Alcohol-induced epigenetic changes in the liver genome

描述（由申请人提供）：酒精是世界上发病率和死亡率的主要原因，影响许多器官，最明显的是肝脏和中枢神经系统。酒精滥用的一个重要的生化后果是S-腺苷甲硫氨酸（SAM）水平的显着降低和S-腺苷同型半胱氨酸（SAH）水平的相应增加。我们的假设是，酒精诱导的肝SAM和SAH水平的改变在改变肝脏表观基因组中起关键作用，导致基因表达改变，导致肝功能障碍，肝毒性和最终肝癌。本研究的长期目标是促进我们对特定基因的表观遗传调控在介导酒精介导的肝功能障碍中的作用的理解。改变的SAM水平还影响细胞氧化还原状态，其影响组蛋白修饰并最终影响基因表达。本研究将使用小鼠启动子微阵列和ChIP芯片分析以及Dnmt 1（主要DNA甲基转移酶）水平降低的亚型小鼠来验证这一假设。具体目的是（1）通过酸-尿素凝胶电泳、凝胶内消化、MALDI-TOF MS和LC/MS-MS分析鉴定在酒精喂养的小鼠的肝脏中体内发生的酒精诱导的组蛋白H4的翻译后修饰，（二）通过蛋白质印迹分析确定DNA甲基化机制的改变，并通过鉴定改变的基因来确定暴露于酒精的小鼠中肝脏表观基因组的变化。分别采用差异甲基化杂交和ChlP-on-chip分析技术检测DNA甲基化状态和组蛋白翻译后修饰谱;（3）研究慢性酒精暴露后Dnmt 1高型小鼠肝脏的病理变化，并确定慢性酒精暴露后Dnmt 1高型小鼠肝脏基因组甲基化谱是否与野生型小鼠不同。预计这项研究将确定发生在酒精诱导的肝脏疾病中的关键表观遗传改变。由于表观遗传变化可以通过DNA甲基转移酶和/或组蛋白去乙酰化酶的小分子抑制剂的药物干预相对容易地逆转，因此这些药物中的一些可以有效地用作酒精诱导的肝损伤的潜在治疗剂。这一建议也非常符合国家酒精滥用和酒精中毒研究所关于"酒精代谢和对组织损伤的表观遗传影响"的使命。

DESCRIPTION (provided by applicant): Alcohol, a leading cause of morbidity and mortality in the world, affects many organs, most notably liver and central nervous system. An important biochemical consequence of alcohol abuse is a significant decrease in S-adenosyl methionine (SAM) level and a corresponding increase in S-adenosyl homocysteine (SAH) level. Our hypothesis is that alcohol-induced alteration in hepatic SAM and SAH levels plays a key role in modifying liver epigenome leading to altered gene expression that causes hepatic dysfunction, liver toxicity and ultimately hepatocarcinogeneis. The long term objective of this study is to advance our understanding of the role of epigenetic regulation of specific genes in mediating alcohol-mediated liver dysfunction. Altered SAM level also affects cellular redox state that influences histone modification and ultimately gene expression. The present study will test the hypothesis using mouse promoter microarray and ChlP-on-Chip analysis as well as Dnmt1 (the major DNA methyltransferase) hypomorphic mice with reduced level of Dnmt1 . The specific aims are (1) identify alcohol-induced post-translational modifications of histone H4 that occur in vivo in the livers of alcohol-fed mice by acid-urea gel electrophoresis, in-gel digestion, MALDI-TOF MS and LC/MS-MS analysis, (2) determine alterations in the DNA methylation machinery by western blot analysis and changes in the liver epigenome that occur in mice exposed to alcohol by identifying genes with altered DNA methylation status and histone post-translational modification profile using differential methylation hybridization and ChlP-on-chip analysis respectively and (3) investigate pathological changes that occur in the livers of Dnmt1 hypermorphic mice after chronic alcohol exposure and determine whether the methylation profile of genome in the livers of these mice is distinct from that of the wild type mice subjected to chronic alcohol exposure. It is anticipated that this study will identify critical epigenetic alterations that occur in alcohol-induced liver diseases. Because epigenetic changes can be reversed with relative ease by pharmacological intervention with small molecule inhibitors of DNA methyltransferase and/or histone deacetylases some of these agents could be used effectively as potential theapeutic agents in alcohol-induced liver injury. This proposal also fits well with the mission of the National Institute of Alcohol Abuse and Alcoholism on "alcohol metabolism and epigenetic effects on tissue injury".