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

药物成瘾综合征严重损害受影响患者及其家人的生活，并对美国造成巨大的经济损失。虽然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相关联，这表明表观遗传机制可能是成瘾表型的基础。最近的证据表明，慢性可卡因暴露调节几个组蛋白修饰酶和转录因子的表达，以及诱导染色质结构的全球变化。例如，在啮齿动物模型和人类患者中，fosB基因的染色质结构和表达在特定的大脑奖赏区域中受到慢性可卡因的调节。这项提案概述了高度创新的使用工程锌指蛋白（ZFP）作为新的分子工具，研究药物成瘾的表观遗传机制。ZFP是DNA结合蛋白，其可以在整个基因组的背景下将转录调控引导至单个基因位点。第一个具体的目的是表征FosB-ZFP在体外和体内对fosB基因表达的调节，使用病毒介导的小鼠腹侧纹状体中的表达，然后进行蛋白质和mRNA表达分析。第二个具体目标是通过分析在测量可卡因敏感性和偏好的范例中表达FosB-ZFP的小鼠的行为来确定FosB-ZFP作为可卡因介导的行为的新型调节剂的用途。第三个具体的目的是表征表观遗传修饰的基础fosB转录调控FosB-ZFP在小鼠腹侧纹状体中表达，使用染色质免疫沉淀，以确定组蛋白修饰和转录因子结合在fosB基因。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博士将获得新的行为和分子生物学技术的广泛培训，这些技术对顶级学术机构的药物滥用研究事业至关重要。拟议的研究地点西奈山医学院是一个最先进的机构，提供开展拟议研究所需的先进技术资源。赞助商Eric Nestler博士是世界知名的药物滥用研究人员，他将提供理想的合作环境，以培训Heller博士，为药物滥用研究的职业生涯做准备。本文提出的研究将为滥用药物的转录调控机制提供新的线索，并展示ZFP作为药物成瘾神经生物学研究中的新工具的用途。

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.