Role of histone demethylases in experience dependent alcohol behavior

描述(由申请人提供)：这项R21拨款提案旨在描述将最初的酒精暴露转化为大脑长期变化的分子机制。我们的工作集中在一个染色质修饰酶家族，组蛋白去甲基酶(HDM)。表观遗传变化越来越被认为与成瘾有关，特定基因组座位上组蛋白甲基化的调节通常控制着基因转录的变化，以响应环境提示。由于其易于基因操作，果蝇是一个强大的平台，在这个平台上研究染色质编程的变化如何导致酒精诱导的行为变化。我们试图测试这一假设，即特定的HDMS调节与酒精接触相关的经验依赖的行为变化。为了了解这些染色质修饰酶在体内对酒精耐受性和消费偏好的作用，我们已经开始系统地敲除果蝇HDM的14个基因中的每一个，所有这些基因都与哺乳动物的同源基因密切相关。为了实现这一目标，我们正在使用尖端的分子技术，如重组工程和同源重组。在这项探索性/发展性研究拨款中，我们提出了以下两个目标：AIM1试图系统地确定携带全部14个HDM基因遗传修饰的果蝇品系的酒精耐受性和消费偏好表型。我们将通过测试每个HDM基因的敲除和过表达株，并通过定量评估它们的酒精耐受性和消费偏好来实现这一目标。在我们的偏好分析中，幼稚的苍蝇倾向于避免含有15%乙醇的食物，而乙醇预暴露将这种回避转化为显著的偏好。我们的初步数据显示，至少有两个HDM基因敲除品系显示出不同的乙醇消费偏好。在AIM2中，我们建议通过使用我们手头已有的HA标记的HDM蛋白进行染色质免疫沉淀和大规模并行深度测序(CHIP-SEQ)来确定功能相关的HDMS在基因组中的结合位置。这项工作将产生一张全面的基因图谱，这些基因在酒精暴露时会受到功能上相关的HDMS的差异结合。确定HDMS在经验依赖的乙醇可塑性中的体内功能对于理解人类成瘾的发展具有很大的潜在影响。这些蛋白质高度保守，代表了一类可药物酶，为未来治疗干预的发展提供了希望。

DESCRIPTION (provided by applicant): This R21 grant proposal aims to characterize the molecular mechanisms that transform initial alcohol exposures into long-lasting changes in the brain. Our work focuses on a family of chromatin modification enzymes, the histone demethylases (HDM). Epigenetic changes are increasingly being recognized as relevant in addiction and modulation of histone methylation at specific genomic loci often governs changes in gene transcription in response to environmental cues. Because of its ease of genetic manipulation, Drosophila represents a powerful platform on which to study how alterations in chromatin programming result in alcohol- induced changes in behavior. We seek to test the hypothesis that specific HDMs regulate the experience- dependent behavioral changes that accompany exposure to ethanol. To understand the in vivo role of these chromatin modification enzymes in alcohol tolerance and consumption preference, we have begun to systematically knock out every one of the 14 Drosophila HDM genes, all of which have closely related mammalian orthologs. To accomplish this goal, we are employing cutting-edge molecular techniques such a recombineering and homologous recombination. In this exploratory/developmental research grant we propose the following two aims: Aim1 seeks to systematically determine the ethanol tolerance and consumption preference phenotypes of Drosophila strains carrying genetic modifications in all 14 HDM genes. We will accomplish this aim by testing knock-out and overexpression lines for each HDM gene, and by quantitatively assessing their ethanol tolerance and consumption preference. In our preference assay, naive flies show a tendency to avoid food containing 15% ethanol, while ethanol pre-exposure transforms this avoidance into a significant preference. Our preliminary data suggests that at least two HDM knock-out lines show altered ethanol consumption preference. In Aim2, we propose to determine where functionally relevant HDMs bind throughout the genome, by performing chromatin immunoprecipitation coupled with massive parallel deep sequencing (ChIP-seq) using HA-tagged HDM proteins we have in hand. This work will yield a comprehensive map of genes differentially bound by functionally relevant HDMs upon a behavior-changing ethanol exposure. Determining the in vivo functions of HDMs in experience-dependent ethanol plasticity has the potential for high impact on understanding the development of human addiction. These proteins are highly conserved and represent a class of druggable enzymes that hold promise for the future development of therapeutic intervention.