Regulation of the CART gene by promoter cis-elements

描述(申请人提供)：这项工作将检测基因启动子区Cre、AP1和Pit1顺式元件调节CART(可卡因和苯丙胺调节的转录本)mRNA表达变化的能力。CART神经肽在基因5‘侧翼区含有6个可能的顺式调节元件，受多种生理过程的高度调控。例如，大量服用可卡因会增加大鼠伏隔核(NAC)中的CART mRNA。我们推测CART基因表达的变化可能受与CART基因顺式调控元件结合的转录因子的调节。为了解决这一假设，将通过凝胶迁移率改变分析(EMSA)和抗体超转移分析(SS)来检测CART基因Cre、AP1和Pit1顺式元件的蛋白结合活性。此外，我们还将通过检测不同突变型和野生型CART启动子驱动的荧光素酶构建体的荧光素酶活性，观察Cre、AP1和Pit1顺式元件在培养细胞中对CART基因转录调控的贡献。最后，我们将通过对交联型大鼠NAC组织进行染色质免疫沉淀来评估Tf CREB对CART基因转录的潜在贡献。

DESCRIPTION (provided by applicant): 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.