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.
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