Keratins 8 (K8) and 18 (K18) are the first intermediate filament proteins to be expressed during mouse and human development and are subsequently restricted to a wide variety of internal, simple epithelial tissues. K8 and K18 are useful markers of carcinoma, as they continue to be expressed even when other more differentiated functions cease. We will address three areas of keratin biology and regulation. First, the possible functions of K8 and K18 in associating with several cell surface receptors will be investigated. The role of K8 snd K18 in trophinin mediate, metal ion independent cell-cell adhesion of endometrial cells will be evaluated in vitro, by defining the domains of keratin which interact with the cytoplasmic bystin adapter protein and testing the functional significance of K8 an K18 on cell- cell adhesion assays. The possible role of keratins in apoptosis will be evaluated in cultured cells and transgenic mice, using targeted mutations, over-expressd mutant keratins, and apoptotic stimuli. Second, the role of DNA methylation of an Ets transcription factor binding site in the K18 intron enhancer on tissue specific expression will be evaluated in DNA methyltransferase mutant mice, by transgenic expression of K18 reporter genes and by identifying proteins which recognize the methylated site. Third, the ability of a K18 derived Alu sequence to provide integration site independent expression in transgenic mice will be compared to its ability to reduce chromatin mediated repression at yeast telomeres. The potential role for RNA polymerase III transcription units to block position effects in both yeast and transgenic mice will be determined. The functional studies of K8 and K18 may provide a basis for the integration of a variety of effects attributed to keratins, and clarify the source of circulating keratin peptides found in cancer patients. The methylation studies will provide a paradigm of the use of DNA methylation for differential gene expression during normal development. Characterization of the mechanism by which K18 flanking sequences protect transgenes from position effects may provide a solution to the problem of sustained expression in gene therapy applications and contribute to the definition of signals necessary to define regulatory domains within the genome.
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