The epidermis of human skin is subject to a variety of physiological and pathological alterations in its pattern and rate of differentiation. In this and other epithelial tissues, differentiation is closely associated with ordered changes in the expression of the keratin class of intermediate filament subunit proteins. Different subsets of keratins appear to not only be markers for epidermal cell subpopulations and for virtually all epithelial cell types, but they also discriminated between cancerous and normal tissues and even between malignant and benign tumors. The connection between keratin expression and epidermal cell type suggests that insights into the control of cell differentiation will be gained by understanding the molecular mechanism of keratin gene regulation. A line of research is proposed, using the mouse as a model, which addresses this problem at the level of chromatin determinants of keratin gene expression. By analyzing the pattern of nuclease hypersensitive sites in the regions of particular genes, this project seeks to identify localized changes in chromatin structure associated with coordinate activation of several mouse keratin genes during epidermal cell differentiation. Nuclei prepared from fractionated epidermal cells, other epithelial cells and nonepithelial tissues will be treated with nuclease, the DNA isolated and the hypersensitive cleavage sites identified by hybridization to labeled cloned keratin gene probes. Transcriptional activity will be determined by cDNA hybridization to isolated RNA. The pattern of nuclease hypersensitive sites will be correlated with cell type and transcriptional activity to select particular sites for further analysis. The nature of these sites will be examined and compared using S1 nuclease and further defined using exonuclease III and by DNA sequencing. Nuclease hypersensitive sites characterized in this study can be used to develop a reconstitution assay to identify tissue-specific proteins involved in their formation. These soluble factors, and the DNA sequences with which they are associated, represent putative keratin gene regulatory elements whose function in tissue-specific gene expression and epidermal cell differentiation will be evaluated in future studies.
