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

描述(申请人提供)：毒瘾综合症严重损害了受影响患者及其家人的生命，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易感性，但很难将这些发现与特定的基因组基因座联系起来，这表明表观遗传机制可能是成瘾表型的基础。最近出现的证据表明，长期接触可卡因可以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整体变化。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都受到慢性可卡因在特定大脑奖赏区域的调节。这项提案概述了工程锌指蛋白(ZFP)作为新的分子工具在研究药物成瘾的表观遗传机制方面的高度创新用途。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控。第一个特定目的是利用病毒介导的小鼠腹侧纹状体表达，然后进行蛋白质和mRNA表达分析，以表征FosB-ZFP在体外和体内对FosB基因表达的调节。第二个具体目的是通过分析表达FosB-ZFP的小鼠在测量可卡因敏感性和偏好的范例中的行为，确定FosB-ZFP作为可卡因中介行为的新调节剂的用途。第三个特定目的是利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，以表征FosB-ZFP在小鼠腹侧纹状体表达的转录调控的表观遗传修饰。通过这项提案中详述的创新和全面的研究战略，申请者伊丽莎白·海勒博士将获得新的行为和分子生物学技术方面的广泛培训，这些技术对在一流学术机构从事药物滥用研究的职业生涯至关重要。拟议的研究地点西奈山医学院是一所最先进的机构，为开展拟议的研究提供必要的先进技术资源。赞助商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，培训Heller博士，为药物滥用研究的Caree做准备。本文提出的研究将为药物滥用的转录调控机制提供新的线索，并证明ZFP作为新的工具用于药物成瘾的神经生物学研究。

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.