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: 8357437
• 负责人: MICHAEL J KUHAR
• 金额: $ 3.29万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8357437
• 关键词: Antibodies; Binding; Biological Assay; CARTPT gene; Cells; Cocaine; Cyclic AMP-Responsive DNA-Binding Protein; Data; Elements; FOS gene; Forskolin; Funding; Genes; Glycine decarboxylase; Goals; Grant; Immunoglobulin G; Injection of therapeutic agent; Messenger RNA; National Center for Research Resources; Nucleus Accumbens; Peptides; Pharmaceutical Preparations; Primates; Principal Investigator; Protein Binding; Rattus; Regulation; Regulatory Element; Research; Research Infrastructure; Resources; Rewards; Site; Source; Testing; United States National Institutes of Health; chromatin immunoprecipitation; cost; promoter

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Both over expression of cyclic AMP response element binding protein (CREB) in the nucleus accumbens (NAc), and intra-accumbal injection of cocaine- and amphetamine-regulated transcript (CART) peptides, have been shown to decrease cocaine reward. Also, over expression of CREB in the rat NAc increased CART mRNA and peptide levels, but it is not known if this was due to a direct action of P-CREB on the CART gene promoter. The goal of this study was to test if CREB and P-CREB bound directly to the CRE site in the CART promoter, using chromatin immunoprecipitation (ChIP) assays. ChIP assay with anti-CREB antibodies showed an enrichment of the CART promoter fragment containing the CRE region over IgG precipitated material, a non-specific control. Forskolin, which was known to increase CART mRNA levels in GH3 cells, was utilized to show that the drug increased levels of P-CREB protein and P-CREB binding to the CART promoter CRE-containing region. A region of the c-Fos promoter containing a CRE cis-regulatory element was previously shown to bind P-CREB, and it was used here as a positive control. These data suggest that the effects of CREB over expression on blunting cocaine reward could be, at least in part, attributed to the increased expression of the CART gene by direct interaction of P-CREB with the CART promoter CRE site, rather than by some indirect action.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 已经证明，在延髓核（NAc）中过表达环AMP反应元件结合蛋白（CREB）和延髓内注射可卡因和安非他明调节转录物（CART）肽都可以降低可卡因奖赏。 此外，CREB在大鼠NAc中的过度表达增加了CART mRNA和肽水平，但尚不清楚这是否是由于P-CREB对CART基因启动子的直接作用。 本研究的目的是使用染色质免疫沉淀（ChIP）测定来测试CREB和P-CREB是否直接结合到CART启动子中的CRE位点。 使用抗CREB抗体的ChIP测定显示含有CRE区域的CART启动子片段相对于IgG沉淀材料（非特异性对照）的富集。 已知毛喉素可增加GH 3细胞中的CART mRNA水平，利用毛喉素来显示该药物增加P-CREB蛋白和P-CREB与CART启动子含CRE区域结合的水平。 含有CRE顺式调节元件的c-Fos启动子区域先前显示结合P-CREB，并且在此将其用作阳性对照。 这些数据表明，CREB过度表达的影响钝化可卡因奖励可能是，至少部分地，归因于CART基因的表达增加的直接相互作用的P-CREB与CART启动子CRE网站，而不是通过一些间接的行动。