Bisphenol A (BPA) is an estrogenic environmental toxin widely used in the production of plastics and ubiquitous human exposure to this chemical has been proposed to be a potential risk to public health. Animal studies suggest that in utero and early postnatal exposure to this compound may produce a broad range of adverse effects, including impaired brain development, sexual differentiation, behavior, and immune function, which could extend to future generations. Molecular mechanisms that underlie the long-lasting effects of BPA continue to be elucidated, and likely involve disruption of epigenetic programming of gene expression during development. Several studies have provided evidence that maternal exposure to BPA results in postnatal changes in DNA methylation status and altered expression of specific genes in offspring. However, further studies are needed to extend these initial findings to other genes in different tissues, and to examine the correlations between BPA-induced epigenetic alterations, changes in gene expression, and various phenotypic outcomes. It will be also important to explore whether the epigenetic effects of BPA are related to its estrogenic activity, and to determine which downstream effector proteins could mediate changes in DNA methylation. In this review, we will highlight research indicating a consequence of prenatal BPA exposure for brain, behavior, and immune outcomes and discuss evidence for the role of epigenetic pathways in shaping these developmental effects. Based on this evidence, we will suggest future directions in the study of BPA-induced epigenetic effects and discuss the transgenerational implications of exposure to endocrine disrupting chemicals.

In recent years, it has become widely recognized that a comprehensive understanding of chromatin biology is necessary to better appreciate its role in a wide range of diseases. The histone code has developed as a new layer upon our appreciation of transcription factor-based mechanisms of gene expression. While epigenetic regulation refers to a host of chromatin modifications that occur at the level of DNA, histones and histone-associated proteins, how this regulation is orchestrated is still incompletely understood. Of those processes that comprise the epigenetic regulatory machinery, DNA methylation and histone acetylation /deacetylation have been the most thoroughly studied. Compounds that act as inhibitors of DNA methyltransferases (DNMTs) or histone deacetylases (HDACs) activate a variety of intracellular signaling pathways that ultimately impact the coordinated expression of multiple genes. The altered patterns of mRNA and protein expression collectively converge on pathways linked to apoptosis and cell cycle arrest, amongst others. This has prompted a widespread search for epigenetic inhibitors that could be used as chemotherapeutic agents and several are undergoing clinical evaluation. More recently, there has been interest in the use of HDAC inhibitors to activate the expression of mRNAs that are down-regulated in various neurological and psychiatric conditions. Considerably less is known regarding the effect these drugs have on post-mitotic cells such as neurons. Before we consider the clinical use of additional HDAC inhibitors to treat schizophrenia or unipolar depression, there are a number of key issues that need to be resolved.

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.

Recent advances in schizophrenia (SZ) research indicate that the telencephalic gamma-aminobutyric acid (GABA)ergic neurotransmission deficit associated with this psychiatric disorder probably is mediated by the hypermethylation of the glutamic acid decarboxylase 67 (GAD(67)), reelin and other GABAergic promoters. A pharmacological strategy to reduce the hypermethylation of GABAergic promoters is to induce a DNA-cytosine demethylation by altering the chromatin remodeling with valproate (VPA). When co-administered with VPA, the clinical efficacy of atypical antipsychotics is enhanced. This prompted us to investigate whether this increase in drug efficacy is related to a modification of GABAergic-promoter methylation via chromatin remodeling. Our previous and present results strongly indicate that VPA facilitates chromatin remodeling when it is associated with clozapine or sulpiride but not with haloperidol or olanzapine. This remodeling might contribute to reelin- and GAD(67)-promoter demethylation and might reverse the GABAergic-gene-expression downregulation associated with SZ morbidity.

The neuronal GABAergic mechanisms that mediate the symptomatic beneficial effects elicited by a combination of antipsychotics with valproate (a histone deacetylase inhibitor) in the treatment of psychosis (expressed by schizophrenia or bipolar disorder patients) are unknown. This prompted us to investigate whether the beneficial action of this combination results from a modification of histone tail covalent esterification or is secondary to specific chromatin remodeling. The results suggest that clozapine, or sulpiride associated with valproate, by increasing DNA demethylation with an unknown mechanism, causes a chromatin remodeling that brings about a beneficial change in the epigenetic GABAergic dysfunction typical of schizophrenia and bipolar disorder patients.

The epigenetic down-regulation of genes is emerging as a possible underlying mechanism of the GABAergic neuron dysfunction in schizophrenia. For example, evidence has been presented to show that the promoters associated with reelin and GAD67 are down-regulated as a consequence of DNMT-mediated hypermethylation. Using neuronal progenitor cells to study this regulation, we have previously demonstrated that DNMT inhibitors coordinately increase reelin and GAD67 mRNAs. Here, we report that another group of epigenetic drugs, HDAC inhibitors, activate these two genes with a comparable dose- and time-dependence. In parallel, both groups of drugs decrease DNMT1, DNMT3A and DNMT3B protein levels, and reduce DNMT enzyme activity. Furthermore, induction of the reelin and GAD67 mRNAs is accompanied by the dissociation of repressor complexes, containing all three DNMTs, MeCP2 and HDAC1, from the corresponding promoters and increased local histone acetylation. Our data imply that drug-induced promoter demethylation is relevant for maximal activation of reelin and GAD67 transcription. The results suggest that HDAC and DNMT inhibitors activate reelin and GAD67 expression through similar mechanisms. Both classes of drugs attenuate, directly or indirectly, the enzymatic and transcriptional repressor activities of DNMTs and HDACs. These data provide a mechanistic rationale for the use of epigenetic drugs, individually or in combination, as a potential novel therapeutic strategy to alleviate deficits associated with schizophrenia.

We have previously described the cloning of the human reelin promoter and provided evidence that it is regulated, in part, through changes in methylation. Results from our current studies provide a more detailed analysis of this promoter and the interactions of the transcription factors Sp1 and paired box gene 6 (Pax6) with their recognition sites. The promoter was studied in NT2 cells which are a neuroprogenitor line that undergoes differentiation in vitro. We examined reelin mRNA and promoter induction following a 6-day treatment of these cells with retinoic acid (RA). Deletion and site-directed mutations showed functionally relevant sequences necessary for regulation. Gel-shift assays demonstrated that the main site of action of the promoter lies within a closely packed ( approximately 25 bp) region in which these transcription factors likely bind, possibly forming a DNA/protein complex. Based on our results, it appears likely that RA-induces reelin expression through a critical Sp1 site that resides adjacent to the Pax6 site within this multisite enhancer region. We show that induction of the reelin promoter with RA is accompanied by higher amounts of Sp1 and Pax6 binding to this region. Finally, we show that while mutations in the Sp1 site prevent the RA-mediated promoter induction, similar mutations in the Pax6 site do not. The data suggest that while the Pax6 site plays a role in modulating reelin expression, it is not absolutely required for induction by RA.

Reelin and GAD67 mRNAs and protein levels are substantially reduced in post-mortem brains of schizophrenia patients. Increasing evidence suggests that the observed down-regulation of reelin and GAD67 gene expression may be caused by the dysfunction of epigenetic regulatory pathways operative in cortical GABAergic interneurons. To explore whether human reelin and GAD67 mRNAs are coordinately regulated through DNA methylation-dependent mechanisms, we studied the effects of DNA methyltransferase inhibitors on reelin and GAD67 expression in NT-2 neuronal precursor cells. Competitive RT-PCR with internal standards was used to quantitate mRNA levels. The data showed that reelin and GAD67 mRNAs are induced in the same dose- and time-dependent manners. We further demonstrated that the activation of these two genes correlated with a reduction in DNA methyltransferase activity and DNMT1 protein levels. Time-course Western blot analysis showed that DNMT1 protein down-regulation occurs temporally prior to the reelin and GAD67 mRNA increase. In addition, ChIP assays demonstrated that the activation of the reelin gene correlates with dissociation of DNMT1 and MeCP2 from the promoter, and an increased acetylation of histones H3 in the region. Collectively, our data strongly imply that human reelin and GAD67 genes are coordinately regulated through epigenetic mechanisms that include the action of DNMT1. Our study also suggests that negative regulation of the reelin gene involves methylation-dependent recruitment of DNMT1, MeCP2, and certain HDACs, which most likely reduce the activity of the promoter by shifting the surrounding chromatin into a more compact state.

A recent report suggests that the down-regulation of reelin and glutamic acid decarboxylase (GAD(67)) mRNAs represents 2 of the more consistent findings thus far described in post-mortem material from schizophrenia (SZ) patients [reviewed in. Neurochemical markers for schizophrenia, bipolar disorder amd major depression in postmortem brains. Biol Psychiatry 57, 252-260]. To study mechanisms responsible for this down-regulation, we have analyzed the promoter of the human reelin gene. Collectively, our studies suggest that SZ is characterized by a gamma-amino butyric acid (GABA)-ergic neuron pathology presumably mediated by promoter hypermethylation facilitated by the over-expression of the methylating enzyme DNA methyltransferase (Dnmt) 1. Using transient expression assays, promoter deletions and co-transfection assays with various transcription factors, we have shown a clear synergistic action that is a critical component of the mechanism of the trans-activation process. Equally important is the observation that the reelin promoter is more heavily methylated in brain regions in patients diagnosed with SZ as compared to non-psychiatric control subjects [Grayson, D. R., Jia, X., Chen, Y., Sharma, R. P., Mitchell, C. P.,& Guidotti, A., et al.(2005). Reelin promoter hypermethylation in schizophrenia. Proc Natl Acad Sci U S A 102, 9341-9346]. The combination of studies in cell lines and in animal models of SZ, coupled with data obtained from post-mortem human material provides compelling evidence that aberrant methylation may be part of a core dysfunction in this psychiatric disease. More interestingly, the hypermethylation concept provides a coherent mechanism that establishes a plausible link between the epigenetic misregulation of multiple genes that are affected in SZ and that collectively contribute to the associated symptomatology.

We investigated the effects of agents that induce reelin mRNA expression in vitro on the methylation status of the human reelin promoter in neural progenitor cells (NT2). NT2 cells were treated with the histone deacetylase inhibitors, trichostatin A (TSA) and valproic acid (VPA), and the methylation inhibitor aza-2'-deoxycytidine (AZA) for various times. All three drugs reduced the methylation profile of the reelin promoter relative to untreated cells. The acetylation status of histones H3 and H4 increased following treatment with VPA and TSA at times as short as 15 min following treatment; a result consistent with the reported mode of action of these drugs. Chromatin immunoprecipitation experiments showed that these changes were accompanied by changes occurring at the level of the reelin promoter as well. Interestingly, AZA decreased reelin promoter methylation without concomittantly increasing histone acetylation. In fact, after prolonged treatments with AZA, the acetylation status of histones H3 and H4 decreased relative to untreated cells. We also observed a trend towards reduced methylated H3 after 18 h treatment with TSA and VPA. Our data indicate that while TSA and VPA act to increase histone acetylation and reduce promoter methylation, AZA acts only to decrease the amount of reelin promoter methylation.