REGULATION OF KIDNEY-SPECIFIC GENE EXPRESSION

该项目的长期目标是确定顺应性代理 调控元件和转录因子负责 肾脏特异性基因表达。建议的研究将检视 肾吸收性钠-钾-氯共转运蛋白(Nkcc2/Slc12a1)作为模型肾脏 特定的基因。在肾脏，NKCC2介导了对氯化钠的主动重吸收 在亨勒环粗大的上升支上，代表着 临床上重要的环状利尿剂速尿和 布美他尼。重要的是，仅观察到Nkcc2转录本的表达 在肾脏里。最近的研究表明，Nkcc2基因在 转录控制和Nkcc2启动子表现出细胞谱系- 特定活动。拟议的研究将检验这一假设 Nkcc2的肾脏特异性转录是由相互作用介导的 肾富集型转录因子与血管紧张素转换酶调控元件的关系 Nkcc2基因。确定负责的监管要素 肾脏特异性启动子活性，近端5‘侧翼片段 Nkcc2基因的区域将连接到一个无启动子的报告基因 并在培养的肾脏细胞中瞬时表达。细胞的证据- 特定类型的启动子活性将通过转染获得 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脏特异性 在转基因小鼠中表达LacZ报告基因。特定的 5‘侧翼区内的增强子和负调控元件 将通过缺失分析和系统发育序列进行鉴定 保护环境。位于基因其他位置的调控元件将是 根据脱氧核糖核酸酶I超敏反应鉴定。候选人是否 包含特定于肾脏的监管元素的区域将由 评估它们对异源启动子转录的影响 在体外和体内都有。电泳迁移率变化分析将是 以确定顺式作用元件是否与富肾药结合 核蛋白。DNA-蛋白质相互作用的特定位置将是 使用体外DNA足迹定义，以及突变的影响 影响蛋白质结合和转录活性的元素将是 评估过了。与相关调控元件结合的转录因子 将通过西南杂交或UV交联法鉴定，以及cDNA 编码这些蛋白的基因将通过筛选表达文库进行克隆 用识别位点探头。识别顺式代理监管机构 转录Nkcc2所需的元素将提供一般 对肾脏特异基因表达机制的见解：将是 对未来研究Nkcc2在状态下的异常表达具有重要意义 肾脏盐处理异常，如巴特综合征和必需的 高血压；并将提供可能对未来 肾脏的活体基因治疗。转录的特征 参与Nkcc2表达的因素也可能在临床上很重要。 由于影响这些蛋白质的突变可能会导致发育， 肾脏的肿瘤性或囊性疾病。

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.