REGULATION OF KIDNEY-SPECIFIC GENE EXPRESSION

肾脏特异性基因表达的调节

• 批准号: 2900229
• 负责人: Peter Igarashi
• 金额: $ 8.89万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-15 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2900229
• 关键词: DNA footprinting; gel mobility shift assay; gene expression; genetic library; genetic mapping; genetic promoter element; genetic regulatory element; genetically modified animals; intermolecular interaction; laboratory mouse; membrane transport proteins; molecular cloning; nucleic acid sequence; renal tubular transport; saluresis; tissue /cell culture; transcription factor; transfection

The long-term objectives of this project are to identify the cis-acting regulatory elements and transcription factors that are responsible for kidney-specific gene expression. The proposed studies will examine the renal absorptive Na-K-Cl cotransporter (Nkcc2/Slc12a1) as a model kidney- specific gene. In the kidney, NKCC2 mediates active reabsorption of NaCl in the thick ascending limb of the loop of Henle and represents the site of action of the clinically important loop diuretics furosemide and bumetanide. Importantly, expression of Nkcc2 transcripts is only observed in the kidney. Recent studies demonstrate that the Nkcc2 gene is under transcriptional control and that the Nkcc2 promoter exhibits cell-lineage- specific activity. The proposed studies will test the hypothesis that kidney-specific transcription of Nkcc2 is mediated by the interaction between kidney-enriched transcription factors and regulatory elements of the Nkcc2 gene. To identify regulatory elements that are responsible for kidney-specific promoter activity, fragments of the proximal 5' flanking region of the Nkcc2 gene will be ligated to a promoterless reporter gene and transiently expressed in cultured kidney cells. Evidence for cell- type-specific promoter activity will be obtained by transfection into cells of differing lineages. It will be important to verify that regulatory elements identified in vitro also confer kidney-specificity upon expression of a lacZ reporter gene in transgenic mice. Specific enhancer and negative regulatory elements within the 5' flanking region will be identified by deletion analysis and phylogenetic sequence conservation. Regulatory elements located elsewhere in the gene will be identified on the basis of DNase I hypersensitivity. Whether candidate regions contain kidney-specific regulatory elements will be determined by evaluating their effects on transcription from a heterologous promoter both in vitro and in vivo. Electrophoretic mobility-shift assays will be performed to determine whether cis-acting elements bind to kidney-enriched nuclear proteins. Specific sites of DNA-protein interaction will be defined using in vitro DNA footprinting, and the effects of mutations of the elements on protein-binding and transcriptional activity will be assessed. Transcription factors that bind to relevant regulatory elements will be identified by Southwestern blotting or UV crosslinking, and cDNAs encoding these proteins will be cloned by screening expression libraries with recognition site probes. Identification of the cis-acting regulatory elements that are required for transcription of Nkcc2 will provide general insights into mechanisms of kidney-specific gene expression: will be important for future studies of dysregulated expression of Nkcc2 in states of abnormal renal salt handling, such as Bartter's syndrome and essential hypertension; and will provide reagents that may be useful for future in vivo gene therapy of the kidney. Characterization of the transcription factors involved in expression of Nkcc2 may also be clinically important since mutations affecting such proteins may cause developmental, neoplastic, or cystic disorders of the kidney.

本项目的长期目标是确定顺式作用的 调控元件和转录因子， 肾脏特异性基因表达。拟议的研究将审查 肾吸收性Na-K-Cl协同转运蛋白（Nkcc 2/Slc 12 a1）作为模型肾- 特定基因在肾脏中，NKCC 2介导NaCl的主动重吸收 在亨利氏袢的粗大上升支中， 临床上重要的袢利尿剂呋塞米和 布美他尼。重要的是，Nkcc 2转录本的表达仅在 在肾脏中。最近的研究表明，Nkcc 2基因是在 转录控制和Nkcc 2启动子表现出细胞谱系， 具体活动。拟议的研究将检验以下假设： Nkcc 2的肾特异性转录是由以下相互作用介导的 肾脏富集的转录因子和 Nkcc 2基因确定负责以下方面的监管要素： 肾特异性启动子活性，近端5'侧翼的片段 将Nkcc 2基因的区域连接到无启动子的报告基因 并在培养的肾细胞中瞬时表达。手机的证据- 型特异性启动子活性将通过转染入 不同谱系的细胞。重要的是要核实 体外鉴定的调节元件也赋予肾特异性 在转基因小鼠中表达lacZ报告基因。具体 增强子和5'侧翼区内的负调控元件 将通过缺失分析和系统发育序列鉴定 节约位于基因其他位置的调控元件将被 根据DNA酶I超敏反应鉴定。候选人是否 包含肾脏特异性调节元件的区域将由以下因素确定 评价它们对从异源启动子转录的影响 无论是在体外还是在体内。电泳迁移率变化分析将 以确定顺式作用元件是否与肾富集的 核蛋白DNA-蛋白质相互作用的特定位点将是 使用体外DNA足迹法定义， 蛋白质结合和转录活性的要素将是 评估。与相关调控元件结合的转录因子 将通过Southwestern印迹或UV交联鉴定， 通过筛选表达文库， 识别位点探针。顺式作用调节因子的鉴定 转录Nkcc 2所需的元件将提供一般的 深入了解肾脏特异性基因表达的机制：将 对于未来研究Nkcc 2在国家中的表达失调很重要 肾盐处理异常，如Bartter综合征和原发性 高血压;并将提供试剂，可能是有用的， 肾脏的体内基因治疗。转录的表征 参与Nkcc 2表达的因子也可能是临床重要的 由于影响这些蛋白质的突变可能引起发育， 肿瘤或肾脏囊性疾病。