REGULATION OF THE CART GENE BY PROMOTER CIS-ELEMENTS

启动子 CIS-元件对 Cart 基因的调控

• 批准号: 8172381
• 负责人: MICHAEL J KUHAR
• 金额: $ 4.39万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8172381
• 关键词: Addictive Behavior; Antibodies; Basic Science; Behavioral; Binding; Biological Assay; Brain; CREB1 gene; Cell Nucleus; Cells; Chronic; Cocaine; Computer Retrieval of Information on Scientific Projects Database; Cultured Cells; Cyclic AMP Response Element; Data; Drug abuse; Elements; Funding; Gene Transfer; Genes; Goals; Grant; Injection of therapeutic agent; Institution; Intake; Life; Mediating; Messenger RNA; Molecular; Mutation; Nucleus Accumbens; Oligonucleotides; Pathway interactions; Peptides; Pharmaceutical Preparations; Pharmacotherapy; Rattus; Regulation; Relapse; Research; Research Personnel; Resources; Rewards; Self Administration; Site; Source; Sum; Transcript; United States National Institutes of Health; Work; addiction; chromatin immunoprecipitation; cocaine- and amphetamine-regulated transcript protein; drug craving; preference; promoter; transcription factor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A major focus of drug abuse research is to elucidate the molecular mechanisms of addiction responsible for long-term behavioral abnormalities causing drug craving and repeated relapse. One goal of that basic research is identifying pharmacotherapy targets to develop medications. Chronic cocaine intake causes long-term genetic alterations in the brain reward pathway that contribute to addictive behaviors by up-regulating CREB transcription factor activity in nucleus accumbens (NAc). Increases in CREB activity in NAc were correlated with decreases in the reinforcing effects of cocaine during conditioned place-preference and drug self-administration assays. The CART gene promoter was found to contain a cAMP response element (CRE) proposed to bind CREB, and CART mRNA was originally identified as a transcript in rat NAc up-regulated after cocaine administration. Intra-NAc injection of CART peptides blunted rewarding effects of cocaine in self administration assays. The overall hypothesis of this dissertation is that CART is a CREB-regulated gene. The data collected during the project period demonstrated by chromatin immunoprecipitation assay that CREB and P-CREB in cultured cells were capable of binding to a region of the CART promoter containing the CRE site in nuclei of living cells. Furthermore, electrophoretic mobility shift and antibody super-shift assays revealed that CREB and P-CREB from rat NAc were able to bind to a short oligonucleotide identical in sequence to the CART promoter CRE site. In rat NAc, over-expression of CREB by virally mediated gene transfer increased CART mRNA and peptide levels. In sum, the body of data from this work strongly suggested that CREB and P-CREB regulated CART mRNA and peptide expression in rat NAc by acting directly at the CRE site in the CART proximal promoter. A mechanism by which CREB blunted the rewarding effects of cocaine may have been, in part, by increased expression of CART peptides in the NAc.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 药物滥用研究的一个主要重点是阐明导致长期行为异常导致药物渴望和反复复发的成瘾分子机制。 该基础研究的目标之一是确定药物治疗靶点以开发药物。 长期摄入可卡因会导致大脑奖赏通路发生长期遗传改变，通过上调伏隔核 (NAc) 中 CREB ​​转录因子的活性而导致成瘾行为。 在条件性位置偏好和药物自我给药试验中，NAc 中 CREB ​​活性的增加与可卡因增强作用的降低相关。 CART 基因启动子被发现含有一个 cAMP 反应元件 (CRE)，拟与 CREB ​​结合，CART mRNA 最初被鉴定为大鼠 NAc 中可卡因给药后上调的转录本。 在自我给药测定中，NAc 内注射 CART 肽会减弱可卡因的奖励作用。 本论文的总体假设是CART是CREB调控的基因。 项目期间收集的数据通过染色质免疫沉淀分析证明，培养细胞中的CREB和P-CREB能够与活细胞核中含有CRE位点的CART启动子区域结合。 此外，电泳迁移率变动和抗体超变动测定表明，来自大鼠 NAc 的 CREB ​​和 P-CREB ​​能够结合与 CART 启动子 CRE 位点序列相同的短寡核苷酸。 在大鼠 NAc 中，通过病毒介导的基因转移过度表达 CREB ​​增加了 CART mRNA 和肽水平。 总之，这项工作的数据强烈表明，CREB ​​和 P-CREB ​​通过直接作用于 CART 近端启动子中的 CRE 位点来调节大鼠 NAc 中的 CART mRNA 和肽表达。 CREB ​​减弱可卡因奖励作用的机制可能部分是通过增加 NAc 中 CART 肽的表达来实现的。