REGULATION OF THE CART GENE BY PROMOTER CIS-ELEMENTS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 伏核(NAC)中环AMP反应元件结合蛋白(CREB)的过度表达，以及伏核内注射可卡因和苯丙胺调节转录物(CART)，都被证明降低了可卡因的奖赏。此外，CREB在大鼠NAC中的过度表达增加了CART的mRNA和多肽水平，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。本研究的目的是用染色质免疫沉淀法(CHIP)检测CREB和P-CREB是否直接与CART启动子中的Cre结合。用抗CREB抗体进行芯片检测显示，含有Cre区的CART启动子片段比免疫球蛋白沉淀物质更富集，这是一种非特异性对照。Forskolin被用来证明该药物增加了CART启动子Cre区域的P-CREB蛋白和P-CREB结合水平，Forsklin已被用来增加GH3细胞中CART mRNA的水平。C-Fos启动子中包含Cre顺式调控元件的区域先前被证明与P-CREB结合，这里将其用作阳性对照。这些数据表明，CREB的过度表达对钝化可卡因奖赏的影响可能至少部分归因于通过P-CREB与CART启动子Cre位点的直接相互作用而增加了CART基因的表达，而不是通过某种间接作用。

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.