DNA-methyltransferase1 (DNMT1) binding to CpG rich GABAergic and BDNF promoters is increased in the brain of schizophrenia and bipolar disorder patients.
ABSTRACT: The down regulation of glutamic acid decarboxylase67 (GAD1), reelin (RELN), and BDNF expression in brain of schizophrenia (SZ) and bipolar (BP) disorder patients is associated with overexpression of DNA methyltransferase1 (DNMT1) and ten-eleven translocase methylcytosine dioxygenase1 (TET1). DNMT1 and TET1 belong to families of enzymes that methylate and hydroxymethylate cytosines located proximal to and within cytosine phosphodiester guanine (CpG) islands of many gene promoters, respectively. Altered promoter methylation may be one mechanism underlying the down-regulation of GABAergic and glutamatergic gene expression. However, recent reports suggest that both DNMT1 and TET1 directly bind to unmethylated CpG rich promoters through their respective Zinc Finger (ZF-CXXC) domains. We report here, that the binding of DNMT1 to GABAergic (GAD1, RELN) and glutamatergic (BDNF-IX) promoters is increased in SZ and BP disorder patients and this increase does not necessarily correlate with enrichment in promoter methylation. The increased DNMT1 binding to these promoter regions is detected in the cortex but not in the cerebellum of SZ and BP disorder patients, suggesting a brain region and neuron specific dependent mechanism. Increased binding of DNMT1 positively correlates with increased expression of DNMT1 and with increased binding of MBD2. In contrast, the binding of TET1 to RELN, GAD1 and BDNF-IX promoters failed to change. These data are consistent with the hypothesis that the down-regulation of specific GABAergic and glutamatergic genes in SZ and BP disorder patients may be mediated, at least in part, by a brain region specific and neuronal-activity dependent DNMT1 action that is likely independent of its DNA methylation activity.
Project description:We have recently reported that mice born from dams stressed during pregnancy (PRS mice), in adulthood, have behavioral deficits reminiscent of behaviors observed in schizophrenia (SZ) and bipolar (BP) disorder patients. Furthermore, we have shown that the frontal cortex (FC) and hippocampus of adult PRS mice, like that of postmortem chronic SZ patients, are characterized by increases in DNA-methyltransferase 1 (DNMT1), ten-eleven methylcytosine dioxygenase 1 (TET1) and exhibit an enrichment of 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC) at neocortical GABAergic and glutamatergic gene promoters. Here, we show that the behavioral deficits and the increased 5MC and 5HMC at glutamic acid decarboxylase 67 (Gad1), reelin (Reln) and brain-derived neurotrophic factor (Bdnf) promoters and the reduced expression of the messenger RNAs (mRNAs) and proteins corresponding to these genes in FC of adult PRS mice is reversed by treatment with clozapine (5 mg kg(-1) twice a day for 5 days) but not by haloperidol (1 mg kg(-1) twice a day for 5 days). Interestingly, clozapine had no effect on either the behavior, promoter methylation or the expression of these mRNAs and proteins when administered to offspring of nonstressed pregnant mice. Clozapine, but not haloperidol, reduced the elevated levels of DNMT1 and TET1, as well as the elevated levels of DNMT1 binding to Gad1, Reln and Bdnf promoters in PRS mice suggesting that clozapine, unlike haloperidol, may limit DNA methylation by interfering with DNA methylation dynamics. We conclude that the PRS mouse model may be useful preclinically in screening for the potential efficacy of antipsychotic drugs acting on altered epigenetic mechanisms. Furthermore, PRS mice may be invaluable for understanding the etiopathogenesis of SZ and BP disorder and for predicting treatment responses at early stages of the illness allowing for early detection and remedial intervention.
Project description:Both Reelin (RELN) and glutamate decarboxylase 67 (GAD1) have been implicated in the pathophysiology of Autism Spectrum Disorders (ASD). We have previously shown that both mRNAs are reduced in the cerebella (CB) of ASD subjects through a mechanism that involves increases in the amounts of MECP2 binding to the corresponding promoters. In the current study, we examined the expression of RELN, GAD1, GAD2, and several other mRNAs implicated in this disorder in the frontal cortices (FC) of ASD and CON subjects. We also focused on the role that epigenetic processes play in the regulation of these genes in ASD brain. Our goal is to better understand the molecular basis for the down-regulation of genes expressed in GABAergic neurons in ASD brains. We measured mRNA levels corresponding to selected GABAergic genes using qRT-PCR in RNA isolated from both ASD and CON groups. We determined the extent of binding of MECP2 and DNMT1 repressor proteins by chromatin immunoprecipitation (ChIP) assays. The amount of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) present in the promoters of the target genes was quantified by methyl DNA immunoprecipitation (MeDIP) and hydroxyl MeDIP (hMeDIP). We detected significant reductions in the mRNAs associated with RELN and GAD1 and significant increases in mRNAs encoding the Ten-eleven Translocation (TET) enzymes 1, 2, and 3. We also detected increased MECP2 and DNMT1 binding to the corresponding promoter regions of GAD1, RELN, and GAD2. Interestingly, there were decreased amounts of 5mC at both promoters and little change in 5hmC content in these same DNA fragments. Our data demonstrate that RELN, GAD1, and several other genes selectively expressed in GABAergic neurons, are down-regulated in post-mortem ASD FC. In addition, we observed increased DNMT1 and MECP2 binding at the corresponding promoters of these genes. The finding of increased MECP2 binding to the RELN, GAD1 and GAD2 promoters, with reduced amounts of 5mC and unchanged amounts of 5hmC present in these regions, suggests the possibility that DNMT1 interacts with and alters MECP2 binding properties to selected promoters. Comparisons between data obtained from the FC with CB studies showed some common themes between brain regions which are discussed.
Project description:Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by symptoms related to altered social interactions/communication and restricted and repetitive behaviors. In addition to genetic risk, epigenetic mechanisms (which include DNA methylation/demethylation) are thought to be important in the etiopathogenesis of ASD. We studied epigenetic mechanisms underlying the transcriptional regulation of candidate genes in cerebella of ASD patients, including the binding of MeCP2 (methyl CpG binding protein-2) to the glutamic acid decarboxylase 67 (GAD1), glutamic acid decarboxylase 65 (GAD2), and Reelin (RELN) promoters and gene bodies. Moreover, we performed methyl DNA immunoprecipitation (MeDIP) and hydroxymethyl DNA immunoprecipitation (hMeDIP) to measure total 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in the same regions of these genes. The enrichment of 5-hmC and decrease in 5-mC at the GAD1 or RELN promoters detected by 5-hmC and 5-mC antibodies was confirmed by Tet-assisted bisulfite (TAB) pyrosequencing. The results showed a marked and significant increase in MeCP2 binding to the promoter regions of GAD1 and RELN, but not to the corresponding gene body regions in cerebellar cortex of ASD patients. Moreover, we detected a significant increase in TET1 expression and an enrichment in the level of 5-hmC, but not 5-mC, at the promoters of GAD1 and RELN in ASD when compared with CON. Moreover, there was increased TET1 binding to these promoter regions. These data are consistent with the hypothesis that an increase of 5-hmC (relative to 5-mC) at specific gene domains enhances the binding of MeCP2 to 5-hmC and reduces expression of the corresponding target genes in ASD cerebella.
Project description:Reduction of prefrontal cortex glutamic acid decarboxylase (GAD67) and reelin (mRNAs and proteins) expression is the most consistent finding reported by several studies of postmortem schizophrenia (SZ) brains. Converging evidence suggests that the reduced GAD67 and reelin expression in cortical GABAergic interneurons of SZ brains is the consequence of an epigenetic hypermethylation of RELN and GAD67 promoters very likely mediated by the overexpression of DNA methyltransferase 1 in cortical GABAergic interneurons. Studies of the molecular mechanisms (DNA methylation plus related chromatin remodeling factors) that cause the down-regulation of reelin and GAD67 in SZ brains have important implications not only to understand the disease pathogenesis but also to improve present pharmacological interventions to treat SZ. The mouse treated with l-methionine models some of the molecular neuropathologies detected in SZ, including the hypermethylation of RELN promoter CpG islands and the down-regulation of reelin and GAD67 expression. We now report that in these mice, RELN and GAD67 promoters express an increased recruitment of methyl-CpG binding domain proteins. In these mice the histone deacetylase inhibitor valproate, which increases acetylated histone content in cortical GABAergic interneurons, also prevents MET-induced RELN promoter hypermethylation and reduces the methyl-CpG binding domain protein binding to RELN and GAD67 promoters. These findings suggest that DNA hypermethylation and the associated chromatin remodeling may be critically important in mediating the epigenetic down-regulation of reelin and GAD67 expression detected in cortical GABAergic interneurons of SZ patients.
Project description:Based on postmortem brain studies, our overarching epigenetic hypothesis is that chronic schizophrenia (SZ) is a psychopathological condition involving dysregulation of the dynamic equilibrium among DNA methylation/demethylation network components and the expression of SZ target genes, including GABAergic and glutamatergic genes. SZ has a natural course, starting with a prodromal phase, a first episode that occurs in adolescents or in young adults, and later deterioration over the adult years. Hence, the epigenetic status at each neurodevelopmental stage of the disease cannot be studied just in postmortem brain of chronic SZ patients, but requires the use of neurodevelopmental animal models. We have directed the focus of our research toward studying the epigenetic signature of the SZ brain in the offspring of dams stressed during pregnancy (PRS mice). Adult PRS mice have behavioral deficits reminiscent of behaviors observed in psychotic patients. The adult PRS brain, like that of postmortem chronic SZ patients, is characterized by a significant increase in DNA methyltransferase 1, Tet methylcytosine dioxygenase 1 (TET1), 5-methylcytosine, and 5-hydroxymethylcytosine at SZ candidate gene promoters and a reduction in the expression of glutamatergic and GABAergic genes. In PRS mice, measurements of epigenetic biomarkers for SZ can be assessed at different stages of development with the goal of further elucidating the pathophysiology of this disease and predicting treatment responses at specific stages of the illness, with particular attention to early detection and possibly early intervention.
Project description:Major psychiatric disorders such as schizophrenia (SZ) and bipolar disorder (BP) with psychosis (BP+) express a complex symptomatology characterized by positive symptoms, negative symptoms, and cognitive impairment. Postmortem studies of human SZ and BP+ brains show considerable alterations in the transcriptome of a variety of cortical structures, including multiple mRNAs that are downregulated in both inhibitory GABAergic and excitatory pyramidal neurons compared with non-psychiatric subjects (NPS). Several reports show increased expression of DNA methyltransferases in telencephalic GABAergic neurons. Accumulating evidence suggests a critical role for altered DNA methylation processes in the pathogenesis of SZ and related psychiatric disorders. The establishment and maintenance of CpG site methylation is essential during central nervous system differentiation and this methylation has been implicated in synaptic plasticity, learning, and memory. Atypical hypermethylation of candidate gene promoters expressed in GABAergic neurons is associated with transcriptional downregulation of the corresponding mRNAs, including glutamic acid decarboxylase 67 (GAD67) and reelin (RELN). Recent reports indicate that the methylation status of promoter proximal CpG dinucleotides is in a dynamic balance between DNA methylation and DNA hydroxymethylation. Hydroxymethylation and subsequent DNA demethylation is more complex and involves additional proteins downstream of 5-hydroxymethylcytosine, including members of the base excision repair (BER) pathway. Recent advances in our understanding of altered CpG methylation, hydroxymethylation, and active DNA demethylation provide a framework for the identification of new targets, which may be exploited for the pharmacological intervention of the psychosis associated with SZ and possibly BP+.
Project description:Prenatal stress (PRS) is considered a risk factor for several neurodevelopmental disorders including schizophrenia (SZ). An animal model involving restraint stress of pregnant mice suggests that PRS induces epigenetic changes in specific GABAergic and glutamatergic genes likely to be implicated in SZ, including the gene for brain-derived neurotrophic factor (BDNF).Studying adult offspring of pregnant mice subjected to PRS, we explored the long-term effects of PRS on behavior and on the expression of key chromatin remodeling factors including DNA methyltransferase 1, ten-eleven-translocation hydroxylases, methyl CpG binding protein 2, histone deacetylases, and histone methyltransferases and demethylase in the frontal cortex and hippocampus. We also measured the expression of BDNF.Adult PRS offspring demonstrate behavioral abnormalities suggestive of SZ and molecular changes similar to changes seen in postmortem brains of patients with SZ. This includes a significant increase in DNA methyltransferase 1 and ten-eleven-translocation hydroxylase 1 in the frontal cortex and hippocampus but not in cerebellum; no changes in histone deacetylases, histone methyltransferases and demethylases, or methyl CpG binding protein 2, and a significant decrease in Bdnf messenger RNA variants. The decrease of the corresponding Bdnf transcript level was accompanied by an enrichment of 5-methylcytosine and 5-hydroxymethylcytosine at Bdnf gene regulatory regions. In addition, the expression of Bdnf transcripts (IV and IX) correlated positively with social approach in both PRS mice and nonstressed mice.Because patients with psychosis and PRS mice show similar epigenetic signature, PRS mice may be a suitable model for understanding the behavioral and molecular epigenetic changes observed in patients with SZ.
Project description:Cortical GABAergic dysfunction, a hallmark of both schizophrenia (SZ) and bipolar (BP) disorder pathophysiologies may relate to the hypermethylation of GABAergic gene promoters (i.e., reelin and GAD67). Benefits elicited by a combination of atypical antipsychotics with valproate (VPA) (a histone deacetylase inhibitor that may also activate brain DNA demethylation) in SZ or BP disorder treatment prompted us to investigate whether the beneficial action of this association depends on induction of a putative DNA demethylase activity. To monitor this activity, we measured the ratio of 5-methyl cytosine to unmethylated cytosine in reelin and GAD67 promoters in the mouse frontal cortex and striatum. We compared normal mice with mice pretreated with l-methionine (5.2 mmol/kg s.c. twice a day for 7 days) to hypermethylate promoters, including reelin and GAD67. Clinically relevant doses of clozapine (CLZ) (3.8 to 15 micromol/kg twice a day s.c. for 3 days) and sulpiride (SULP) (12.5 to 50 micromol/kg twice a day for 3 days) but not clinically relevant doses of haloperidol (HAL) (1.3 to 4 micromol/kg twice a day s.c. for 3 days) or olanzapine (OLZ) (4 to 15 micromol/kg twice a day for 3 days) exhibited dose-related increases in the cortical and striatal demethylation of hypermethylated reelin and GAD67 promoters. These effects of CLZ and SULP were dramatically potentiated by a clinically relevant VPA dose (0.5 mmol/kg twice a day for 3 days). By activating a DNA demethylase, the association of CLZ or SULP with VPA may facilitate a chromatin remodeling that normalizes the GABAergic gene expression down-regulation detected in the telencephalic regions of SZ and BP patients.
Project description:KRAS activation drives DNA methylation and silencing of specific tumor suppressor genes (TSGs). We previously showed that the ERK pathway induces transcriptional repression of TET1, which results in conversion of TSG promoters from a hydroxymethylated, active state to a hypermethylated and silenced state. Here we identified miR-29b as a KRAS-induced molecule that represses TET1 expression. In KRAS-transformed cells, ectopic miR-29b inhibition restores expression of TET1, thereby reactivating TSGs by reducing methylation and restoring hydroxymethylation. Mining gene expression data of lung cancer cell lines identified additional TSGs suppressed by KRAS signaling whose expression was restored by inhibition of miR-29b and re-expression of TET1. Because KRAS changes TSG promoters from hydroxymethylated to hypermethylated with miR-29b-dependent silencing of TET1, we demonstrate a model in which DNMT1 is present on target promoters prior to KRAS transformation. In addition, we propose miR-29b as a potential circulating biomarker and target for rational treatment of specific malignancies.
Project description:Reduced expression of the Gad1 gene-encoded 67-kDa protein isoform of glutamic acid decarboxylase (GAD67) is a hallmark of schizophrenia. GAD67 downregulation occurs in multiple interneuronal sub-populations, including the parvalbumin-positive (PVALB+) cells. To investigate the role of the PV-positive GABAergic interneurons in behavioral and molecular processes, we knocked down the Gad1 transcript using a microRNA engineered to target specifically Gad1 mRNA under the control of Pvalb bacterial artificial chromosome. Verification of construct expression was performed by immunohistochemistry. Follow-up electrophysiological studies revealed a significant reduction in ?-aminobutyric acid (GABA) release probability without alterations in postsynaptic membrane properties or changes in glutamatergic release probability in the prefrontal cortex pyramidal neurons. Behavioral characterization of our transgenic (Tg) mice uncovered that the Pvalb/Gad1 Tg mice have pronounced sensorimotor gating deficits, increased novelty-seeking and reduced fear extinction. Furthermore, NMDA (N-methyl-d-aspartate) receptor antagonism by ketamine had an opposing dose-dependent effect, suggesting that the differential dosage of ketamine might have divergent effects on behavioral processes. All behavioral studies were validated using a second cohort of animals. Our results suggest that reduction of GABAergic transmission from PVALB+ interneurons primarily impacts behavioral domains related to fear and novelty seeking and that these alterations might be related to the behavioral phenotype observed in schizophrenia.