Psychiatric Institute, Department of Psychiatry, 1601 West Taylor Street, M/C 912, College of Medicine, University of Illinois, Chicago, IL 60612, USA.
Reln mRNA and protein levels are reduced by approximately 50% in various cortical structures of post-mortem brain from patients diagnosed with schizophrenia or bipolar illness with psychosis. To study mechanisms responsible for this down-regulation, we have analyzed the promoter of the human reelin gene. We show that the reelin promoter directs expression of a reporter construct in multiple human cell types: neuroblastoma cells (SHSY5Y), neuronal precursor cells (NT2), differentiated neurons (hNT) and hepatoma cells (HepG2). Deletion constructs confirmed the presence of multiple elements regulating Reln expression, although the promoter activity is promiscuous, i.e. activity did not correlate with expression of the endogenous gene as reflected in terms of reelin mRNA levels. Co-transfection of the -514 bp human reelin promoter with either Sp1 or Tbr1 demonstrated that these transcription factors activate reporter expression by 6- and 8.5-fold, respectively. Within 400 bp of the RNA start site there are 100 potential CpG targets for DNA methylation. Retinoic acid (RA)-induced differentiation of NT2 cells to hNT neurons was accompanied by increased reelin expression and by the appearance of three DNase I hypersensitive sites 5' to the RNA start site. RA-induced differentiation was also associated with demethylation of the reelin promoter. To test if methylation silenced reelin expression, we methylated the promoter in vitro prior to transfection. In addition, we treated NT2 cells with the methylation inhibitor aza-2'-deoxycytidine and observed a 60-fold increase in reelin mRNA levels. The histone deacetylase inhibitors trichostatin A (TSA) and valproic acid also induced expression of the endogenous reelin promoter, although TSA was considerably more potent. These findings indicate that one determinant responsible for regulating reelin expression is the methylation status of the promoter. Our data also raise the interesting possibility that the down-regulation of reelin expression documented in psychiatric patients might be the consequence of inappropriate promoter hypermethylation.
Latest citations:
PLoS One. 2011 ;6 (5):e19955
21603580
Department of Genetics, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.
Reelin plays an important role in the development and function of the brain and has been linked to different neuropsychiatric diseases. To further clarify the connection between reelin and psychiatric disorders, we studied the factors that influence the expression of reelin gene (RELN) and its different isoforms. We examined the total expression of RELN, allelic expression, and two alternative RELN isoforms in postmortem brain samples from patients with schizophrenia and bipolar disorder, as well as unaffected controls. We did not find a significant reduction in the total expression of RELN in schizophrenia or bipolar disorder. However, we did find a significant reduction of the proportion of the short RELN isoform, missing the C-terminal region in bipolar disorder, and imbalance in the allelic expression of RELN in schizophrenia. In addition, we tested the association between variation in RELN expression and rs7341475, an intronic SNP that was found to be associated with schizophrenia in women. We did not find an association between rs7341474 and the total expression of RELN either in women or in the entire sample. However, we observed a nominally significant effect of genotype-by-sex interaction on the variation in microexon skipping. Women with the risk genotype of rs7341475 (GG) had a higher proportion of microexon skipping, which is the isoform predominant in tissues outside the brain, while men had the opposite trend. Finally, we tested 83 SNPs in the gene region for association with expression variation of RELN, but none were significant. Our study further supports the connection between RELN dysfunction and psychiatric disorders, and provides a possible functional role for a schizophrenia associated SNP. Nevertheless, the positive associations observed in this study needs further replication as it may have implications for understanding the biological causes of schizophrenia and bipolar disorder.
The Psychiatric Institute, University of Illinois at Chicago, 1601 W. Taylor St., Chicago, IL 60612, USA.
Background. Studies have implicated abnormalities in epigenetic gene regulation in schizophrenia. Presentation. We hypothesize that identifying abnormalities in chromatin structure and the epigenetic machinery in peripheral blood mononuclear cells (PBMC) from schizophrenia patients could (a) help characterize a subset of schizophrenia patients and (b) lead to targeted pharmacological interventions. Testing. Investigate the relationship between clinical symptoms, demographics, hormonal fluctuations, substance abuse, disease characteristics across the major mental illnesses, and epigenetic parameters in PBMC. In addition, examine the effects of individual antipsychotics, mood stabilizers, as well as experimental agents both as clinically prescribed as well as in cultured PBMC to understand the effects of these agents on chromatin. Implications. If PBMC could serve as a reliable model of overall epigenetic mechanisms then this could lead to a "biomarker" approach to revealing pathological chromatin state in schizophrenia. This approach may provide an informed method for selecting chromatin modifying agents for psychiatric disorders.
Department of Biology, University of Bologna, Bologna, Italy. antonio.contestabile@unibo.it.
Valproic acid (VPA, 2-propylpentanoic acid) has been widely used as an antiepileptic drug and for the therapy of bipolar disorders for several years. Its mechanism of action was initially found to be primarily related to neurotransmission and modulation of intracellular pathways. More recently, it emerged as an anti-neoplastic agent as well, by acting on cell growth, differentiation and apoptosis. Here, it mainly exerts its effect by regulating gene expression at the molecular level, through epigenetic mechanisms. In particular, it has been demonstrated the effect of VPA in chromatin remodeling, as VPA directly inhibits histone deacetylases (HDACs) activity. Interestingly, it has been observed that these biochemical and molecular pathways are involved not only in beneficial effect of VPA against epilepsy and malignancies, but they are also responsible for more general neuroprotective mechanisms. In particular, it has been demonstrated that VPA is neuroprotective in several models of neurodegenerative diseases. Moreover, due to the involvement of the VPA-affected mechanisms in complex behaviors, VPA is increasingly used as a psychotherapeutic agent. This review summarizes the more recent data on VPA neuroprotective mechanisms at the biochemical, molecular and epigenetic levels, focusing on both in vitro and in vivo models of neurodegenerative diseases. In particular, attention is paid to mechanisms by which VPA affects neuronal survival/apoptosis and proliferation/differentiation balance, as well as synaptic plasticity, by acting both directly on neurons and indirectly through glial cells. Perspective applications of the VPA neuroprotective potential in human neurodegenerative diseases are discussed, when relevant.
Br J Pharmacol. 2009 Dec 15;:
20015091
Cit:4
Department of Pharmacology and the National Research Centre for Growth and Development, The University of Auckland, Auckland, New Zealand.
Epigenetics is a rapidly growing field and holds great promise for a range of human diseases, including brain disorders such as Rett syndrome, anxiety and depressive disorders, schizophrenia, Alzheimer disease and Huntington disease. This review is concerned with the pharmacology of epigenetics to treat disorders of the epigenome whether induced developmentally or manifested/acquired later in life. In particular, we will focus on brain disorders and their treatment by drugs that modify the epigenome. While the use of DNA methyl transferase inhibitors and histone deacetylase inhibitors in in vitro and in vivo models have demonstrated improvements in disease-related deficits, clinical trials in humans have been less promising. We will address recent advances in our understanding of the complexity of the epigenome with its many molecular players, and discuss evidence for a compromised epigenome in the context of an ageing or diseased brain. We will also draw on examples of species differences that may exist between humans and model systems, emphasizing the need for more robust pre-clinical testing. Finally, we will discuss fundamental issues to be considered in study design when targeting the epigenome.
Neuroreport. 2009 Dec 1;:
19952965
Cit:2
aLaboratory of Molecular Psychiatry and Neurogenetics, University 'Campus Bio-Medico' bThe Laboratory of Molecular Psychiatry and Psychiatric Genetics, Department of Experimental Neurosciences, I.R.C.C.S.'Fondazione Santa Lucia', Rome, Italy.
Reelin plays a pivotal role in neurodevelopment. Excessive RELN promoter methylation and/or decreased RELN gene expression have been described in schizophrenia and autism. We assessed RELN promoter methylation in post-mortem temporocortical tissue (Brodmann Area 41/42) of three prepuberal and six postpuberal normal individuals. The former display very little or no methylation, whereas most postpuberal individuals are heavily methylated, especially at CpG positions located between -131 and -98 bp (prepuberal vs. postpuberal, P<0.05). Sex hormones thus seemingly boost DNA methylation at the RELN promoter. This physiological change could significantly contribute to the onset of schizophrenia and the worsening of autistic behaviors, both typically occurring at puberty.
Biol Psychiatry. 2009 Nov 16;:
19922905
Cit:5
Juho Wedenoja,
Annamari Tuulio-Henriksson,
Jaana Suvisaari,
Anu Loukola,
Tiina Paunio,
Timo Partonen,
Teppo Varilo,
Jouko Lönnqvist,
Leena Peltonen
Institute for Molecular Medicine Finland FIMM, University of Helsinki and National Institute for Health and Welfare, Helsinki University Central Hospital, Helsinki, Finland; Department of Medical Genetics, Helsinki University Central Hospital, Helsinki, Finland.
BACKGROUND: The challenges in gene identification for psychiatric disorders have awakened interest toward quantitative traits and endophenotypes that are potentially more closely related to the underlying biology and provide more power in the linkage and association analyses. Previously, we successfully replicated schizophrenia linkage on chromosome 7q21-32 in Finnish families and demonstrated that an intragenic short tandem repeat (STR) allele of the regional Reelin (RELN) gene is associated with multiple cognitive traits representing central cognitive functions regarded as valid endophenotypes for schizophrenia. METHODS: Here, we used an extended sample of 290 Finnish families with schizophrenia and 375 control subjects in an association analysis between 96 SNPs and three STRs in RELN and diagnostic categories, clinical disorder features, as well as central cognitive functions impaired in schizophrenia. RESULTS: We replicated the original association between RELN intragenic STR allele and working memory in individuals (n = 342) not overlapping with the previous study. This risk allele remained central in the whole study sample by being associated with impaired cognitive functioning and more severe positive and negative symptoms of schizophrenia (p =.0005-.00002). Additionally, multiple SNPs indicated association with the severity of positive symptoms of schizophrenia and together showed potential additive effect on the severity of the symptoms (p =.0000001). However, no significant associations with clinical diagnostic categories emerged. CONCLUSIONS: The strongest effects on cognitive functions were detected among the affected individuals. We thus propose a particular role for RELN as a modifier gene of the pathogenesis of schizophrenia.
J Neural Transm. 2009 Sep 26;:
19784753
Cit:5
Laboratory of Psychobiology, Department of Psychiatry, Rambam Medical Center and Faculty of Medicine, Rappaport Family Institute for Research in the Medical Sciences, Technion IIT, Haifa, Israel, shachar@tx.technion.ac.il.
Schizophrenia is currently believed to result from variations in multiple genes, each contributing a subtle effect, which combines with each other and with environmental stimuli to impact both early and late brain development. At present, schizophrenia clinical heterogeneity as well as the difficulties in relating cognitive, emotional and behavioral functions to brain substrates hinders the identification of a disease-specific anatomical, physiological, molecular or genetic abnormality. Mitochondria play a pivotal role in many essential processes, such as energy production, intracellular calcium buffering, transmission of neurotransmitters, apoptosis and ROS production, all either leading to cell death or playing a role in synaptic plasticity. These processes have been well established as underlying altered neuronal activity and thereby abnormal neuronal circuitry and plasticity, ultimately affecting behavioral outcomes. The present article reviews evidence supporting a dysfunction of mitochondria in schizophrenia, including mitochondrial hypoplasia, impairments in the oxidative phosphorylation system (OXPHOS) as well as altered mitochondrial-related gene expression. Abnormalities in mitochondrial complex I, which plays a major role in controlling OXPHOS activity, are discussed. Among them are schizophrenia specific as well as disease-state-specific alterations in complex I activity in the peripheral tissue, which can be modulated by DA. In addition, CNS and peripheral abnormalities in the expression of three of complex I subunits, associated with parallel alterations in their transcription factor, specificity protein 1 (Sp1) are reviewed. Finally, this review discusses the question of disease specificity of mitochondrial pathologies and suggests that mitochondria dysfunction could cause or arise from anomalities in processes involved in brain connectivity.
Eur J Neurosci. 2009 Jun 10;:
19508697
Cit:17
Brain Research Institute, Medical Faculty of the University of Zürich, and Department of Biology, Swiss Federal Institute of Technology, Zürich, Switzerland.
Epigenetic mechanisms are not only essential for biological functions requiring stable molecular changes such as the establishment of cell identity and tissue formation, they also constitute dynamic intracellular processes for translating environmental stimuli into modifications in gene expression. Over the past decade it has become increasingly clear that both aspects of epigenetic mechanisms play a pivotal role in complex brain functions. Evidence from patients with neurodegenerative and neurodevelopmental disorders such as Alzheimer's disease and Rett syndrome indicated that epigenetic mechanisms and chromatin remodeling need to be tightly controlled for proper cognitive functions, and their dysregulation can have devastating consequences. However, because they are dynamic, epigenetic mechanisms are also potentially reversible and may provide powerful means for pharmacological intervention. This review outlines major cognitive disorders known to be associated with epigenetic dysregulation, and discusses the potential of 'epigenetic medicine' as a promising cure.
Centre for Nutrition and Food Safety, School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK.
The cost of psychiatric illness to the UK economy was recently estimated at pound77 billion annually. Despite years of research no firm aetiological explanation exists, and with no physiological or biochemical markers diagnosis is made entirely on a behavioural basis. All current pharmacological therapies are associated with serious long-term side effects. Substantial evidence supports the involvement of one-carbon cycle dysregulation in psychiatric illness, but this is not currently used as a basis for diagnosis or treatment. The present paper reviews the evidence for one-carbon cycle dysregulation in schizophrenic, bipolar, depressed and autistic patients. Also presented are novel findings from the field of epigenetics, which demonstrate how the one-carbon cycle-derived methyl donor S-adenosylmethionine influences the expression of key genes in the brain affecting memory, learning, cognition and behaviour, genes whose expression is reduced to varying degrees in these patient groups. Clinical evidence that nutritional supplements can rectify one-carbon cycle activity, and restore normal gene expression, suggests a novel approach to the development of biochemical tests and simple, non-harmful treatments for some psychiatric patients. Conversely, evidence from animal studies highlights the dangers of exposing the unborn fetus to very high dietary levels of folic acid, a one-carbon cycle cofactor. Fetal adaptations to a high-folate environment may interfere with folate metabolism postnatally, with serious consequences for the epigenetic regulation of gene expression. The public health implications of these diverse scenarios indicate an urgent need for further research in this field.
Mol Pharmacol. 2008 Nov 24;:
19029285
Cit:15
University of Illinois @ Chicago.
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.
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Pharmacol Ther. 2006 Mar 28;:
16574235
Cit:16
Dennis R Grayson,
Ying Chen,
Erminio Costa,
Erbo Dong,
Alessandro Guidotti,
Marija Kundakovic,
Rajiv P Sharma
The Psychiatric Institute, Department of Psychiatry, College of Medicine, University of Illinois at Chicago, 1601 W. Taylor St., Chicago, IL 60612, United States.
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.
Dennis R Grayson,
Xiaomei Jia,
Ying Chen,
Rajiv P Sharma,
Colin P Mitchell,
Alessandro Guidotti,
Erminio Costa
Psychiatric Institute, Department of Psychiatry, College of Medicine, University of Illinois, 1601 West Taylor Street, Chicago, IL 60612, USA. dgrayson@psych.uic.edu
Reelin mRNA and protein levels are reduced by approximately 50% in various cortical structures of postmortem brain from patients diagnosed with schizophrenia or bipolar illness with psychosis. In addition, the mRNA encoding the methylating enzyme, DNA methyltransferase 1, is up-regulated in the same neurons that coexpress reelin and glutamic acid decarboxylase 67. We have analyzed the extent and pattern of methylation within the CpG island of the reelin promoter in genomic DNA isolated from cortices of schizophrenia patients and nonpsychiatric subjects. Ten (The Stanley Foundation Neuropathology Consortium) and five (Harvard Brain Collection) schizophrenia patients and an equal number of nonpsychiatric subjects were selected from each brain collection. Genomic DNA was isolated, amplified (from base pair -527 to base pair +322) after bisulphite treatment, and sequenced. The results show that within the promoter region there were interesting regional variations. There was increased methylation at positions -134 and 139, which is particularly important for regulation, because this portion of the promoter is functionally competent based on transient transfection assays. This promoter region binds a protein present in neuronal precursor nuclear extracts that express very low levels of reelin mRNA; i.e., an oligonucleotide corresponding to this region and that contains methylated cytosines binds more tightly to extracts from nonexpressing cells than the nonmethylated counterpart. Collectively, the data show that this promoter region has positive and negative properties and that the function of this complex cis element relates to its methylation status.
Jai Sung Noh,
Rajiv P Sharma,
Marin Veldic,
Alain A Salvacion,
Xiaomei Jia,
Ying Chen,
Erminio Costa,
Alessandro Guidotti,
Dennis R Grayson
Psychiatric Institute, Department of Psychiatry, College of Medicine, University of Illinois, 1601 West Taylor Street, Chicago, IL 60612, USA.
The polygenic nature of complex psychiatric disorders suggests a common pathway that may be involved in the down-regulation of multiple genes through an epigenetic mechanism. To investigate the role of methylation in down-regulating the expression of mRNAs that may be associated with the schizophrenia phenotype, we have adopted a cell-culture model amenable to this line of investigation. We have administered methionine (2 mM) to primary cultures of cortical neurons prepared from embryonic day 16 mice and show that this treatment down-regulated reelin and glutamic acid decarboxylase 67 (GAD67) mRNA expression but not that corresponding to neuron-specific enolase mRNA. Moreover, methionine increased methylation of the reelin promoter, suggesting a possible mechanism for the observed change. These cultures contain a mixed population of neurons and glia. Approximately 83% of the neurons are GABAergic based on GAD immunoreactivity, and these neurons coexpress high levels of reelin and DNA methyltransferase (Dnmt) 1 immunoreactivity. To examine whether Dnmt1 regulates reelin gene expression, we used an antisense approach to reduce (knock down) Dnmt1 expression. The reduced Dnmt1 mRNA and protein were accompanied by increased reelin mRNA expression. More importantly, the Dnmt1 knockdown blocked the methionine-induced reelin and GAD67 mRNA down-regulation. These data support the hypothesis that the reduced amounts of reelin and GAD67 mRNAs documented in postmortem schizophrenia brain may be the consequence of a Dnmt1-mediated hypermethylation of the corresponding promoters.
Erminio Costa,
Ying Chen,
Erbo Dong,
Dennis R Grayson,
Marija Kundakovic,
Ekrem Maloku,
William Brad Ruzicka,
Rosalba Satta,
Marin Veldic,
Adrian Zhubi,
Alessandro Guidotti
Professor and Director, Psychiatric Institute, Department of Psychiatry, University of Illinois at Chicago, 1601 Taylor, Chicago, IL 60612, USA. ecosta@psych.uic.edu , Department of Psychiatry, University of Illinois at Chicago, 1601 Taylor, Chicago, IL 60612, USA. ychen@psych.uic.edu , Department of Psychiatry, University of Illinois at Chicago, 1601 Taylor, Chicago, IL 60612, USA. edong@psych.uic.edu , Department of Psychiatry, University of Illinois at Chicago, 1601 Taylor, Chicago, IL 60612, USA. dgrayson@psych.uic.edu , Department of Psychiatry, University of Illinois at Chicago, 1601 Taylor, Chicago, IL 60612, USA. mkundakovic@psych.uic.edu , Department of Psychiatry, University of Illinois at Chicago, 1601 Taylor, Chicago, IL 60612, USA. emaloku@psych.uic.edu , Department of Psychiatry, University of Illinois at Chicago, 1601 Taylor, Chicago, IL 60612, USA. wruzicka@psych.uic.edu , Department of Psychiatry, University of Illinois at Chicago, 1601 Taylor, Chicago, IL 60612, USA. rsatta@psych.uic.edu , Department of Psychiatry, University of Illinois at Chicago, 1601 Taylor, Chicago, IL 60612, USA. mveldic@psych.uic.edu , Department of Psychiatry, University of Illinois at Chicago, 1601 Taylor, Chicago, IL 60612, USA. azhubi@psych.uic.edu , Scientific Director, Psychiatric Institute, Department of Psychiatry, University of Illinois at Chicago, 1601 Taylor, Chicago, IL 60612, USA. aguidotti@psych.uic.edu.
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.
Mol Pharmacol. 2006 Oct 25;:
17065238
Cit:30
DNA Methyltransferase Inhibitors Coordinately Induce Expression of the Human Reelin and GAD67 Genes.
University of Illinois at Chicago.
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.
Schizophr Res. 2006 Sep 25;:
16996718
Cit:17
Rajiv P Sharma,
Cherise Rosen,
Saritha Kartan,
Alessandro Guidotti,
Erminio Costa,
Dennis R Grayson,
Kayla Chase
The Psychiatric Institute, University of Illinois at Chicago, 1601 West Taylor Street, Chicago, IL 60612, USA.
Levels of acetylated Histone 3 and 4 proteins are strongly predictive of a chromatin structure that is conducive to gene expression. In cell and animal studies, valproic acid is a potent inhibitor of histone deactylating enzymes, and consequently results in increased levels of acetylated Histone 3 (acH3) and acetylated Histone 4 proteins (acH4). To examine this effect in a clinical setting, 14 schizophrenic and bipolar patients were treated with valproic acid (Depakote ER(R)), either as monotherapy or in combination with antipsychotics, over a period of 4 weeks. AcH3 and acH4 levels from lymphocyte nuclear protein extracts were measured by Western Blot. Treatment with Depakote ER resulted in a significant increase of acH3 and a trend-level increase of acH4. Levels of valproic acid were positively and significantly correlated with percent increase in acH3 but not acH4. Schizophrenia patients were significantly less likely to increase their acH3 and acH4 levels after 4 weeks on Depakote ER. The authors consider these results in the context of future application of HDAC inhibitors to the treatment of psychiatric disorders.
The Psychiatric Institute, University of Illinois at Chicago, Chicago, IL 60612, USA. rsharma@psych.uic.edu
The accessibility of cognate binding sites within a gene promoter can be modified by the condensation or relaxation of local chromatin structure. Local chromatin structure is in turn programmed by covalent modifications of cytosine bases in DNA and amino acid residues in histone protein tails. These chemical and physical adaptations around gene promoters can significantly change levels of mRNA expression. Furthermore, linear patterns of covalent modification of histone protein tails are emerging as a distinct regulatory code--another form of cellular memory. Because chromatin structure can be modified by conventional pharmacologic therapy, a novel approach to the regulation of neuronal gene expression in clinical populations is possible.
J Neurochem. 2005 Apr ;93:483-92
15816871
Cit:13
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.
J Comp Neurol. 2011 Dec 1;:
22134929
The Psychiatric Institute, Department of Psychiatry, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA.
DNA methylation is an epigenetic regulatory mechanism commonly associated with transcriptional silencing. DNA methyltransferases (DNMTs) are a family of distantly related proteins that both catalyze the de novo formation of 5-methylcytosine and maintain these methylation marks in cell-specific patterns in virtually all mitotic cells of the body. In the adult brain, methylation occurs in progenitor cells of the neurogenic zones and in post-mitotic neurons. Of the DNMTs, DNMT1 and DNMT3a are most highly expressed in post-mitotic neurons. While it has been commonly thought all post-mitotic neurons and glia express DNMTs at comparable levels, the co-expression of selected DNMTs with markers of distinct neurotransmitter phenotypes has not been previously examined in detail in the mouse. To this end, we analyzed the expression of DNMT1 and DNMT3a along with GAD67 in the brains of the GAD67-GFP knockin mice. After first confirming that GFP immunopositive neurons were also GAD67-positive, we showed that in the motor cortex, piriform cortex, striatum, CA1 region of the hippocampus, dentate gyrus, and basolateral amygdala (BLA), GFP immunofluorescence coincided with the signal corresponding to DNMT1 and DNMT3a. A detailed examination of cortical neurons, particularly in cortical layers III to V, showed that ∼30% of NeuN immunopositive neurons were also DNMT1 positive. These data do not exclude the expression of DNMT1 or DNMT3a in glutamatergic neurons and glia. However, they suggest that their expression is very low compared with the levels present in GABAergic neurons.
Epigenetics. ;5 (8):730-5
20716949
Cit:4
Psychiatric Institute, Department of Psychiatry, College of Medicine, University of Illinois, Chicago, IL, USA. Edong@psych.uic.edu
The methylation and demethylation of CpG dinucleotides that are embedded in promoters play an important role in controlling gene transcription. In the mammalian brain, CpG promoter methylation is a postreplicative process mediated by a group of DNA methyltransferases (DNMT), such as DNMT1 and DNMT3a, DNMT3b. Several studies demonstrate that in addition to DNMTs, promoter methylation in the brain can be regulated by a putative DNA demethylation process that specifically removes the methyl group from the carbon-5 of cytosines. To test the existence of a possible active DNA demethylation activity in postmitotic neuronal or glial cells, we incubated an SssI methylated mouse reelin (Reln) promoter fragment (-720 to +140) with nuclear extracts from the mouse frontal cortex (FC). We observed the presence of DNA demethylation activity, which was increased in FC nuclear extracts from mice treated with valproate (VPA, 2.2 mmol/kg, twice a day for 3 days). VPA not only reduces anxiety, and cognitive deficits, and other symptoms in bipolar disorder (BP) disorder and schizophrenia (SZ) patients but also upregulates Reln and glutamic acid decarboxylase 67 (Gad67) mRNA/protein expression by reducing the methylation of their promoters. We believe that the identification of an enzyme in brain that facilitates DNA-demethylation and an understanding of how drugs induce DNA demethylation are crucial to progress in a new line of pharmacological interventions to treat neurodevelopment, neuropsychiatric, and neurodegenerative diseases.
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Department of Pharmacology and Toxicology, University of Magdeburg, Magdeburg, Germany.
OBJECTIVE The aim of this study was to investigate the effect of the epigenetic modifiers trichostatin A and 5-aza-2'-deoxycytidine on the expression of the cannabinoid receptors CB1 and CB2 and μ-opioid receptors in human SH SY5Y neuroblastoma cells and human Jurkat T lymphocytes. METHODS Using quantitative real-time RT-PCR, mRNA specific for the aforementioned receptors was determined. The functionality of the induced receptors was determined by analyzing the effect of the ligands to regulate intracellular cAMP. RESULTS We demonstrated that treatment of SH SY5Y cells, which endogenously express μ-opioid receptors and CB1, but not CB2, resulted in de novo induction of CB2, while mRNA levels of CB1 and μ-opioid receptors were not significantly altered. In contrast, treatment of Jurkat lymphocytes, which endogenously express CB2, but not CB1 and μ-opioid receptors, resulted in de novo induction of CB1 and μ-opioid receptors, while mRNA levels of CB2 were not significantly altered. Furthermore, the functionality of the induced μ-opioid receptors and CB1 in the Jurkat cells was demonstrated. CONCLUSIONS Our data suggest an epigenetically regulated expression of cannabinoid receptors and μ-opioid receptors. Their induction by epigenetic modifiers in distinct cells of the nervous and immune system might result in increased effects of the cognate drugs on neuronal and immune functions. Such modifications might be useful for novel therapies for various disorders, e.g. multiple sclerosis, where the elevated transmission of cannabinoid or opioid signals is beneficial.
Gene. 2011 Sep 21;:
21963386
SVEP1 gene encodes a cell adhesion molecule (CAM) that was previously shown to be expressed by cells related to skeletal tissues. Here we focus on SVEP1 expression regulation in pre-osteoblastic MBA-15 and mammary adenocarcinoma DA3 cells. We show that SVEP1 message and protein are highly expressed by MBA-15 when compared with DA3 cells. DNA methylation of CpGs sites is an epigenetic mechanism associated with gene silencing. Therefore, we analyzed the methylation status of a region potentially harbors SVEP1 promoter and further activity alterations induced by estrogen (17βE(2)) and TNFα. We also mapped in silico the transcription binding sites namely TFIIB, NF-κB, ERE, AP1 and Sp1 at the putative promoter. Treatments with demethylation reagents, 5'-aza-deoxy-Cytidine (5'-aza-dC), or histone deacetylase inhibitor, Trichostatin A (TSA) resulted with an elevation of SVEP1 mRNA expression in both cell types. Methylation levels of specific CpGs islets located at transcription binding sites were assessed using sodium bisulfite genomic DNA sequencing, methylated DNA immunoprecipitation (meDIP) and Methylation-Specific PCR (MSP). Our results show that the putative promoter of SVEP1 is hypermethylated in DA3- compared with MBA-15 cells, thus regulating SVEP1 expression levels. In addition, by affecting SVEP1 promoter methylation status, 17βE(2) and TNFα regulate its mRNA expression. Our data shed a light on understanding the cell-type specific promoter status for regulation of the SVEP1. Since SVEP1 protein mediates cellular adhesion, this data might be beneficial for the future characterization of SVEP1 expression in the interactions existing in bone.
Int J Oncol. 2011 Jun ;38 (6):1713-8
21424117
Unité MEDyC, UMR URCA-CNRS 6237, IFR53, Facultés de Pharmacie et de Médecine, Université de Reims, 1 avenue du Maréchal Juin, 51096 Reims, France.
The matrix metalloproteinase (MMP) family members play an important role in various physiological and pathological processes. Although MMP-1 (collagenase-1) has been shown to be involved in tumor invasiveness, the regulation of its expression is still not fully elucidated and could implicate epigenetic mechanisms. The aim of this study was to analyze the effects of the Histone Deacetylase Inhibitor (HDI) trichostatin A (TSA) and the inhibitor of DNA methylation 5-aza-2'-deoxycytidine (5-azadC) on the proMMP-1 expression in the human HT1080 fibrosarcoma cell line. Real-time RT-PCR revealed that 5-azadC or 5-azadC + TSA but not TSA alone, despite global histone H4 hyperacetylation, increased proMMP-1 mRNA levels. This transcription activation was correlated with chromatin decondensation determined by nuclear texture image analysis technique. Western blot analysis of cell culture conditioned media revealed a significant increase in proMMP-1 secretion after 5-azadC or 5-azadC + TSA treatment compared to untreated cells. These results suggested that epigenetic mechanisms could be involved in proMMP-1 gene expression including chromatin supra-organization changes. Indeed, although the proMMP-1 gene promoter does not appear to contain CpG islands, its expression can be induced by the demethylating agent 5-azadC. Further experiments revealed that inhibition of protein neosynthesis by cycloheximide decreased 5-azadC-induced proMMP-1 mRNA, suggesting that epigenetically regulated intermediate molecules could be involved in proMMP-1 expression regulation in these cells.
Epigenetics. 2011 Apr ;6 (4):478-89
21364323
UMR M100 IFREMER-UCBN, Physiologie et Ecophysiologie des Mollusques Marins, Esplanade de la Paix, Université de Caen Basse-Normandie, Caen, France. guillaume.riviere@unicaen.fr
Somatic angiotensin-converting enzyme (sACE) is crucial in cardiovascular homeostasis and displays a tissue-specific profile. Epigenetic patterns modulate genes expression and their alterations were implied in pathologies including hypertension. However, the influence of DNA methylation and chromatin condensation state on the expression of sACE is unknown. We examined whether such epigenetic mechanisms could participate in the control of sACE expression in vitro and in vivo. We identified two CpG islands in the human ace-1 gene 3 kb proximal promoter region. Their methylation abolished the luciferase activity of ace-1 promoter/reporter constructs transfected into human liver (HepG2), colon (HT29), microvascular endothelial (HMEC-1) and lung (SUT) cell lines (p < 0.001). Bisulphite sequencing revealed a cell-type specific basal methylation pattern of the ace-1 gene -1,466/+25 region. As assessed by RT-qPCR, inhibition of DNA methylation by 5-aza-2'-deoxycytidine and/or of histone deacetylation by trichostatin A highly stimulated sACE mRNA expression cell-type specifically (p < 0.001 vs. vehicle treated cells). In the rat, in vivo 5-aza-cytidine injections demethylated the ace-1 promoter and increased sACE mRNA expression in the lungs and liver (p = 0.05), but not in the kidney. In conclusion, the expression level of somatic ACE is modulated by CpG-methylation and histone deacetylases inhibition. The basal methylation pattern of the promoter of the ace-1 gene is cell-type specific and correlates to sACE transcription. DNMT inhibition is associated with altered methylation of the ace-1 promoter and a cell-type and tissue-specific increase of sACE mRNA levels. This study indicates a strong influence of epigenetic mechanisms on sACE expression.
Department of Microbiology, College of Natural Sciences, Pusan National University, Busan, Republic of Korea.
Despite current molecular evidence suggesting that hepatitis B virus (HBV) X protein (HBx) plays an important role during HBV-mediated hepatocarcinogenesis, the detailed mechanism is still controversial. Here, it was shown that HBx overcomes cellular senescence provoked by all-trans retinoic acid (ATRA) in HepG2 cells, as demonstrated by the impaired induction of irreversible G(1) arrest and senescence-associated β-galactosidase activity by ATRA in the presence of HBx. The anti-senescence effect of HBx was also observed in another human hepatoma cell line, Hep3B, but not in Huh-7 cells in which the p16 and p21 proteins are absent. In addition, HBx suppressed ATRA-mediated induction of p16 and p21 in HepG2 cells via promoter hypermethylation, resulting in inactivation of retinoblastoma protein. Furthermore, the ability of HBx to overcome ATRA-induced cellular senescence almost completely disappeared when the levels of p16 and p21 in the HBx-expressing cells became similar to those in the control cells by complementation in the former by exogenous expression, knockdown of their expression in the latter using specific small interfering RNA or treatment with a DNA methylation inhibitor, 5-Aza-2'-deoxycytidine. These results suggest that HBx executes its potential by downregulating levels of p16 and p21 via DNA methylation. As cellular senescence is a tumour-suppression process, the present study provides a new strategy by which HBV promotes hepatocarcinogenesis.
BMC Cancer. 2011 ;11 :66
21314941
Helena Carén,
Anna Djos,
Maria Nethander,
Rose-Marie Sjöberg,
Per Kogner,
Camilla Enström,
Staffan Nilsson,
Tommy Martinsson
Department of Clinical Genetics, Institute of Biomedicine, University of Gothenburg, Sahlgrenska University Hospital, SE-41345 Gothenburg, Sweden. helena.caren@clingen.gu.se
Epigenetic mechanisms such as DNA methylation and histone modifications are important regulators of gene expression and are frequently involved in silencing tumor suppressor genes. In order to identify genes that are epigenetically regulated in neuroblastoma tumors, we treated four neuroblastoma cell lines with the demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-dC) either separately or in conjunction with the histone deacetylase inhibitor trichostatin A (TSA). Expression was analyzed using whole-genome expression arrays to identify genes activated by the treatment. These data were then combined with data from genome-wide DNA methylation arrays to identify candidate genes silenced in neuroblastoma due to DNA methylation. We present eight genes (KRT19, PRKCDBP, SCNN1A, POU2F2, TGFBI, COL1A2, DHRS3 and DUSP23) that are methylated in neuroblastoma, most of them not previously reported as such, some of which also distinguish between biological subsets of neuroblastoma tumors. Differential methylation was observed for the genes SCNN1A (p < 0.001), PRKCDBP (p < 0.001) and KRT19 (p < 0.01). Among these, the mRNA expression of KRT19 and PRKCDBP was significantly lower in patients that have died from the disease compared with patients with no evidence of disease (fold change -8.3, p = 0.01 for KRT19 and fold change -2.4, p = 0.04 for PRKCDBP). In our study, a low methylation frequency of SCNN1A, PRKCDBP and KRT19 is significantly associated with favorable outcome in neuroblastoma. It is likely that analysis of specific DNA methylation will be one of several methods in future patient therapy stratification protocols for treatment of childhood neuroblastomas.
T Angrisano,
S Sacchetti,
F Natale,
A Cerrato,
R Pero,
S Keller,
S Peluso,
B Perillo,
V E Avvedimento,
A Fusco,
C B Bruni,
F Lembo,
M Santoro,
L Chiariotti
Dipartimento di Biologia e Patologia Cellulare e Molecolare and Istituto di Endocrinologia ed Oncologia Sperimentale CNR, Università degli Studi di Napoli 'Federico II' 80131 Naples, Italy.
Although it is well known that RET gene is strongly activated by retinoic acid (RA) in neuroblastoma cells, the mechanisms underlying such activation are still poorly understood. Here we show that a complex series of molecular events, that include modifications of both chromatin and DNA methylation state, accompany RA-mediated RET activation. Our results indicate that the primary epigenetic determinants of RA-induced RET activation differ between enhancer and promoter regions. At promoter region, the main mark of RET activation was the increase of H3K4me3 levels while no significant changes of the methylation state of H3K27 and H3K9 were observed. At RET enhancer region a bipartite chromatin domain was detected in unstimulated cells and a prompt demethylation of H3K27me3 marked RET gene activation upon RA exposure. Moreover, ChIP experiments demonstrated that EZH2 and MeCP2 repressor complexes were associated to the heavily methylated enhancer region in the absence of RA while both complexes were displaced during RA stimulation. Finally, our data show that a demethylation of a specific CpG site at the enhancer region could favor the displacement of MeCP2 from the heavily methylated RET enhancer region providing a novel potential mechanism for transcriptional regulation of methylated RA-regulated loci.
Shanghai OB/GYN Hospital, Shanghai Medical College, Fudan University, Shanghai, China.
Adenomyosis is a fairly common gynecologic disease with unknown pathogenesis. We sought to investigate as to whether the promoter of progesterone receptor isoform B (PR-B) is hypermethylated in adenomyosis and to investigate the treatment of ectopic endometrial stromal cells with trichostatin A (TSA), a histone deacetylase inhibitor (HDI), and 5-aza-2-deoxycytidine (ADC), a demethylation agent, on PR-B gene and protein expression, and on cell viability. Ectopic endometrial tissue specimens were obtained from 9 women with adenomyosis whereas control endometrial tissue samples were obtained from 8 women with surgically diagnosed benign ovarian cysts but without any clinical history of endometriosis/adenomyosis/ myoma. Endometrial stromal cells were isolated, purified, cultured, and analyzed by methylation-specific polymerase chain reaction (PCR), real-time reverse transcriptase PCR (RT-PCR), and Western blot analysis, cell viability assays, and fluorescence-activated cell sorting. We found that none of the normal endometrial stromal cells had PR-B promoter methylation. In contrast, 2 out of 3 ectopic endometrial stromall cells had PR-B hypermethylation (P <.05). The treatment with both TSA and ADC elevated PR-B gene and protein expression in ectopic, but not in normal, endometrial stromal cells. Both TSA and ADC treatment dose-dependently reduced cell viability of ectopic endometrial stromal cells. Trichostatin A and ADC treatment also suppressed the cell cycle progression in ectopic endometrial stromal cells. Thus, this study provides the first piece of evidence that adenomyosis has epigenetic aberration and may also be an epigenetic disease amenable to rectification by pharmacological means. This perspective may shed new light onto the pathogenesis of adenomyosis and lead to novel ways to treat the disease.
Pancreas. 2010 Feb 12;:
20173668
Eliane Angst,
David W Dawson,
Anne Nguyen,
Jenny Park,
Vay L W Go,
Howard A Reber,
Oscar Joe Hines,
Guido Eibl
From the *Hirshberg Laboratories for Pancreatic Cancer Research, Department of Surgery, daggerUCLA Center for Excellence in Pancreatic Diseases, and double daggerDepartment of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA.
OBJECTIVES:: N-myc downstream-regulated gene 1 (NDRG1), important in tumor growth and metastasis, has recently gained interest as a potential therapeutic target. Loss of NDRG1 expression is generally associated with poor clinical outcome in pancreatic cancer (PaCa) patients. As the NDRG1 gene possesses a large promoter CpG island, we sought to determine whether its repression is epigenetically mediated in PaCa cells. METHODS:: Pancreatic cancer cells were treated with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor trichostatin A. Promoter methylation was assessed by genomic bisulfite sequencing and by combined bisulfite restriction analyses. RESULTS:: Treatment with 5-aza-2'-deoxycytidine and trichostatin A enhanced NDRG1 protein expression, implicating epigenetic regulation of NDRG1. However, there was no significant DNA methylation of the NDRG1 promoter CpG island, as determined by genomic bisulfite sequencing of HPAF-II cells. We further confirmed the lack of promoter methylation in 6 PaCa cell lines by combined bisulfite restriction analyses. CONCLUSIONS:: These findings indicate that NDRG1 gene reactivation in PaCa cell lines by pharmacologic reversal of DNA methylation and histone deacetylation occurs via an indirect mechanism. This may occur via the altered expression of genes involved in the regulation of NDRG1 transcription or NDRG1 protein stability in PaCa cells.
BMC Cancer. 2010 Feb 4;10 (1):32
20132554
Cit:5
ABSTRACT: BACKGROUND: Epigenetic changes associated with promoter DNA methylation results in silencing of several tumor suppressor genes that lead to increased risk for tumor formation and for progression of the cancer. METHODS: Methylation specific PCR (MSP) and bisulfite sequencing were used for determination of proapoptotic gene Caspase 8 (CASP8) and the tumor suppressor gene maspin promoter methylation in four breast cancer and two nontumorigenic breast cell lines. Involvement of histone H3 methylation in those cell lines were examined by CHIP assay. RESULTS: The CpG sites in the promoter region of CASP8 and maspin were methylated in all four breast cancer cell lines but not in two non-tumorigenic breast cell lines. Demethylation agent 5-aza-2'-deoxycytidine (5-aza-dc) selectively inhibits DNA methyltransferases, DNMT3a and DNMT3b, and restored CASP8 and maspin gene expression in breast cancer cells. 5-aza-dc also reduced histone H3k9me2 occupancy on CASP8 promoter in SKBR3cells, but not in MCF-7 cells. Combination of histone deacetylase inhibitor Trichostatin A (TSA) and 5-aza-dc significant decrease in nuclear expression of Di-methyl histone H3-Lys27 and slight increase in acetyl histone H3-Lys9 in MCF-7 cells. CASP8 mRNA and protein level in MCF-7 cells were increased by the 5-aza-dc in combination with TSA. Data from our study also demonstrated that treatment with 5-FU caused a significant increase in unmethylated CASP8 and in CASP8 mRNA in all 3 cancer lines. CONCLUSIONS: CASP8 and maspin expression were reduced in breast cancer cells due to promoter methylation. Selective application of demethylating agents could offer novel therapeutic opportunities in breast cancer.
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