Epigenetic regulation of alcoholic steatohepatitis in a mouse model

描述(由申请人提供)：这份为期2年的R03提案的总体目标是证明酒精性脂肪性肝炎(ASH)的发病机制是由调控基因甲基化的表观遗传变化所介导的，这些甲基化是由于暴露在乙醇中导致肝脏蛋氨酸代谢异常而导致的。这项研究将使用胱硫醚β合成酶(CBS)缺陷的蛋氨酸代谢异常小鼠模型，在该模型中，野生型(+/+)和杂合子(+/-)小鼠被喂以对照或含有乙醇的饲料，这些饲料将在4周内添加或不添加甲基供体甜菜碱。肝脏将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的转录本和蛋白质水平，包括那些涉及脂肪生成、脂肪酸氧化和脂肪输出的基因。随后的表观遗传学研究将包括通过DNA亚硫酸氢盐测序以及通过组蛋白残基的甲基化和乙酰化来分析单个基因的DNA甲基化。组蛋白研究将包括对凋亡和脂肪变性基因启动子区的染色质免疫沉淀(ChIP)分析，这些基因已被发现受到酒精喂养、基因型和甜菜碱补充的影响。如果假设是正确的，酒精喂养的小鼠组将出现ASH的组织病理学，伴随着甲基供体S-腺苷蛋氨酸水平的降低，甲基转移酶抑制剂S-腺苷同型半胱氨酸的增加，以及与脂肪变性有关的基因的激活或抑制及其表观遗传调节。这一假设的确凿证据将是证明通过饮食补充甲基供体甜菜碱来预防所有变化。该项目的意义将是定义和证明改变的肝脏蛋氨酸代谢的机制作用及其在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，这是基于对调节其相关基因的表观遗传机制的纠正。这些研究将为随后的R01应用奠定基础，以扩展实验方法，分析ASH中与酒精性肝损伤和纤维化的其他途径有关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.