Histone Modifications by Ethanol: Mechanism & Consequence

描述(由申请人提供)：乙醇的细胞和亚细胞效应的机制还不是很清楚。新的证据突显了组蛋白修饰在基因表达中的重要性。我们观察到乙醇选择性地促进组蛋白H3在Lys 9(H3-Lys9)的乙酰化，而不是在Lys 14、18或23。令人惊讶的是，增幅高达10倍，甚至可以在低至5 mM的乙醇中检测到。在原代培养的肝细胞和体内注射乙醇的大鼠肝脏中都注意到了这一点。基于这些有希望的原始数据，我们假设“乙醇通过调节组蛋白乙酰转移酶(HAT)来促进肝脏中特定基因的转录激活，从而促进组蛋白乙酰化”。长期的目标是确定乙酰化的机制，确定所涉及的HAT的身份，并研究组蛋白乙酰化与染色质中受乙醇影响的基因的特定DMA结构域的关联，以及这些变化与细胞凋亡和肝损伤的关系。目的1：确定组蛋白乙酰化的机制和调控：乙醇衍生的磷脂酰乙醇的作用；乙酸乙酯和乙醇对乙酰化的影响；它与细胞损伤和凋亡的关系；氧化应激在乙酰化中的作用和选择的HAT基因敲除(KO)小鼠[ADH1(-/-)；SOD1(-/-)；SOD-2(-/-；)]目的2：确定HAT的同一性：HAT抗体的免疫沉淀研究；区分HAT表达上调与其激活的差异；使用siRNA方法和选择的HAT KO小鼠(PCAF-/-；GCN5)。目的3：确定乙酰化在转录过程中的意义：体外和体内研究；染色质免疫沉淀(ChIP)法确定ADH1基因启动子与乙酰化H3-lys9的相互作用部位；分析乙酰化组蛋白与所选基因的特定DMA结构域(如诱导型一氧化氮合酶、细胞色素P42E1、肿瘤坏死因子α)的相关性；确定乙酰化组蛋白与转录因子(如NFkB、P53)的相关性；利用TF蛋白阵列。这个项目涉及酒精研究的一条新途径。关于乙醇引起的组蛋白乙酰化的机制和后果的数据将对构建乙醇所致肝损伤的“分子图谱”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也将对受乙醇影响的其他细胞/系统的研究产生横向影响。

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.