Epigenetic regulation of alcoholic steatohepatitis in a mouse model

描述（由申请方提供）：本2年R 03提案的总体目标是证明以下假设：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导致的调节基因甲基化的表观遗传变化介导的。该研究将使用异常甲硫氨酸代谢的胱硫醚β合酶（CbS）缺陷小鼠模型，其中野生型（+/+）和杂合型（+/-）小鼠喂食对照或含乙醇的饮食，所述饮食将或将不补充甲基供体甜菜碱超过4周。肝脏将用于分级组织病理学和甲硫氨酸代谢物以及与细胞凋亡和脂肪变性相关的基因（包括参与脂肪生成、脂肪酸氧化和脂质输出的基因）的转录物和蛋白质水平的测量。随后的表观遗传学研究将包括通过DNA亚硫酸氢盐测序和组蛋白残基的甲基化和乙酰化分析单个基因DNA甲基化。组蛋白研究将包括细胞凋亡和脂肪变性基因的启动子区域的染色质免疫沉淀（ChIP）分析，这些基因已被发现受到乙醇喂养，基因型和甜菜碱补充剂的影响。如果假设是正确的，乙醇喂养的小鼠组将出现ASH的组织病理学，以及甲基供体S-腺苷甲硫氨酸（SAM）水平的降低，甲基转移酶抑制剂S-腺苷高半胱氨酸（SAH）的增加，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。这一假设的确切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证明。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对ASH发病机制的表观遗传作用。此外，这些研究将指出这些发现与ASH治疗的潜在相关性，基于调节其相关基因的表观遗传机制的校正。这些研究将成为后续R 01应用的基础，以扩展实验方法，分析参与其他途径的酒精性肝损伤和纤维化的基因的表达和表观遗传调控，以及相关DNA和组蛋白甲基转移酶，乙酰化酶和脱乙酰酶参与这些途径的表观遗传调控。

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.