Alcohol-induced epigenetic changes in the liver genome

描述(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生化后果是S-腺苷蛋氨酸水平显著降低，而S-腺苷同型半胱氨酸水平相应升高。我们的假设是，酒精诱导的肝脏SAM和SAH水平的改变在修饰肝脏表观基因组导致基因表达改变，从而导致肝功能障碍、肝脏毒性和最终肝癌的发生中起关键作用。这项研究的长期目标是促进我们对特定基因的表观遗传调节在介导酒精介导的肝功能障碍中的作用的理解。SAM水平的改变也会影响细胞的氧化还原状态，从而影响组蛋白的修饰和最终的基因表达。本研究将利用小鼠启动子芯片和ChlP-on-Chip分析以及Dnmt1(主要DNA甲基转移酶)低构型小鼠的Dnmt1水平降低来验证这一假说。其具体目的是(1)通过酸尿素凝胶电泳、凝胶内消化、MALDI-TOF MS和LC/MS-MS分析，鉴定酒精引起的组蛋白H4在酒精喂养的小鼠肝脏中的翻译后修饰，(2)通过蛋白质印迹杂交和ChlP-on-Chip分析分别鉴定DNA甲基化状态发生改变的基因和组蛋白翻译后修饰谱基因，以确定酒精暴露小鼠肝脏DNA甲基化机制的变化和肝脏表观基因组的变化；(3)研究Dnmt1高多态性小鼠慢性酒精暴露后肝脏的病理变化，并确定这些小鼠肝脏基因组的甲基化谱是否与长期酒精暴露野生型小鼠的肝脏甲基化谱有显著差异。预计这项研究将确定在酒精诱导的肝病中发生的关键的表观遗传学改变。由于通过小分子DNA甲基转移酶和/或组蛋白脱乙酰酶抑制剂的药物干预可以相对容易地逆转表观遗传学改变，其中一些药物可以有效地作为潜在的酒精性肝损伤的治疗药物。这项建议也非常符合国家酒精滥用和酒精中毒研究所关于“酒精新陈代谢和表观遗传对组织损伤的影响”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".