In Vivo Gene-Specific Regulation Using Engineered ZFPs in Drug Abuse

描述（由申请人提供）：药物成瘾综合症严重损害了受影响的患者及其家人的生活，并给美国造成了巨大的经济损失。尽管科学数据已经证明了药物滥用的遗传倾向，但很难将这些发现与特定的基因组位点联系起来，这表明表观遗传机制可能是成瘾表型的基础。最近有证据表明，慢性可卡因暴露可调节几种组蛋白修饰酶和转录因子的表达，并诱导染色质结构的全局变化。例如，在啮齿类动物模型和人类患者中，fosB基因的染色质结构和表达受到慢性可卡因在特定大脑奖励区域的调节。本提案概述了工程锌指蛋白（ZFPs）作为研究药物成瘾的表观遗传机制的新分子工具的高度创新使用。zfp是一种dna结合蛋白，可以在整个基因组的背景下直接对单个基因位点进行转录调节。第一个具体目的是利用病毒介导小鼠腹侧纹状体的表达，然后分析蛋白和mRNA的表达，表征fosB - zfps在体外和体内对fosB基因表达的调控。第二个具体目的是通过分析表达FosB-ZFPs的小鼠在测量可卡因敏感性和偏好的范式中的行为，确定FosB-ZFPs作为可卡因介导行为的新调节剂的使用。第三个具体目标是表征小鼠腹侧纹状体中表达的fosB - zfps对fosB转录调控的表观遗传修饰，使用染色质免疫沉淀来鉴定fosB基因的组蛋白修饰和转录因子结合。通过本提案中详细介绍的创新和全面的研究策略，申请人Elizabeth Heller博士将在新的行为和分子生物学技术方面获得广泛的培训，这对于在顶级学术机构从事药物滥用研究至关重要。拟议的研究地点是西奈山医学院，这是一个最先进的机构，为开展拟议的研究提供了必要的技术先进资源。主办者Eric Nestler博士是一位世界知名的药物滥用研究人员，他将提供理想的合作环境来训练Heller博士，为他在药物滥用研究方面的职业生涯做准备。本研究将揭示药物滥用对转录调控的新机制，并证明zfp作为药物成瘾神经生物学研究的新工具。

DESCRIPTION (provided by applicant): The syndrome of drug addiction severely damages the lives of affected patients and their families, and exacts an enormous economic toll on the United States. Although scientific data has demonstrated a hereditary predisposition to drug abuse it has been difficult to correlate these findings with specific genomic loci, suggesting epigenetic mechanisms may underlie the addicted phenotype. Recent evidence has emerged that chronic cocaine exposure regulates expression of several histone-modifying enzymes and transcription factors, as well as induces global changes in chromatin structure. For example, the chromatin structure and expression of the fosB gene is regulated by chronic cocaine in specific brain reward regions in both rodent models and human patients. This proposal outlines the highly innovative use of engineered zinc-finger proteins (ZFPs) as novel molecular tools to study the epigenetic mechanisms underlying drug addiction. ZFPs are DNA-binding proteins that can direct transcriptional regulation to a single gene locus in the context of the whole genome. The first specific aim is to characterize the regulation of fosB gene expression by FosB-ZFPs in vitro and in vivo, using viral-mediated expression in the mouse ventral striatum followed by protein and mRNA expression analysis. The second specific aim is to determine the use of FosB-ZFPs as novel modulators of cocaine-mediated behavior by analyzing the behavior of mice expressing FosB-ZFPs in paradigms measuring cocaine sensitivity and preference. The third specific aim is to characterize the epigenetic modifications underlying fosB transcriptional regulation by FosB-ZFPs expressed in mouse ventral striatum, using chromatin-immunoprecipitation to identify histone modifications and transcription factor binding at the fosB gene. Through the innovative and comprehensive research strategy detailed in this proposal, the applicant, Dr. Elizabeth Heller, will gain extensive training in new behavioral and molecular biology techniques, which are vital to a career in drug abuse research at a top academic institution. The proposed site of research, the Mount Sinai School of Medicine, is a state-of-the-art institution, providing the technologically advanced resources necessary to carry out the proposed research. The sponsor, Dr. Eric Nestler, is a world-renowned drug abuse researcher, who will provide the ideal collaborative environment to train Dr. Heller in preparation for a caree in drug abuse research. The research proposed here will shed new light on the mechanisms of transcriptional regulation by drugs of abuse, as well as demonstrate the use of ZFPs as novel tools in the study of the neurobiology of drug addiction.