Histone Modifications by Ethanol: Mechanism & Consequence

描述（由申请人提供）：乙醇的细胞和亚细胞作用机制尚不清楚。新出现的证据强调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在赖氨酸9位点（H3- lys9）增加组蛋白H3乙酰化，但在赖氨酸14、18或23位点没有。引人注目的是，这种增加高达10倍，并且可以在低至5毫米乙醇的情况下检测到。这在肝细胞的原代培养和体内给药乙醇的大鼠肝脏中都有发现。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制，确定所涉及的HAT的身份，并研究乙酰化组蛋白与受乙醇影响的染色质中特定基因的DMA结构域的关联，并将这些改变与细胞凋亡和肝损伤联系起来。目的1：确定组蛋白乙酰化的机制和调控；乙醇衍生磷脂酰乙醇的作用；乙酸和乙醇对乙酰化反应的影响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性敲除（KO）小鼠的使用[ADH1 (-/-)]；SOD 1 (- / -);Sod-2 （-/-;）目的2：确定所涉HAT的身份：HAT抗体的免疫沉淀研究；区分HAT的增加是由于表达上调还是其激活；使用SiRNA方法并选择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）测定ADH1基因启动子与乙酰化H3-lys9相互作用位点；分析乙酰化组蛋白与特定基因（如iNOS， Cyp2E1,TNFa）的DMA结构域之间的关系；测定乙酰化组蛋白与转录因子（如NFkB、p53）的关联；使用TF-蛋白阵列。这个项目涉及酒精研究的新途径。获得的关于乙醇引起组蛋白乙酰化的机制和结果的数据对于构建乙醇诱导肝损伤的“分子图谱”非常重要，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其他受乙醇影响的细胞/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.