The hippocampus is a primary region of the brain controlling the formation of memories and learned behaviours. The ability to learn or form a memory requires a neuron to translate a transient signal into gene expression changes that have a long-lasting effect on synapse activity and connectivity. Numerous studies over the past decade have detailed changes in epigenetic modifications under various learning paradigms to support a role for chromatin remodelling in these processes. Moreover, the identification of mutations in epigenetic regulators as the cause of mental retardation or intellectual disability (MR/ID) disorders further strengthens their importance to learning and memory. Animal models for many of these disorders are emerging and advancing our understanding of the molecular mechanisms linking epigenetic regulation and cognitive function. Here, we review how chromatin remodelling proteins implicated in MR/ID contribute to the development of the hippocampus and memory formation.

In CNS, GABA(A) receptor-mediated responses switch from depolarization to hyperpolarization during postnatal development. This switch is mediated by developmental down-regulation of inwardly directed Na(+)-K(+)-2Cl(-) co-transporter type 1 (NKCC1) and up-regulation of outwardly directed K(+)-Cl(-) co-transporter type 2. While several factors have been shown to regulate K(+)-Cl(-) co-transporter type 2 expression, little is known about the mechanisms by which the expression of NKCC1 is regulated during postnatal development. Here, we report a novel epigenetic mechanism underlying the developmental regulation of NKCC1 gene expression in the rat cerebral cortex. In vitro DNA methylation of the NKCC1 promoter region, which contains a high density of cytosine-phosphodiester-guanine islands, significantly decreased the expression of NKCC1 mRNA, and the degree of methylation of the NKCC1 promoter region significantly increased during postnatal development. In addition, treatment with 5-aza-2'-deoxycytidine, a specific DNA methyltransferase inhibitor, elicited an increase in the expression of NKCC1 mRNA and protein in cortical slice cultures. Focal ischemic injury induced by the occlusion of the middle cerebral artery led to the re-expression of NKCC1 mRNA and protein even in the mature rat cortex. The re-expression of NKCC1 mRNA and protein in the injured cerebral cortex was related to a decrease in the methylation status of the NKCC1 promoter region. Our results indicate that epigenetic mechanisms, such as DNA methylation, might be involved in the regulation of NKCC1 gene expression during postnatal development as well as under pathological conditions.

BACKGROUND: Insulin is a critical component of metabolic control, and as such, insulin gene expression has been the focus of extensive study. DNA sequences that regulate transcription of the insulin gene and the majority of regulatory factors have already been identified. However, only recently have other components of insulin gene expression been investigated, and in this study we examine the role of DNA methylation in the regulation of mouse and human insulin gene expression. METHODOLOGY/PRINCIPAL FINDINGS: Genomic DNA samples from several tissues were bisulfite-treated and sequenced which revealed that cytosine-guanosine dinucleotide (CpG) sites in both the mouse Ins2 and human INS promoters are uniquely demethylated in insulin-producing pancreatic beta cells. Methylation of these CpG sites suppressed insulin promoter-driven reporter gene activity by almost 90% and specific methylation of the CpG site in the cAMP responsive element (CRE) in the promoter alone suppressed insulin promoter activity by 50%. Methylation did not directly inhibit factor binding to the CRE in vitro, but inhibited ATF2 and CREB binding in vivo and conversely increased the binding of methyl CpG binding protein 2 (MeCP2). Examination of the Ins2 gene in mouse embryonic stem cell cultures revealed that it is fully methylated and becomes demethylated as the cells differentiate into insulin-expressing cells in vitro. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that insulin promoter CpG demethylation may play a crucial role in beta cell maturation and tissue-specific insulin gene expression.

The role of methylation in the history of psychiatry has traversed a storied path. The original trans-methylation hypothesis was proposed at a time when chlorpromazine had been synthesized but not yet marketed as an antipyschotic (Thorazine). The premise was that abnormal metabolism led to the methylation of biogenic amines in the brains of schizophrenia patients and that these hallucinogenic compounds produced positive symptoms of the disease. At the time, psychiatry was very interested in drugs such as mescaline and lysergic acid diethyl amide that replicated clinical symptoms and understood that these compounds might provide a biological basis for psychosis. The amino acid methionine (MET) was given to patients in the hopes of confiriming the transmethylation hypothesis. However with time, many realized that the hunt for an endogenous psychotropic compound would remain elusive. We now believe that the MET studies may have produced a toxic reaction in susceptible patients by disrupting epigenetic regulation in the brain. The focus of the current review is on the coordinate regulation of multiple promoters expressed in neurons that may be modulated through methylation. While certainly the identification of genes and promoters regulated epigenetically has been steadily increasing over the years, there have been few studies that examine methylation changes as a consequence of increased levels of a dietary amino acid such as methionine (MET). We suggest that the MET mouse model may provide information regarding the indentification of genes that are regulated by epigenetic perturbations. In addition to our studies with the reelin and GAD67 promoters, we also have evidence that additional promoters expressed in select neurons of the brain are similarly affected by MET administration. We suggest that to expand our knowledge of epigenetically-responsive promoters using MET might allow for a better appreciation of global methylation changes occurring in selected brain regions.

Global alterations in gene expression have been observed in different traumatic brain injury (TBI) models and are considered of crucial importance to the development of subsequent tissue injury and repair. Cytosine methylation is a well-known process of endogenous DNA modification in mammals and the primary mechanism responsible for changes in epigenetic gene expression. Here we have investigated the early global spatio-temporal changes of the status of cellular DNA methylation in a rat TBI model by immunohistochemistry and analyzed the effects of dexamethasone on these changes. Global cellular hypomethylation was seen as early as day 1 in pannecrosis and day 2 in peripannecrosis following TBI. A sub-population of reactive microglia/macrophages was identified as the major source of hypomethylated cells by double-staining experiments. Further, peripheral administration of dexamethasone suppressed this lesional hypomethylation at day 2 post-injury. In sum, our data suggest that lesional hypomethylation defines a sub-population of activated microglia/macrophages involved in the early processes following traumatic brain injury.

Histone deactylase enzymes are responsible for the deacetylation of histone tails, and consequently influence gene regulation through their ability to modify chromatin structure surrounding promoter regions. We analyzed the microarray collection of the National Brain Databank to investigate differential expression of these enzymes in the prefrontal cortices of control, schizophrenia and bipolar subjects. HDAC1 expression levels were significantly higher in schizophrenia versus normal subjects. The mRNA expression level of an epigenetically regulated schizophrenia candidate gene GAD67 was strongly and negatively correlated with the mRNA expression levels of HDAC1, HDAC3 and HDAC4 levels. These findings provide additional support for the proposal that epigenetic factors are operative in the brain pathology of patients with schizophrenia.

Dysfunction of prefrontal cortex in schizophrenia includes changes in GABAergic mRNAs, including decreased expression of GAD1, encoding the 67 kDa glutamate decarboxylase (GAD(67)) GABA synthesis enzyme. The underlying molecular mechanisms remain unclear. Alterations in DNA methylation as an epigenetic regulator of gene expression are thought to play a role but this hypothesis is difficult to test because no techniques are available to extract DNA from GAD1 expressing neurons efficiently from human postmortem brain. Here, we present an alternative approach that is based on immunoprecipitation of mononucleosomes with anti-methyl-histone antibodies differentiating between sites of potential gene expression as opposed to repressive or silenced chromatin. Methylation patterns of CpG dinucleotides at the GAD1 proximal promoter and intron 2 were determined for each of the two chromatin fractions separately, using a case-control design for 14 schizophrenia subjects affected by a decrease in prefrontal GAD1 mRNA levels. In controls, the methylation frequencies at CpG dinucleotides, while overall higher in repressive as compared to open chromatin, did not exceed 5% at the proximal GAD1 promoter and 30% within intron 2. Subjects with schizophrenia showed a significant, on average 8-fold deficit in repressive chromatin-associated DNA methylation at the promoter. These results suggest that chromatin remodeling mechanisms are involved in dysregulated GABAergic gene expression in schizophrenia.