Regulation of the CART gene by promoter cis-elements

启动子顺式元件对 CART 基因的调控

• 批准号: 7294892
• 负责人: GEORGE A ROGGE
• 金额: $ 2.82万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-30 至 2009-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7294892
• 关键词: 5' Flanking Region; Address; Amphetamines; Animal Experimentation; Animals; Antibodies; Area; Base Pairing; Behavioral; Binding; Biological Assay; Brain; CARTPT gene; CREB1 gene; Cells; Chemistry; Chromatin; Cocaine; Cocaine Dependence; Complex; Cultured Cells; Cyclic AMP-Responsive DNA-Binding Protein; DNA; DNA Sequence; Dissection; Drug Addiction; Education; Electrophoretic Mobility Shift Assay; Elements; Exhibits; Family; Fellowship; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Human; Indium; Individual; Laboratories; Link; Luciferases; Measures; Messenger RNA; Methodology; Methods; Modeling; Molecular; Molecular Biology; Names; Neuropeptides; Nuclear Protein; Nuclear Proteins; Nucleus Accumbens; Overdose; Pathway interactions; Peptides; Physiological Processes; Physiology; Pituitary Gland; Polymerase Chain Reaction; Promoter Regions; Property; Protein Binding; Proteins; Psychological reinforcement; Rate; Rattus; Reaction; Regulation; Regulatory Element; Rewards; Saline; Time; Tissues; Training; Transcription Factor AP-1; Transcriptional Regulation; Treatment Protocols; Universities; Western Blotting; Work; chromatin immunoprecipitation; crosslink; drug of abuse; feeding; in vivo; insight; mRNA Expression; mutant; paraform; promoter; research study; response; transcription factor

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.

描述（由申请人提供）：这项工作将研究基因启动子区域CRE， AP1和Pit1顺式元件调节CART（可卡因和安非他明调节转录本）mRNA表达变化的能力。CART神经肽在基因5'侧区含有六个假定的顺式调控元件，并受到各种生理过程的高度调控。例如，暴食可卡因可增加大鼠伏隔核（NAc）的CART mRNA。我们假设CART mRNA表达的变化可能受到与CART基因顺式调控元件结合的转录因子（tf）的调控。为了验证这一假设，CART基因CRE、AP1和Pit1顺式元件将通过电泳迁移位移法（EMSAs）和抗体超位移法（SS）检测其蛋白结合活性。此外，我们将通过测量各种突变型和野生型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顺式元件对培养细胞中CART基因转录调控的贡献。最后，我们将通过对交联大鼠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。