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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 在伏隔核（NAC）中的环状AMP反应元件结合蛋白（CREB）的表达均过多，以及可卡因和苯丙胺调节的转录物（CART）肽的良心内注射均可减少可卡因奖励。 同样，在大鼠NAC中，CREB的表达增加了CART mRNA和肽水平，但尚不清楚这是否是由于P-Creb在CART基因启动子上的直接作用。 这项研究的目的是使用染色质免疫沉淀（CHIP）测定法测试CREB和P-CREB是否直接与CRE启动子的CRE位点结合。 用抗CREB抗体的CHIP测定法显示了含有CRE区域的CRE区域的富集，该碎片含有IgG沉淀物材料（一种非特异性对照）。 已知福斯科蛋白会增加GH3细胞中的CART mRNA水平，以表明该药物增加了P-CREB蛋白的水平和P-CREB与含CART启动子CRE的区域的结合。 先前显示出包含CIS调节元件的C-FOS启动子的区域，以结合P-CREB，在这里被用作阳性对照。 这些数据表明，Creb对表达对可卡因奖励的影响至少部分归因于CART基因的表达增加，而不是通过P-CREB与CART启动子CRE位点的直接相互作用，而不是通过某些间接作用。