Alcohol-Induced Epigenetic Changes in Stem Cells

描述（由申请人提供）：酒精消费导致多个器官损伤，包括肝脏和大脑，乙醇可以通过修饰DNA甲基化和修饰组蛋白来影响细胞的表观遗传状态。这些表观遗传变化可能损害组织的再生，使细胞易于发生肿瘤转化，和/或改变干细胞或分化细胞的功能。在测试乙醇对小鼠胚胎干（ES）细胞的影响的试验中，我们观察到乙醇和乙醇的代谢产物乙醛对基因表达和组蛋白修饰的一些显著影响。事实上，我们已经有数据表明酒精影响Polycomb抑制复合物2的活性，这是一种多蛋白复合物，是干细胞分化的中心负表观遗传调节因子。基于这些初步结果，我们的假设是酒精和乙醛诱导干细胞的表观遗传变化，影响这些干细胞进行正常分化的能力。在目标（1）中，我们提出 通过使用染色质免疫沉淀测序（ChIP-Seq）进行全基因组分析，并通过还原型亚硫酸氢盐测序（RRBS）定量DNA甲基化的全基因组变化，确定酒精和乙醛对小鼠胚胎干（ES）细胞表观遗传状态的总体影响。在目标（2）中，我们将通过使用RNA-seq技术进行全基因组分析并根据染色质结构的变化解释这些变化，确定酒精和乙醛诱导的表观遗传状态的变化如何影响基因表达和ES细胞的分化（目标1）。我们将使用全反式视黄酸（RA），一种诱导ES细胞分化的信号分子，作为Aim（1）中的阳性对照，因为我们和其他人已经证明RA可以改变ES细胞的表观遗传状态。由于酒精导致组织损伤，需要成年组织中的干细胞反复增殖和分化以试图修复损伤，因此我们的研究将深入了解酒精导致组织损伤和损害对组织损伤的反应的机制。我们推测酒精引起的表观遗传学变化可能是成瘾行为、肿瘤形成和肝硬化等多种疾病的基础。此外，与改变DNA序列并且通常是永久性的基因突变不同，表观遗传变化可以逆转，因此是治疗酒精诱导的组织损伤的合理靶点。 公共卫生相关性：酗酒是一个重大的全球问题，会导致组织损伤和疾病。我们建议在干细胞中进行实验，以描述酒精及其代谢产物乙醛对细胞中DNA周围的组蛋白的影响。我们从这个项目中获得的新信息对于理解酒精相关的组织损伤至关重要，因为干细胞在整个生命过程中不断分化， 并可能导致新型的“表观遗传”药物治疗酒精相关的组织损伤。

DESCRIPTION (provided by applicant): Alcohol consumption causes damage to multiple organs, including the liver and brain, and ethanol can affect the epigenetic state of cells by modifying DNA methylation and modifying histones. These epigenetic changes may compromise regeneration of tissues, predispose cells to become neoplastically transformed, and/or alter the function of stem cells or differentiated cells. In pilot experiments testing the effects of ethanol on murine embryonic stem (ES) cells, we observed some striking effects of ethanol and acetaldehyde, a metabolite of ethanol, on both gene expression and on histone modifications. Indeed, we already have data suggesting that alcohol influences the activity of the Polycomb Repressive Complex 2, a multi-protein complex that is a central negative epigenetic regulator of stem cell differentiation. Based on these preliminary results, our hypothesis is that alcohol and acetaldehyde induce epigenetic changes in stem cells, influencing the ability of such stem cells to undergo normal differentiation. In Aim (1) we propose to determine the global effects of alcohol and acetaldehyde on the epigenetic state of murine embryonic stem (ES) cells by performing genome-wide analysis using chromatin immunoprecipitation-sequencing (ChIP-Seq) and quantifying genome-wide changes in DNA methylation by Reduced-Representation Bi-sulfite Sequencing (RRBS). In Aim (2) we will determine how changes in the epigenetic state induced by alcohol and acetaldehyde affect gene expression and the differentiation of ES cells by performing genome-wide analysis using RNA-seq technology and by interpreting these changes in light of the changes in chromatin structure (Aim 1). We will use all-trans retinoic acid (RA), a signaling molecule that induces ES cell differentiation, as a positive control in Aim (1) since we and others have shown that RA can change the epigenetic state of ES cells. Since alcohol causes tissue injury, requiring stem cells in adult tissues to proliferate and differentiate repeatedly in an attempt to repair the damage, ou studies will lead to insights into the mechanisms by which alcohol both causes tissue injury and compromises response to tissue injury. We hypothesize that alcohol-induced epigenetic changes may underlie disorders as diverse as addictive behavior, tumor formation, and cirrhosis of the liver. Moreover, unlike genetic mutations that change the DNA sequence and are generally permanent, epigenetic changes can be reversed and therefore are reasonable targets for therapies to treat alcohol-induced tissue injury. PUBLIC HEALTH RELEVANCE: Alcohol abuse is a major global problem, resulting in tissue injury and disease. We propose to perform experiments in stem cells that will delineate the effects of alcohol and a metabolite of alcohol, acetaldehyde, on histones, proteins that surround DNA in cells. The new information we gain from this project will be critical for understanding alcohol- associated tissue injury since stem cells are continually differentiating throughout life, and may lead to new types of "epigenetic" drug treatments for alcohol-associated tissue injury.