Regulation of the CART gene by promoter cis-elements

启动子顺式元件对 CART 基因的调控

基本信息

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
项目类别：
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&apos 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

项目摘要

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）：这项工作将检查基因启动子区域 CRE、AP1 和 Pit1 顺式元件调节 CART（可卡因和安非他明调节转录物）mRNA 表达变化的能力。 CART 神经肽在基因 5' 侧翼区域包含六个假定的顺式调节元件，并受到各种生理过程的高度调节。例如，大量服用可卡因会增加大鼠伏核 (NAc) 中的 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) 的调节。为了解决这一假设，将通过电泳迁移率变动分析 (EMSA) 和抗体超位移分析 (SS) 来分析 CART 基因 CRE、AP1 和 Pit1 顺式元件的蛋白质结合活性。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，我们将通过对交联的大鼠 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。