DNA Methylation/Demethylation Mechanisms in AUD

AUD 中的 DNA 甲基化/去甲基化机制

基本信息

批准号：
10380654
负责人：
ALESSANDRO GUIDOTTI
金额：
$ 19.6万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10380654
关键词：
Aberrant DNA Methylation Alcohol abuse Alcohol consumption Alcohol dependence Alcohol withdrawal syndrome Alcohols Animal Model Antibodies Anxiety Australia Autopsy Binding Bioinformatics Biological Assay Biological Process Brain Chromatin Chromatin Structure Chronic Clinical Research Clustered Regularly Interspaced Short Palindromic Repeats Cytosine DNA DNA Methylation DNA Modification Methylases DNA Modification Process Data Data Set Development Down-Regulation Environmental Risk Factor Enzymes Epigenetic Process Ethanol Exons Female Functional disorder Funding Gene Expression Gene Expression Profile Gene Expression Regulation Genes Genetic Transcription Genomics Glucocorticoid Receptor Glucocorticoids Goals Human Individual Investigation Limbic System Link Location Maintenance Measures Mediating Methods Methylation Modeling NR3C1 gene Neurons New South Wales Pathway interactions Patients Pattern Play Precipitation Prefrontal Cortex Process Proteins Quantitative Reverse Transcriptase PCR RNA analysis Rattus Reaction Recording of previous events Regulation Regulatory Element Research Role Sampling Self Administration Site Stress Techniques Testing Tetanus Helper Peptide Thinking Translating Transposase Universities alcohol abuse therapy alcohol exposure alcohol research alcohol use disorder anxiety-like behavior bead chip behavioral phenotyping bisulfite cohort demethylation disorder control drinking behavior epigenome gene network genome-wide genome-wide analysis mRNA Expression male member methyl group new therapeutic target novel oxidation preclinical study promoter receptor tissue resource transcription factor transcriptome transcriptome sequencing translational approach translocase whole genome

项目摘要

Project Summary Environmental factors, including alcohol abuse and stress, cause long-lasting changes in the regulation of gene expression in the brain via epigenetic mechanisms, such as DNA methylation. Similar to stress, alcohol stimulates glucocorticoid release that bind to specific receptors, i.e., the glucocorticoid receptor (encoded by NR3C1). As of today, little is known on the role of epigenetic DNA modifications in regulating the transcriptome in the human prefrontal cortex (PFC, BA10) and rat PFC during chronic alcohol exposure and withdrawal. The goal of research component #4 is to interrogate genome-wide changes in DNA methylation of novel gene networks, including the NR3C1 gene network in AUD patients. Additional goals are to study whether and how altered DNA methylation and/or hydroxymethylation marks underlie the pathophysiology of AUD. The genome- wide DNA methylation approach (Infinium MethylationEPICBeadChip, Illumina) will be used in prefrontal cortex samples obtained from 30 pairs of controls and AUD subjects from the New South Wales Tissue Resource Centre (University of Sydney, Australia). In preliminary studies we identified a differential pattern of total DNA methylation in AUD for 5,254 genes. However, this technique does not differentiate 5-methyl-cytosine (5mC) from 5-hydroxymethyl-cytosine (5hmC). Here, we propose to investigate the genome–wide distribution of 5mC and 5hmC using the TET bisulfite conversion method followed by the Human MethylationEPIC BeadChip assay. Hence, we will examine the enrichment of 5hmC/5mC in association with changes in novel gene expression measured by RNA-seq. Chromatin accessibility in association with previously identified epigenetic marks will be assessed by Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq). Integration of different whole-genome approaches, i.e. genome-wide DNA methylation, RNA-seq and ATAC-seq will allow an in-depth investigation of the status of the epigenome in AUD. Additionally, using a reverse-translational approach, we propose to mechanistically investigate downstream effects of DNA methylation on neuronal function in PFC and on anxiety-like behaviors and escalation of alcohol self-administration in rats treated chronically with alcohol or following a 24 h alcohol-withdrawal. Because we observed an increase of DNA methylation associated with a downregulation of TET expression (the enzyme that catalyzes the conversion of 5mC to 5hmC), we propose the use of a dCas9-Tet1-mediated protein approach to correct methylation deficits at the levels of NR3C1 and other gene promoters in the PFC of rats and determine their effect on gene expression, anxiety and drinking behaviors. The proposed study will help to identify in the human and rat brain novel epigenetic mechanisms that may provide new therapeutic targets for the treatment of AUD.

项目摘要环境因素，包括酗酒和压力，导致基因调节的持久变化通过表观遗传机制（例如DNA甲基化）在大脑中的表达。与压力相似，酒精刺激与特定受体结合的糖皮质激素释放，即糖皮质激素受体（由 NR3C1）。截至当今在慢性酒精暴露和戒断期间，在人前额叶皮层（PFC，BA10）和大鼠PFC中。这研究成分4的目标是询问全基因甲基的全基因组变化的新基因的变化网络，包括AUD患者的NR3C1基因网络。其他目标是研究是否以及如何 DNA甲基化改变和/或羟甲基化标志是AUD的病理生理。基因组 - 宽DNA甲基化方法（Infinium甲基化Epicbeadchip，Illumina）将用于额叶皮层从新南威尔士组织资源的30对对照和AUD受试者获得的样品中心（澳大利亚悉尼大学）。在初步研究中，我们确定了总DNA的差异模式 5,254个基因的AUD中的甲基化。但是，该技术并未区分5-甲基 - 胞嘧啶（5MC）来自5-羟基甲基 - 胞嘧啶（5HMC）。在这里，我们建议研究5MC的基因组范围的分布 5HMC使用TET Bisulfite转化方法，然后是人类甲基化珠芯片测定法。因此，我们将检查5HMC/5MC的富集与新基因表达的变化有关通过RNA-seq测量。与先前确定的表观遗传标记相关的染色质可及性将是通过测定评估转座酶可访问的染色质测序（ATAC-SEQ）。不同的整合全基因组方法，即全基因组DNA甲基化，RNA-SEQ和ATAC-SEQ将允许深入调查AUD中表观基因组的状态。此外，使用反向翻译方法，我们提出机械研究DNA甲基化对PFC中神经元功能的下游影响的建议关于焦虑症的行为和酒精自我给药的升级，在长期用酒精或酒精治疗的大鼠中在24小时饮酒之后。因为我们观察到与A相关的DNA甲基化增加 TET表达的下调（催化5MC转化为5HMC的酶），我们提出使用DCAS9-TET1介导的蛋白质方法纠正甲基化定义在NR3C1和其他的水平上大鼠PFC中的基因启动子，并确定其对基因表达，动画和饮酒行为的影响。拟议的研究将有助于确定可能提供的新型表观遗传机制用于治疗AUD的新治疗靶标。