Epigenetic dissection of functional ethanol tolerance and dependence

功能性乙醇耐受和依赖的表观遗传学剖析

• 批准号: 8760028
• 负责人: NIGEL S ATKINSON
• 金额: $ 33.78万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8760028
• 关键词: Acetylation; Adult; Alcohol dependence; Alcohol withdrawal syndrome; Alcoholic Intoxication; Alcoholism; Alcohols; Amygdaloid structure; Animals; Anti-Anxiety Agents; Anxiety; Appearance; Behavior; Behavioral; Behavioral Assay; Benzyl Alcohols; Candidate Disease Gene; ChIP-seq; Chromatin; Cognitive; Consumption; Data; Data Set; Dependence; Diagnostic; Dissection; Doctor of Philosophy; Drosophila genus; Drosophila melanogaster; Epigenetic Process; Ethanol; Ethanol dependence; Future; Gene Expression; Genes; Genetic; Genomics; Goals; Head; Histone Deacetylase; Histone H3; Histone H4; Histones; Human; Individual; Larva; Link; Mammals; Mediating; Memory; Meta-Analysis; Methylation; Modification; Molecular; Monitor; Mutation; Nervous system structure; Neurons; Pattern; Pharmaceutical Preparations; Phenotype; Physiological; Play; Population; Postdoctoral Fellow; Predisposition; Promoter Regions; Property; Public Health; RNA; RNA Interference; Rattus; Research; Role; Sedation procedure; Seizures; Surveys; Symptoms; System; Testing; Time; Training; Translating; United States; Variant; Withdrawal; Withdrawal Symptom; Work; addiction; alcohol abstinence; alcohol effect; alcohol exposure; alcohol related problem; alcohol response; base; behavioral genomics; classical conditioning; cohort; cost; drinking; epigenetic marker; epigenome; experience; fly; genome wide association study; histone acetyltransferase; histone modification; innovation; interest; meetings; mutant; neuroadaptation; novel; problem drinker; public health relevance; response; tool; transcriptome sequencing; undergraduate student

DESCRIPTION (provided by applicant): The potential of genomics to identify genes important for alcoholism has not been completely realized because ethanol produces a surfeit of changes in gene expression, most of which do not seem to make any meaningful contribution to the ethanol response of interest. A simple and effective way to pan for genomic changes relevant for an alcoholism phenotype would be invaluable. The problem is solved by focusing on genes that show common changes produced by two chemically and metabolically dissimilar drugs, ethanol and benzyl alcohol, which produce cross-tolerance by a mechanism with a shared molecular origin. Genes that respond in the same way to both alcohols will be identified by genomically surveying epigenetic histone modifications and gene expression. These will be functionally tested by mutant analysis in the fruit fly Drosophila melanogaster, an animal that has ethanol responses remarkably similar to humans. The alcohol response genes will be tested for a functional role in ethanol tolerance and two withdrawal/dependence symptoms - alcohol withdrawal induced hyperexcitability and cognitive ethanol dependence. Parts of this response have already been shown to be conserved in mammals. To directly bridge our work to human alcoholism the data will be collaboratively compared to human alcoholic datasets. The advantage of using Drosophila is that candidate genes can be rapidly and functionally tested by mutant analysis for their role in producing these ethanol responses. In Aim 1, chromatin and RNA will be prepared from the heads of flies treated with ethanol or benzyl alcohol. Epigenetic and gene expression changes common to both drugs will be identified by ChIP-seq to identify changes in histone H3 and H4 acetylation and methylation (H3K36me1 and H3K36me3). These histone marks have tight linkage to changes in gene activity. RNA-seq will be used to monitor changes in gene expression. These candidates and pre-existing candidates from an epigenetic screen will be tested in Aim 2 and 3 by mutant, RNAi, and mis-expression analysis. In Aim 2, candidate genes will be tested for a role in functional tolerance to ethanol sedation. In Aim 3, th effects of mutations on alcohol-withdrawal will be determined electrophysiologically in adults and behaviorally in larvae. Adult flies show alcohol-withdrawal hyperexcitability that increases the susceptibility for seizures. While in ethanol-adapted larvae, the capacity for associative learning declines during withdrawal and is restored after ethanol reinstatement-demonstrating functional dependence. This contribution will be significant because it will define the origins of functional tolerance phenotype and establish its relationship to dependence phenotypes. The proposed research is innovative because it uses 1) a shared drug response to focus a genomic survey on ethanol-response genes, 2) epigenetic histone modifications to help identify genes that transcriptionally respond to ethanol intoxication, and 3) new behavioral assays that allow us to detect tolerance and physiological dependence in flies. Over its 5 year span, this project will be used to train 2 post-docs, 2 graduate (Ph.D) and 10 undergraduate students.

描述（由申请人提供）：基因组学鉴定对酒精中毒重要的基因的潜力尚未完全实现，因为乙醇会产生基因表达的变化，其中大多数似乎并没有对感兴趣的乙醇反应做出任何有意义的贡献。与酒精中毒表型相关的基因组变化的一种简单有效的方法是无价的。通过关注两种化学和代谢不同的药物，乙醇和苄醇产生的常见变化的基因来解决该问题，这些基因通过具有共同的分子起源的机制产生交叉耐受性。通过基因组测量表观遗传组蛋白的修饰和基因表达来鉴定以相同方式对两种醇的反应的基因。这些将通过突变分析在果蝇果蝇中的突变分析在功能上进行测试，果蝇果蝇（Melanogaster）的动物具有与人类非常相似的乙醇反应。酒精反应基因将在乙醇耐受性和两种戒断/依赖性症状中的功能作用 - 酒精戒断诱导的过度兴奋性和认知乙醇依赖性。该反应的一部分已经显示在哺乳动物中是保守的。为了将我们的工作直接桥接到人类酒精中毒，将与人类酒精数据集进行协作。使用果蝇的优点是，可以通过突变分析在产生这些乙醇反应中的作用来迅速和功能地测试候选基因。在AIM 1中，将从用乙醇或苄醇处理的苍蝇的头部制备染色质和RNA。两种药物的表观遗传和基因表达变化将通过CHIP-SEQ鉴定，以鉴定组蛋白H3和H4乙酰化和甲基化的变化（H3K36ME1和H3K36ME3）。这些组蛋白标记与基因活性变化有紧密的联系。 RNA-seq将用于监测基因表达的变化。这些候选者和来自表观遗传筛查的候选者将通过突变体，RNAi和错误表达分析在AIM 2和3中测试。在AIM 2中，将测试候选基因在对乙醇镇静功能耐受性中的作用。在AIM 3中，突变对酗酒的影响将在成人和行为上在幼虫中进行电生理确定。成年苍蝇显示出酗酒的过度兴奋性，从而增加了癫痫发作的敏感性。在乙醇适应的幼虫中，联想学习的能力在退出期间下降，并在恢复乙醇恢复功能依赖性后恢复。这种贡献将是重要的，因为它将定义功能耐受性表型的起源并建立与依赖表型的关系。拟议的研究具有创新性，因为它使用1）共同的药物反应将基因组调查集中在乙醇反应基因上，2）表观遗传组蛋白修饰，以帮助鉴定转录转录响应乙醇中毒的基因，3）3）新的行为测定方法，使我们能够检测到不能触及苍蝇的耐受性和物理依赖性。在其5年范围内，该项目将用于培训2个毕业后，2个毕业生（博士）和10名本科生。