REGULATION OF THE CART GENE BY PROMOTER CIS-ELEMENTS

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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.