This work will examine the ability of gene promoter region CRE, AP1 and Pit1 cis-elements to regulate changes in CART (cocaine- and amphetamine-regulated transcript) mRNA expression. CART neuropeptides contain six, putative cis-regulatory elements in the gene 5' flanking region and are highly regulated by various physiological processes. For example, binge cocaine administration increases CART mRNA in the nucleus accumbens (NAc) of rats. We hypothesize that changes in CART mRNA expression may be regulated by transcription factors (TFs) binding to CART gene cis-regulatory elements. To address this hypothesis, the CART gene CRE, AP1, and Pit1 cis-elements will be assayed by electrophoretic mobility shift assays (EMSAs) and antibody super shift assays (SS) for their protein binding activities. In addition, we will observe the contributions of CRE, AP1 and Pit1 cis-elements to transcriptional regulation of the CART gene in cultured cells by measuring the luciferase activities of various mutant and wild-type CART promoter-driven luciferase constructs. Finally, we will evaluate the potential contribution of the TF CREB to CART gene transcription by performing chromatin immunoprecipitaion on cross-linked rat NAc tissue. ? ? ?
| Rogge, George A; Shen, Li-Ling; Kuhar, Michael J (2010) Chromatin immunoprecipitation assays revealed CREB and serine 133 phospho-CREB binding to the CART gene proximal promoter. Brain Res 1344:1-12 |
