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

在药物滥用中使用工程 ZFP 进行体内基因特异性调控

• 批准号: 8518823
• 负责人: Elizabeth A Heller
• 金额: $ 5.39万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8518823
• 关键词: Affect; Behavior; Behavioral; Binding; Brain; Brain region; Cell physiology; Chromatin; Chromatin Structure; Chronic; Cocaine; DNA Binding Domain; DNA-Binding Proteins; Data; Drug Addiction; Drug Exposure; Drug abuse; Economics; Engineering; Environment; Enzymes; Epigenetic Process; Family; Functional disorder; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic Transcription; Genome; Genomics; Global Change; Histones; Human; In Vitro; Inherited; Institution; Knowledge; Laboratories; Light; Measures; Mediating; Mediator of activation protein; Modification; Molecular; Molecular Biology Techniques; Molecular Probes; Morphine; Mus; Nature; Neurobiology; Nucleus Accumbens; Patients; Phenotype; Platelet Factor 4; Predisposition; Preparation; Protein Isoforms; Protein Overexpression; Proteins; Proteome; Public Health; Regulation; Reporting; Research; Research Personnel; Research Proposals; Resources; Rewards; Rodent Model; Site; Specificity; Syndrome; Technology; Training; Transcriptional Regulation; United States; Ventral Striatum; Viral; Zinc Fingers; addiction; career; chromatin immunoprecipitation; chromatin modification; cocaine exposure; drug of abuse; drug sensitivity; histone modification; in vivo; innovation; interest; mRNA Expression; medical schools; novel; overexpression; preference; promoter; protein expression; public health relevance; research study; response; tool; transcription factor

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.

描述（由申请人提供）：吸毒成瘾综合症严重损害了受影响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药物滥用具有遗传倾向，但很难将这些发现与特定的基因组位点关联起来，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，长期接触可卡因会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的特定大脑奖励区域中，fosB 基因的染色质结构和表达受到慢性可卡因的调节。该提案概述了工程化锌指蛋白（ZFP）作为新型分子工具的高度创新用途，用于研究药物成瘾背后的表观遗传机制。 ZFP 是 DNA 结合蛋白，可以指导整个基因组中单个基因位点的转录调控。第一个具体目标是利用小鼠腹侧纹状体中病毒介导的表达，然后进行蛋白质和 mRNA 表达分析，来表征 FosB-ZFP 在体外和体内对 fosB 基因表达的调节。第二个具体目标是通过分析在测量可卡因敏感性和偏好的范式中表达 FosB-ZFP 的小鼠的行为，确定 FosB-ZFP 作为可卡因介导行为的新型调节剂的用途。第三个具体目标是通过在小鼠腹侧纹状体中表达的 FosB-ZFP 来表征 fosB 转录调控的表观遗传修饰，使用染色质免疫沉淀来识别 fosB 基因上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，申请人伊丽莎白·海勒博士将获得新行为和分子生物学技术方面的广泛培训，这对于在顶级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，提供开展拟议研究所需的先进技术资源。赞助商埃里克·内斯特勒博士是一位世界著名的药物滥用研究人员，他将为海勒博士提供理想的合作环境，为他从事药物滥用研究的职业生涯做好准备。这里提出的研究将为滥用药物的转录调节机制提供新的线索，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。