylation levels. These regional differences in global methylation and expression of the DNA methylation machinery in the adult brain are consistent with the emerging hypothesis that DNA methylation may play a dynamic physiological role in the adult brain.

Acetylation-induced transcription is required for active DNA demethylation in methylation-silenced genes.

ABSTRACT A hallmark of vertebrate genes is that actively transcribed genes are hypomethylated in critical regulatory sequences. However, the mechanisms that link gene transcription and DNA hypomethylation are unclear. Using a trichostatin A (TSA)-induced replication-independent demethylation assay with HEK 293 cells, we show that RNA transcription is required for DNA demethylation. Histone acetylation precedes but is not sufficient to trigger DNA demethylation. Following histone acetylation, RNA polymerase II (RNAP II) interacts with the methylated promoter. Inhibition of RNAP II transcription with actinomycin D, α-amanitin, or CDK7-specific small interfering RNA inhibits DNA demethylation. H3 trimethyl lysine 4 methylation, a marker of actively transcribed genes, was associated with the cytomegalovirus promoter only after demethylation. TSA-induced demethylation of the endogenous cancer testis gene GAGE follows a similar sequence of events and is dependent on RNA transcription as well. These data suggest that DNA demethylation follows rather than precedes early transcription and point towards a novel function for DNA demethylation as a memory of actively transcribed genes.

Maternal behavior regulates long-term hippocampal expression of BAX and apoptosis in the offspring

Naturally occurring variations in maternal care influence hippocampal development in the rat. In the present study we found that variations in maternal licking/grooming (LG) during the first week of life are associated with altered hippocampal expression of BAX (group‐1 tumor necrosis factor family mediated cell death effector) in 90‐day‐old male offspring. BAX‐like immunoreactivity on western blots is significantly increased in the adult offspring of low‐level LG mothers. There is no effect of maternal care on levels of either B‐cell lymphoma‐2 (BCL‐2) (group‐II mitochondria mediated cell death suppressor) or BAD (group‐III endoplasmic reticulum mediated cell death effector). The most striking biochemical event in apoptosis is DNA fragmentation. Terminal deoxynucleotidyl transerferase (Tdt)‐mediated dUTP‐biotin nick‐end labeling (TUNEL) and 4′,6′‐diamidino‐2‐phenylindole hydrochloride (DAPI) staining showed that the number of TUNEL‐positive cells in both the dentate gyrus and CA1 region of the hippocampus is significantly increased in the adult offspring of low‐level LG mothers. In conclusion, we propose that hippocampal neurons in the offspring of low‐level LG mothers may be more vulnerable to loss through apoptosis.