DNA Methylation/Demethylation Mechanisms in AUD

AUD 中的 DNA 甲基化/去甲基化机制

• 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
• 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

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，会导致基因调控的长期变化 通过表观遗传机制在大脑中表达，如DNA甲基化。与压力类似，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(编码为 NR3C1)。到目前为止，关于表观遗传dna修饰在调节转录组中的作用还知之甚少。 在慢性酒精暴露和戒断过程中人的前额叶皮质(PFC，BA10)和大鼠的PFC。这个 研究部分4的目标是询问新基因DNA甲基化的全基因组变化 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 改变的DNA甲基化和/或羟甲基化标记是AUD的病理生理学基础。基因组- 广泛的DNA甲基化方法(Infinium MethylationEPICBeadChip，Illumina)将用于前额叶皮质 来自新南威尔士组织资源的30对对照和AUD受试者的样本 中心(悉尼大学，澳大利亚)。在初步研究中，我们确定了总DNA的不同模式 5,254个基因的AUD甲基化。然而，这项技术不能区分5-甲基胞嘧啶(5mC) 由5-羟甲基胞嘧啶(5HmC)合成。在这里，我们建议研究5mC的全基因组分布 和5HmC，使用Tet亚硫酸氢盐转换法，然后是人类甲基化EPIC珠片法。 因此，我们将研究5hmC/5mC的丰富与新基因表达的变化 以RNA-seq.与先前确定的表观遗传标记相关的染色质可及性将是 通过转座酶可及染色质测序分析(ATAC-SEQ)进行评估。集成不同的 全基因组方法，即全基因组DNA甲基化、RNA-SEQ和ATAC-SEQ将允许深入的 AUD中表观基因组状态的调查。此外，使用反向翻译方法，我们 建议从机制上研究DNA甲基化对PFC和PFC神经元功能的下游影响 慢性酒精中毒大鼠焦虑样行为与酒精自我给药的升级 在戒酒24小时后。因为我们观察到DNA甲基化的增加与 为了下调Tet的表达(催化5mC到5hmC转化的酶)，我们建议 使用dCas9-Tet1介导的蛋白质方法纠正NR3C1和其他基因水平的甲基化缺陷 基因启动子在大鼠前额叶皮质中的表达，并确定它们对基因表达、焦虑和饮酒行为的影响。 这项拟议的研究将有助于在人类和大鼠的大脑中识别可能提供 AUD治疗的新靶点。