REGULATION OF SODIUM/INOSITOL COTRANSPORTER

肌醇在保护细胞免受高渗应激中起重要作用。 正常情况下，肾髓质是高渗的;高渗的程度取决于水化状态。在某些病理状态下，所有组织都会经历高渗并使用与肾髓质相似的机制来适应不利环境。 钠/肌醇协同转运蛋白的调节在肾上皮细胞、脑和眼睛中肌醇的保护性积累中至关重要。 当细胞暴露于高渗环境时，编码协同转运蛋白的基因的转录被显著刺激，导致协同转运蛋白的活性增加和肌醇的细胞积累。参与高渗诱导的共转运蛋白基因转录刺激的张力应答增强子分布在基因上游的广泛区域。 我们假设，有特殊的结构特征，在染色质中，允许所有的增强子与启动子相互作用。 为了直接对此进行测试，我们将首先制备含有共转运蛋白基因座的100 kb基因组DNA片段-整个基因和含有所有增强子的60 kb侧翼序列。 利用酵母遗传学方法，通过选择性删除增强子并改变其位置来制备基因座的变异。 将野生型和变异基因座DNA引入细胞中，并研究基因的调节以评估变化的影响。所获得的信息将提供新的见解的作用，在基因调控的染色质的远程相互作用。将在动物中研究与张力响应增强子特异性相互作用的转录因子的作用。将产生表达转录因子的显性阴性形式的转基因小鼠系。 我们将检查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到损伤。 为了进一步探索这种转录因子的作用，将使用基因打靶技术产生转录因子缺陷的小鼠品系。 将仔细检查这些动物的表型。 这项工作将增加有关细胞如何适应高渗应激的知识。

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.