Epigenetic regulation of alcoholic steatohepatitis in a mouse model

描述（由申请人提供）：这个为期2年的R03提案的总体目标是证明酒精性脂肪性肝炎（ASH）的发病机制是由暴露于乙醇诱导异常肝脏蛋氨酸代谢引起的调节基因甲基化的表观遗传变化介导的。该研究将使用蛋氨酸代谢异常的胱硫氨酸-合成酶（CbS）缺陷小鼠模型，其中野生型（+/+）和杂合型（+/-）小鼠被喂食含有对照或乙醇的饮食，这些饮食将在4周内补充或不补充甲基供体甜菜碱。肝脏将被用于分级组织病理学和蛋氨酸代谢物的测量，以及与细胞凋亡和脂肪变性相关的基因转录物和蛋白质水平的测量，包括那些涉及脂肪生成、脂肪酸氧化和脂质输出的基因。随后的表观遗传学研究将包括通过DNA亚硫酸酯测序和组蛋白残基甲基化和乙酰化分析个体基因DNA甲基化。组蛋白研究将包括染色质免疫沉淀（ChIP）分析细胞凋亡和脂肪变性基因的启动子区域，这些区域已被发现受到乙醇喂养、基因型和甜菜碱补充的影响。如果这一假设是正确的，乙醇喂养的小鼠组将出现ASH的组织病理学，同时甲基供体S-腺苷蛋氨酸（SAM）水平降低，甲基转移酶抑制剂S-腺苷同型半胱氨酸（SAH）水平增加，以及参与脂肪变性及其表观遗传调控的基因的激活或抑制。该假设的最终证据将是通过膳食补充甲基供体甜菜碱来预防所有变化的论证。该项目的意义将是定义和证明肝脏蛋氨酸代谢改变的机制作用及其在ASH发病机制中的表观遗传效应。此外，这些研究将指出这些发现与ASH治疗的潜在相关性，基于对调节其相关基因的表观遗传机制的纠正。这些研究将为后续的R01申请奠定基础，将实验方法扩展到分析ASH中涉及酒精性肝损伤和纤维化其他途径的基因的表达和表观遗传调控，以及参与这些途径的表观遗传调控的相关DNA和组蛋白甲基转移酶、乙酰化酶和去乙酰化酶。

DESCRIPTION (provided by applicant): The overall objective of this 2-year R03 proposal is to prove the hypothesis that the pathogenesis of alcoholic steatohepatitis (ASH) is mediated by epigenetic changes in the methylation of regulatory genes that result from the induction of aberrant hepatic methionine metabolism by exposure to ethanol. The study will use the cystathionine beta synthase (CbS) deficient mouse model of aberrant methionine metabolism in which wildtype (+/+) and heterozygous (+/-) mice with be fed control or ethanol containing diets that will or will not be supplemented with the methyl donor betaine over 4 weeks. Livers will be used for graded histopathology and measurements of methionine metabolites and of transcripts and protein levels of genes relevant to apoptosis and steatosis including those involved in lipogenesis, fatty acid oxidation, and lipid export. Subsequent epigenetic studies will include analyses of individual gene DNA methylation by DNA bisulfite sequencing and by methylation and acetylation of histone residues. Histone studies will include chromatin immunoprecipitation (ChIP) analysis of promoter regions of apoptosis and steatosis genes that have been found to be affected by ethanol feeding, genotype, and betaine supplementation. If the hypothesis is correct, the ethanol fed mouse groups will develop the histopathology of ASH, together with reductions in levels of the methyl donor S- adenosylmethionine (SAM), increase in the methyltransferase inhibitor S-adenosylhomocysteine (SAH), together with activation or suppression of genes involved in steatosis and their epigenetic regulations. The definitive proof of the hypothesis will be demonstration of the prevention of all changes by dietary supplementation with the methyl donor betaine. The significance of the project will be definition and proof of the mechanistic role of altered hepatic methionine metabolism and its epigenetic effects on the pathogenesis of ASH. Furthermore, the studies will point to the potential relevance of the findings to the treatment of ASH, based on correction of the epigenetic mechanisms that regulate its relevant genes. The studies will form the basis for a subsequent R01 application to extend the experimental approach to analysis of the expressions and epigenetic regulation of genes involved in other pathways of alcoholic liver injury and fibrosis in ASH and of relevant DNA and histone methyltransferases, acetylases and de-acetylases that are involved in the epigenetic regulation of these pathways. PUBLIC HEALTH RELEVANCE: The proposed R03 will use ethanol fed and genetically modified mice to study the effects of altered liver methionine metabolism on the genes that contribute to steatosis in alcoholic liver disease and their epigenetic regulation by the methyl donor betaine. The results will form the basis for a more extensive and comprehensive proposal to study mechanisms of epigenetic regulation of other gene pathways for the development of alcoholic liver disease.