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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 药物滥用研究的一个主要重点是阐明成瘾的分子机制，导致长期的行为异常，导致药物渴望和反复复发。 基础研究的一个目标是确定药物治疗目标以开发药物。 慢性可卡因摄入导致大脑奖赏途径的长期遗传改变，通过上调丘脑核（NAc）中的CREB转录因子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强化作用的减少相关。 CART基因启动子被发现含有一个cAMP反应元件（CRE），建议结合CREB，CART mRNA最初被确定为可卡因给药后在大鼠NAc上调的转录本。 在自我施用测定中，CART肽的NAc内注射减弱了可卡因的奖励作用。 本论文的总体假设是，CART是一个CREB调控的基因。 在项目期间收集的数据通过染色质免疫沉淀试验证明，培养细胞中的CREB和P-CREB能够结合到活细胞核中含有CRE位点的CART启动子区域。 此外，电泳迁移率变化和抗体超位移测定显示，CREB和P-CREB从大鼠NAc能够结合到一个短的寡核苷酸序列相同的CART启动子CRE网站。 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，来自这项工作的大量数据强烈表明，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和肽的表达。 CREB减弱可卡因奖赏效应的机制可能部分是通过增加NAc中CART肽的表达。