Histone Modifications by Ethanol: Mechanism & Consequence

乙醇对组蛋白的修饰：机制

• 批准号: 7813978
• 负责人: Shivendra D Shukla
• 金额: $ 32.96万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-15 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7813978
• 关键词: Acetates; Acetylation; Affect; Alcohol dehydrogenase; Alcoholic Liver Diseases; Antibodies; Apoptosis; Biological Assay; CYP2E1 gene; Cells; Chromatin; DNA; Data; Enzymes; Epigenetic Process; Ethanol; Event; Future; Gel; Gene Expression; Genes; Genetic Transcription; Grant; Hepatocyte; Histone Acetylation; Histone H3; Histones; Immunoprecipitation; In Vitro; Injury; Investigation; JUN gene; Knock-out; Knockout Mice; Lateral; Lipids; Liver; Maps; Methodology; Modeling; Modification; Molecular; Mus; Oxidative Stress; PCAF gene; Physiological; Process; Protein Array; Proteins; Rattus; Regulation; Research; Research Personnel; Role; SRE-1 binding protein; Site; Superoxide Dismutase; System; TP53 gene; Therapeutic; Transcriptional Activation; Transferase; Up-Regulation; Work; alcohol effect; alcohol research; base; cell injury; chromatin immunoprecipitation; histone modification; in vivo; insight; phosphatidylethanol; programs; promoter; therapeutic development; tool; transcription factor

DESCRIPTION (provided by applicant): Mechanisms for the cellular and subcellular effects of ethanol are not well understood. Emerging evidence highlight the importance of histone modifications in gene expressions. We have observed that ethanol increases histone H3 acetylation selectively at Lys 9 (H3-Lys9) but not at Lys 14, 18 or 23. Strikingly, the increases were up to 10 fold and could be detected at as low as 5 mM ethanol. This was noted both in primary cultures of hepatocytes and in the liver of rats administered ethanol in vivo. Based on these promising original data it is hypothesized that 'ethanol increases histone acetylation via modulation of histone acetyl transferase (HAT) leading to transcriptional activation of specific genes' in the liver. The long term objective is to identify the mechanism of the acetylation, determine the identity of the HAT involved and investigate the association of the acetylated histone with specific DMA domains of genes affected by ethanol in the chromatin and correlate these alterations to apoptosis and liver damage. There are three specific aims: Aim 1: Determine mechanism and regulation of the histone acetylation: Role of ethanol derived phosphatidylethanol; effect of acetate versus ethanol on acetylation; its correlation to cell damage and apoptosis; role of oxidative stress in acetylation and use of selected knockout (KO) mice [ ADH1 (-/-); SOD 1(-/-); SOD-2 (-/-; )] Aim 2: Determine identity of HAT involved: Immunoprecipitation studies with HAT antibodies; differentiate HAT increase due to upregulation of expression versus its activation; use of SiRNA methodology and selected HAT KO mice (PCAF -/-; GCN5 ). Aim 3: Determine significance of the acetylation in transcriptional process: In vitro and in vivo studies; chromatin immunoprecipitation (CHIP) assays to determine site of interaction between the promoter of ADH1 gene and acetylated H3-lys9; analyze association between acetylated histone with specific DMA domains of selected genes (e.g. iNOS, Cyp2E1,TNFa); determine association of acetylated histone with transcription factor(s) (e.g. NFkB, p53); use of TF- protein arrays. This project deals with a new avenue in alcohol research. The data obtained on the mechanisms and consequences of histone acetylations by ethanol will be important in the construction of the 'molecular map' of ethanol induced liver injury and will help develop therapeutic tools targeting specific molecules/steps. Furthermore, study of these epigenetic changes will also have 'lateral impact1 on investigations in other cells/systems affected by ethanol.

描述（由申请人提供）：乙醇的细胞和亚细胞效应的机制尚不清楚。新兴的证据突出了组蛋白在基因表达中的重要性。我们已经观察到乙醇在Lys 9（H3-Lys9）下选择性地增加了组蛋白H3乙酰化，但在Lys 14、18或23中不选择乙醇。令人惊讶的是，增加的增加高达10倍，可以在低至5 mm的乙醇中检测到。这在肝细胞的原发性培养物和体内施用乙醇的肝脏中都被指出。基于这些有希望的原始数据，假设“乙醇通过调节组蛋白乙酰转移酶（HAT）增加组蛋白乙酰化，从而导致肝脏中特定基因的转录激活。长期目标是确定乙酰化的机理，确定所涉及的帽子的身份，并研究乙酰化组蛋白与染色质乙醇影响的基因的特定基因的特定DMA结构域的关联，并将这些改变与凋亡和肝损伤相关。有三个特定的目的：目标1：确定组蛋白乙酰化的机理和调节：乙醇衍生的磷脂酰乙醇的作用；乙酸与乙醇对乙酰化的影响；它与细胞损伤和凋亡的相关性；氧化应激在乙酰化和使用选定敲除（KO）小鼠的使用中的作用[ADH1（ - / - ）;草1（ - / - ）; SOD-2（ - / - ;）]目标2：确定涉及的帽子的身份：使用HAT抗体的免疫沉淀研究；由于表达的上调与其激活而区分帽子的增加；使用siRNA方法论和精选的HAT KO小鼠（PCAF - / - ; GCN5）。目标3：确定乙酰化在转录过程中的重要性：体外和体内研究；染色质免疫沉淀（CHIP）测定，以确定ADH1基因启动子与乙酰化H3-Lys9之间相互作用的位点；分析乙酰化组蛋白与选定基因的特定DMA结构域之间的关联（例如Inos，Cyp2e1，TNFA）；确定乙酰化组蛋白与转录因子的关联（例如NFKB，p53）；使用TF-蛋白阵列。该项目介绍了酒精研究的新途径。乙醇对组蛋白乙酰化的机制和后果获得的数据在构建乙醇诱导的肝损伤的“分子图”中很重要，并将有助于开发针对特定分子/步骤的治疗工具。此外，对这些表观遗传变化的研究还将对其他受乙醇影响的细胞/系统的研究产生'横向影响。