REGULATION OF THE CART GENE BY PROMOTER CIS-ELEMENTS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 物质滥用研究确定其表达水平因滥用药物而改变的基因，目的是确定体内每个基因转录和/或翻译调节的分子机制。CART(可卡因和安非他明调节转录本)基因定位于大脑奖赏通路，并受可卡因调节。我们实验室研究了先前发现的CART基因启动子DNA顺式调控元件。实验室解决的具体问题是：1)启动子顺式调控元件是否有助于大鼠NAC、垂体和培养细胞中CART基因的转录调控；2)可卡因如何影响大鼠NAC和垂体中CART的转录调控；以及3)CREB是否调控大鼠NAC中CART基因的表达。 到目前为止，我们已经通过DNA-蛋白质相互作用分析确定，从大鼠NAC、垂体和培养细胞中分离的CREB和Pit-1蛋白能够以特定的和浓度依赖的方式与其结合的CART启动子顺式调节元件结合。这些数据结合以前的体外实验结果，表明CART基因启动子中的Cre和Pit-1DNA顺式元件很可能具有功能，并在体内调节CART基因的表达和产物。为了研究CREB对大鼠NAC中CART基因表达的潜在调节作用，给大鼠外周注射可卡因，但对CREB蛋白水平的影响很小，对CART的表达没有影响。通过在NAC内注射非复制型、高表达CREB的病毒载体，CREB被人为地增加，原位杂交和Western印迹分析分别观察到CART mRNA和多肽水平的显著增加。

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. Substance abuse research identifies genes whose expression levels are altered by drugs of abuse in the brain reward pathway with a goal to determine an in vivo, molecular mechanism for each gene's transcriptional and/or translational regulation. The CART (cocaine- and amphetamine-regulated transcript) gene is localized in the brain reward pathway and regulated by cocaine. Our laboratory investigates previously identified, CART gene promoter DNA cis-regulatory elements. The specific questions the laboratory addresses are: 1) do promoter cis-regulatory elements contribute to CART gene transcriptional regulation in the rat NAc, the pituitary and cultured cells; 2) how does cocaine affect the transcriptional regulation of CART in the rat NAc and pituitary; and 3) does CREB regulate CART gene expression in the rat NAc. To date, we have determined by DNA-protein interaction assays that CREB and Pit-1 proteins isolated from the rat NAc, pituitary and cultured cells can bind in specific and concentration-dependant manners to their conjugate CART promoter cis-regulatory elements. These data, in conjunction with previous in vitro findings, indicate that the CRE and Pit-1 DNA cis-elements in the CART gene promoter are most likely functional and can regulate the expression of CART mRNA and production in vivo. To investigate CREB's potential regulatory role on CART gene expression in the rat NAc, cocaine was peripherally administered to rats with a negligible effect on CREB protein levels and no changes in CART expression. CREB was increased artificially by intra-NAc injections of non-replicative, viral vectors over expressing CREB and a significant effect increase in both CART mRNA and peptide levels were observed by in situ hybridization and Western blot analyses, respectively.