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. A major focus of drug abuse research is to elucidate the molecular mechanisms of addiction responsible for long-term behavioral abnormalities causing drug craving and repeated relapse. One goal of that basic research is identifying pharmacotherapy targets to develop medications. Chronic cocaine intake causes long-term genetic alterations in the brain reward pathway that contribute to addictive behaviors by up-regulating CREB transcription factor activity in nucleus accumbens (NAc). Increases in CREB activity in NAc were correlated with decreases in the reinforcing effects of cocaine during conditioned place-preference and drug self-administration assays. The CART gene promoter was found to contain a cAMP response element (CRE) proposed to bind CREB, and CART mRNA was originally identified as a transcript in rat NAc up-regulated after cocaine administration. Intra-NAc injection of CART peptides blunted rewarding effects of cocaine in self administration assays. The overall hypothesis of this dissertation is that CART is a CREB-regulated gene. The data collected during the project period demonstrated by chromatin immunoprecipitation assay that CREB and P-CREB in cultured cells were capable of binding to a region of the CART promoter containing the CRE site in nuclei of living cells. Furthermore, electrophoretic mobility shift and antibody super-shift assays revealed that CREB and P-CREB from rat NAc were able to bind to a short oligonucleotide identical in sequence to the CART promoter CRE site. In rat NAc, over-expression of CREB by virally mediated gene transfer increased CART mRNA and peptide levels. In sum, the body of data from this work strongly suggested that CREB and P-CREB regulated CART mRNA and peptide expression in rat NAc by acting directly at the CRE site in the CART proximal promoter. A mechanism by which CREB blunted the rewarding effects of cocaine may have been, in part, by increased expression of CART peptides in the NAc.