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. Pathologically, the chronic intake of drugs of abuse causes permanent genetic alterations in the "brain reward pathway." A major focus of drug abuse research is to identify target genes affected by drugs of abuse in this brain region and to ascribe the precise role that gene product plays in the addicted phenotype. One target gene localized in the brain reward pathway, and regulated by cocaine, is the CART (cocaine- and amphetamine-regulated transcript) gene, originally identified as an inducible gene whose mRNA product is up regulated in the rat nucleus accumbens (NAc) after psychostimulant administration. The specific questions our laboratory will address are: 1) how is CART transcriptionally regulated in the rat NAc, 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 in the rat NAc? The aims of the project are--Aim 1: to determine: 1) if transcription factors (TFs) from rat NAc and pituitary bind CART promoter CRE, AP1, and Pit1 cis-regulatory elements; and 2) the effect of binge cocaine treatment on CREB, Pit1 and AP1 TF binding to the above cis-elements (4x20mg/kg, 2hrs). Aim 2: to use wild-type and mutant HSV-CREB constructs to determine if over expression of CREB in the rat NAc contributes to increases in CART peptide levels due to increased P-CREB binding to the CART CRE cis-element. Aim 3: to determine if CREB and P-CREB bind to the CART promoter CRE site in vivo, in the rat NAc by chromatin immunoprecipitation. To date, aim 1 is complete and our binge cocaine regimen does not affect CART gene transcriptional regulation. Aim 2 is nearly complete and HSV-CREB does cause increases in CART peptide and that increase coincides with increased levels of CREB protein as detected by Western blotting.