Associate editor: B.L. RothThe human reelin gene: Transcription factors (+), repressors (−) and the methylation switch (+/−) in schizophrenia
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.
References (133)
NuRD and SIN3 histone deacetylase complexes in development
Trends Genet
(2000)- et al.
The effects of l-methionine (without MAOI) in schizophrenia
J Psychiatry Res
(1971) - et al.
Modulation of synaptic plasticity and memory by reelin involves differential splicing of the lipoprotein receptor Apoer2
Neuron
(2005) - et al.
GABAergic interneurons: implications for understanding schizophrenia and bipolar disorder
Neuropsychopharmacology
(2001) - et al.
Up-regulation of GABAA receptor binding on neurons of the prefrontal cortex in schizophrenic subjects
Neuroscience
(1996) - et al.
T-brain-1: a homologue of Brachyury whose expression defines molecularly distinct domains within the cerebral cortex
Neuron
(1995) - et al.
DNA methyltransferases get connected to chromatin
Trends Genet
(2002) - et al.
Enhanced dizocilpine efficacy in heterozygous reeler mice relates to GABA turnover downregulation
Neuropharmacology
(2004) - et al.
Demethylase activity is directed by histone acetylation
J Biol Chem
(2001) - et al.
Sp1: regulation of gene expression by phosphorylation
Gene
(2005)
Reelin downregulation and dendritic spine hypoplasia as putative vulnerability factors in schizophrenia
Neurobiol Dis
Neurochemical basis for an epigenetic vision of synaptic organization
Int Rev Neurobiol
Role of reelin in the control of brain development
Brain Res Brain Res Rev
A truncated reelin protein is produced but not secreted in the ‘Orleans’ reeler mutation (Relnrl-Orl)
Mol Brain Res
Valproate induces replication-independent active DNA demethylation
J Biol Chem
Reelin binds alpha3beta1 integrin and inhibits neuronal migration
Neuron
Reelin signaling is impaired in autism
Biol Psychiatry
CpG islands in vertebrate genomes
J Mol Biol
Sp5, a new member of the Sp1 family, is dynamically expressed during development and genetically interacts with Brachyury
Dev Biol
Tbr1 regulates differentiation of the preplate and layer 6
Neuron
Direct binding of reelin to VLDL receptor and ApoE receptor 2 induces tyrosine phosphorylation of disabled-1 and modulates tau phosphorylation
Neuron
Methyl-CpG-binding protein, MeCP2, is a target molecule for maintenance DNA methyltransferase, Dnmt1
J Biol Chem
Olfactory learning deficit in heterozygous reeler mice
Brain Res
The RXR heterodimers and orphan receptors
Cell
DNA methylation and silencing of gene expression
TEM
Histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen
J Biol Chem
Hypermethylation of the reelin (RELN) promoter in the brain of schizophrenic patients: a preliminary report
Am J Med Genet B Neuropsychiatr Genet
Chromatin alterations associated with down-regulated metabolic gene expression in the prefrontal cortex of subjects with schizophrenia
Arch Gen Psychiatry
Regional and cellular patterns of reelin mRNA expression in the forebrain of the developing and adult mouse
J Neurosci
Rett syndrome: methyl-CpG-binding protein 2 mutations and phenotype-genotype correlations
Am J Med Genet
Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2
Nat Genet
Increased GABAA receptor binding in superficial layers of cingulate cortex in schizophrenics
J Neurosci
Epigenetics-an epicenter of gene regulation: histones and histone-modifying enzymes
Angew Chem Int Ed Engl
Chromosomal regulation by MeCP2: structural and enzymatic considerations
Cell Mol Life Sci
MeCP2 in neurons: closing in on the cause of Rett syndrome
Hum Mol Genet
A decade of molecular biology of retinoic acid receptors
FASEB J
Deficiency of methyl-CpG binding protein-2 in CNS neurons results in a Rett-like phenotype in mice
Nat Genet
On the epigenetic regulation of the human reelin promoter
Nucleic Acids Res
REELIN and schizophrenia: a disease at the interface of the genome and the epigenome
Mol Interv
Epigenetic downregulation of GABAergic function in schizophrenia: potential for pharmacological intervention?
Mol Interv
GABAergic cortical neuron chromatin as a putative target to treat schizophrenia vulnerability
Crit Rev Neurobiol
Methylation matters
J Med Genet
Apoer2: a reelin receptor to remember
Neuron
A protein related to extracellular matrix proteins deleted in the mouse mutant reeler
Nature
The human reelin gene: isolation, sequencing, and mapping on chromosome 7
Genome Res
A reelin-integrin receptor interaction regulates Arc mRNA translation in synapto-neurosomes
Proc Natl Acad Sci U S A
Reelin and glutamic acid decarboxylase67 promoter remodeling in an epigenetic methionine-induced mouse model of schizophrenia
Proc Natl Acad Sci U S A
Interstioal white matter neurons express less reelin and are abnormally distributed in schizophrenia: towards an integration of molecular and morphologic aspects of the neurodevelopmental hypothesis
Mol Psychiatry
Dnmt cooperativity-the developing links between methylation, chromatin structure and cancer
BioEssays
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2017, Biochimica et Biophysica Acta - Molecular Basis of DiseaseCitation Excerpt :Reports showing up-regulation of reelin following tissue injury in several organs [22–29] proposed a role for reelin in tissue repair and our previous observations, revealing reelin involvement in the homeostasis of the small intestine epithelium [5], are consistent with this role. Several genetic and epigenetic mechanisms regulate reelin gene expression (see Grayson et al. [30] for a review). The reelin promoter region contains recognition sites for the transcription factors Sp1 and Tbr1 [30] and both factors up-regulate reelin expression in brain [31].