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.

项目概要环境因素，包括酗酒和压力，会导致基因调控发生长期变化通过表观遗传机制（例如 DNA 甲基化）在大脑中表达，与压力、酒精类似。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（由迄今为止，人们对表观遗传 DNA 修饰在转录组调控中的作用知之甚少。在慢性酒精暴露和戒断过程中，人类前额皮质（PFC，BA10）和大鼠 PFC 中的变化。研究部分 #4 的目标是探究新基因 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进行。 DNA 甲基化和/或羟甲基化标记的改变是 AUD 病理生理学的基础。广泛的 DNA 甲基化方法（Infinium MmethylationEPICBeadChip，Illumina）将用于前额皮质样本取自新南威尔士州组织资源中心的 30 对对照者和 AUD 受试者中心（澳大利亚悉尼大学）在初步研究中我们发现了总 DNA 的差异模式。 5,254 个基因的 AUD 甲基化然而，该技术无法区分 5-甲基胞嘧啶 (5mC)。来自 5-羟甲基-胞嘧啶 (5hmC) 在这里，我们建议研究 5mC 的全基因组分布。和 5hmC，使用 TET 亚硫酸氢盐转化方法，然后进行 Human MmethylationEPIC BeadChip 测定。因此，我们将检查 5hmC/5mC 的富集与新基因表达变化的关系通过 RNA-seq 测量染色质可及性与先前鉴定的表观遗传标记的相关性。通过转座酶可及染色质测序（ATAC-seq）的不同整合进行评估。全基因组方法，即全基因组 DNA 甲基化、RNA-seq 和 ATAC-seq 将允许深入研究此外，我们使用逆翻译方法研究了 AUD 的表观基因组状态。提议从机制上研究 DNA 甲基化对 PFC 和神经功能的下游影响长期饮酒或治疗的大鼠的焦虑样行为和酒精自我给药的升级因为我们观察到与 24 小时戒酒相关的 DNA 甲基化增加。 TET 表达（催化 5mC 转化为 5hmC 的酶）的下调，我们提出使用 dCas9-Tet1 介导的蛋白质方法来纠正 NR3C1 和其他水平的甲基化缺陷大鼠 PFC 中的基因启动子，并确定它们对基因表达、焦虑和饮酒行为的影响。拟议的研究将有助于在人类和大鼠大脑中识别新的表观遗传机制，这些机制可能提供治疗AUD的新治疗靶点。