REGULATION OF SODIUM/INOSITOL COTRANSPORTER

肌醇在保护细胞免受高渗应激方面起着重要作用。肾髓质通常是高渗的；高渗程度的变化取决于水合状态。在某些病理状态下，所有组织都经历高渗，并利用类似于肾髓质的机制来适应不利的环境。钠/肌醇共转运体的调节对肾上皮细胞、脑和眼睛肌醇的渗透保护性积累至关重要。当细胞暴露于高渗环境时，编码共转运蛋白的基因转录被显著刺激，导致共转运蛋白活性增加和细胞肌醇积累。参与高张力诱导的共转运体基因转录刺激的张力响应增强子分布在该基因上游的广泛区域。我们假设染色质中存在特殊的结构特征，允许所有增强子与启动子相互作用。为了直接验证这一点，我们将首先准备一个包含整个基因共转运位点的100 kb基因组DNA片段和包含所有增强子的60 kb侧翼序列。利用酵母遗传方法，通过选择性地删除增强子并改变其位置来制备位点的变异。将野生型和变异位点DNA引入细胞，并研究基因的调控以评估变化的影响。获得的信息将为染色质在基因调控中的远程相互作用提供新的见解。与张力反应增强子特异性相互作用的转录因子的作用将在动物中进行研究。将产生表达显性阴性形式转录因子的转基因小鼠系。我们将研究这些动物的肾髓质是否如预期的那样由于缺乏对高渗的适应而受损。为了进一步探索该转录因子的作用，将利用基因靶向技术产生缺乏该转录因子的小鼠品系。这些动物的表型将被仔细研究。这项工作将增加对细胞如何适应高渗应激的认识。

Inositol plays an important role in protecting cells from hypertonic stress. The renal medulla is normally hypertonic; the degree of hypertonicity changes depending on hydration status. In certain pathologic states all tissues experience hypertonicity and adapt to the adverse environment using mechanisms similar to those of the renal medulla. Regulation of the sodium/inositol cotransporter is critical in osmo-protective accumulation of inositol in renal epithelial cells, brain, and eyes. When cells are exposed to a hypertonic environment, transcription of the gene coding for the cotransporter is markedly stimulated resulting in increased activity of the cotransporter and cellular accumulation of inositol. Tonicity-responsive enhancers involved in the hypertonicity- induced stimulation of the transcription of the cotransporter gene are spread over a wide region upstream of the gene. We hypothesize that there are special structural features in the chromatin that allow all the enhancers to interact with the promoter. To test this directly we will first prepare a 100 kb genomic DNA fragment containing the cotransporter locus - the entire gene and 60 kb of flanking sequence containing all the enhancers. Taking advantage of yeast genetic methods, variations of the locus will be prepared by selectively deleting the enhancers and changing their location. Wildtype and variant locus DNA will be introduced into cells and regulation of the gene will be studied to assess the effects of the changes. Information obtained will provide novel insight into the role of long-range interactions of the chromatin in gene regulation. The role of a transcription factor that specifically interacts with tonicity-responsive enhancers will be studied in animals. Transgenic mouse lines expressing the dominant negative form of the transcription factor will be generated. We will examine whether the renal medullas of these animals are injured as expected due to the lack of adaptation to hypertonicity. To explore the role of this transcription factor further, mouse lines deficient in the transcription factor will be generated using the gene targeting technique. Phenotypes of these animals will be scrutinized. This work will enhance knowledge regarding how cells adapt to the hypertonic stress.