Associate editor: B.L. Roth
The human reelin gene: Transcription factors (+), repressors (−) and the methylation switch (+/−) in schizophrenia

https://doi.org/10.1016/j.pharmthera.2005.01.007Get rights and content

Abstract

A recent report suggests that the down-regulation of reelin and glutamic acid decarboxylase (GAD67) mRNAs represents 2 of the more consistent findings thus far described in post-mortem material from schizophrenia (SZ) patients [reviewed inTorrey, E. F., Barci, B. M., Webster, M. J., Bartko, J. J., Meador-Woodruff, J. H., & Knable, M. B. (2005). 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.

Introduction

The starting point for the studies presented herein was the seminal observations regarding the down-regulation of reelin and glutamic acid decarboxylase (GAD67) expression in cortical gamma-amino butyric acid (GABA)-ergic interneurons of post-mortem brain samples from patients diagnosed with schizophrenia (SZ) compared to similar material obtained from patients with other psychiatric diagnoses (Guidotti et al., 2000). These initial studies which showed some selectivity for psychoses were independently replicated at the immunocytochemical level in the hippocampi of patients with SZ and bipolar disorder (BP) (Fatemi et al., 2000). The observation that reelin mRNA is reduced in patients with SZ was also established using in situ hybridization histochemistry of cortical slices prepared post-mortem (Impagnatiello et al., 1998, Eastwood & Harrison, 2003). While beyond the context of the current review, reelin mRNA and protein levels have also been examined in autistic patients and these studies suggest a common link relating the down-regulation of reelin to abnormal neurobiological function (Fatemi et al., 2005a). In fact, these data suggest the possibility that psychosis and autism might share common features related to information and or sensory processing with a major difference being the age of onset at which these deficits occur.

The above studies establish that reelin and GAD67 mRNA and protein levels are reduced by approximately 50% in nearly every region of the cerebral cortex, as well as, in the hippocampus and basal ganglia of patients diagnosed with SZ (Impagnatiello et al., 1998, Fatemi et al., 2000, Guidotti et al., 2000, Eastwood & Harrison, 2003). The reduced levels of cortical reelin and GAD67 in SZ and BP patients are probably not due to a loss of GABAergic neurons but are more likely due to a decreased expression in defective GABAergic neurons. This is supported by the lack of concomitant changes in the levels of GAD65 immunoreactivity and mRNA which are expressed in the same GABAergic interneurons that express the reelin and GAD67 downregulation. In addition, the levels of neuron specific enolase mRNA are not reduced in SP (Guidotti et al., 2000). This finding supports the concept that the reduced reelin and GAD67 are not the result of a reduced number of neurons. This also suggests the possibility that the genes encoding reelin and GAD67 may be coordinately regulated and that a defect in a ‘master’ regulatory protein may compromise the expression of sets of genes expressed in these neurons. This master regulator may be involved in controlling the expression of multiple promoters contributing to the GABAergic neuron phenotype in SZ. In fact, the compromised promoters could easily be extended to include the NR2A (Woo et al., 2004) GAT1 and GAT 3 (Schleimer et al., 2004) and parvalbumin promoters and other promoters expressed in GABAergic neurons (Lewis et al., 2005). While the main focus of this review is the study of the reelin promoter and its regulation, in this context, many of the observations quoted apply equally well to other genes expressed in GABAergic neurons including GAD67.

We have begun to decode the reelin gene regulatory program by focusing our attention on the reelin promoter (Chen et al., 2002). Our long-term goal has been to elucidate mechanisms operative in regulating the expression of the human reelin promoter, so as to provide an appropriate framework for the development of hypotheses relevant to the reduced reelin mRNA expression documented in various psychiatric disorders (see Guidotti et al., 2000, Costa et al., 2003a, Costa et al., 2003b, Costa et al., 2004 for recent reviews). Data obtained thus far indicate that multiple sequence elements within the human reelin promoter are important for expression in various human cell lines (Chen et al., 2002). We have reported data showing that the human reelin promoter is sensitive to methylation and that when the promoter is hypermethylated, reelin expression is silenced. More recently, we have shown that DNA methyltransferase (Dnmt) 1 mRNA is overexpressed in those same GABAergic neurons in which reelin, as well as, GAD67 are reduced suggesting a common negative regulatory mechanism that likely involves the coordinated hypermethylation of the corresponding promoters (Veldic et al., 2004, Veldic et al., 2005). Central to our hypothesis is that biologically accurate patterns of reelin and also GAD67 expression are due to the positive action of specific transcription factors and that access of these factors to their recognition sites is modified through temporal- and spatial-dependent modifications in the methylation status of the corresponding promoter. In addition, it seems equally likely that methyl CpG binding proteins play a role by binding sites within the methylated promoter(s) and further reinforcing the negative regulation.

Section snippets

Reelin expression in the CNS

Reelin is expressed in Cajal-Retzius neurons of the marginal zone during cortical development and when secreted, the protein interacts with the products of several other genes, including the cell membrane receptors VLDL/Apo ER2 and mouse disabled 1, and serves to regulate neuronal positioning during telencephalic development (reviewed in Rice & Curran, 2001). The mouse reelin gene encodes a large (388 kD) glycoprotein expressed mainly in the central nervous system (CNS). The homozygous null

Structure of the reelin gene

The gene encoding reelin was identified and characterized after a transgene insertion into the reeler locus produced a phenotype identical to that of the reeler mice (Miao et al., 1994, D'Arcangelo et al., 1995). Subsequent work led directly to the isolation of a large mRNA that was disrupted in these transgenic mice and it was subsequently shown that the mRNA encodes the protein reelin. The murine reelin protein is translated from a mRNA that contains an open reading frame of 10,383 bp in

Analysis of the human reelin promoter–transcription factors

As a starting point for our studies, we mapped the exon/intron structure of the human reelin gene to various BAC sequences present in the human database (Chen et al., 2002). The 5′ portion of the human mRNA and upstream sequence maps to human BAC clone RG126M09 which contains 163 kb of human genomic DNA. We obtained this DNA (Research Genetics) and verified the location of many restriction sites. The first exon maps to a region that is located near the terminal 3′ end of the BAC. We have since

Regulation by methylation

In the course of these studies, we conducted experiments to evaluate the role of DNA methylation in regulating reelin promoter expression. These experiments were motivated by observations that (1) the cloned reelin promoter behaves promiscuously in different human cell lines in vitro; (2) the promoter is embedded in a large CpG island that surrounds the 5′ flanking sequence and first exon of the gene (Chen et al., 2002); and (3) agents that alter methylation patterns increase reelin mRNA

Gene expression in GABAergic neurons

With respect to the possible coordinate regulation of GAD67 and reelin expression, GAD67 mRNA levels are reduced in multiple brain regions of SZ patients and in the heterozygous reeler (+/−) mice (HRM; Liu et al., 2001, Veldic et al., 2004). In contrast, reelin mRNA levels are normal in GAD67 (+/−) mice. This finding is consistent with the possibility that reelin signaling in the prefrontal cortex might influence GAD67 expression. That is, alterations in the levels of reelin may impact either

The methylation switch and schizophrenia

More recently, it has been shown that the acetylation state of histone H4 in hippocampal pyramidal neurons is altered following pilocarpine injection providing evidence for 1 mechanism of chromatin remodeling associated with seizure-induced changes in gene expression (Huang et al., 2002). Additional findings show that histone lysine methylation at gene promoters is involved in the developmental regulation and maintenance of region-specific expression patterns of both ligand-gated and G-protein

Perspectives for an epigenetic treatment of reelin dysfunction in schizophrenia

Based on the evidence that in cortical GABAergic neurons an epigenetic methylation switch may be at the heart of the neuropathologies detected in SZ, a rationale approach to correct reelin expression deficiency in this disorder should be the use of drugs that correct the DNA methylation patterns. We have reported (Tremolizzo et al., 2002, Tremolizzo et al., 2005, Mitchell et al., 2005) that the transcriptionally silent hypermethylated reelin promoter can be reactivated by either Dnmt1

Significance

Experiments described in this review are designed to provide information relevant to the mechanism(s) responsible for the coordinate regulation of human reelin and GAD67 gene expression. More specifically, we have attempted to establish a role for methylation, as well as histone acetylation in the context of transcriptional regulation of the respective promoters. As tissue-specific and developmental expression patterns are accompanied by distinct alterations in chromatin structure and DNA

Acknowledgments

This work was supported by Grant R01 MH62682 from the National Institutes of Health.

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