Our long-term goal is to elucidate the pathways and controls of epidermal keratin synthesis in health and disease. During the next project period we shall focus on the control of expression of genes for the six major epidermal keratins: K14 and K5 of basal cells, K10 and K1 of suprabasal differentiated cells and K16 and K6 of hyperproliferative epidermis.
Our specific aims are to characterize the cis-active regulatory regions of the keratin genes important for transcriptional control, to define the transacting factors which interact with their regulatory regions, to evaluate the changes in transcriptional control associated with hyperproliferative diseases such as psoriasis and to determine the interactions among the regulatory elements involved in coordinate expression of keratin gene expression in vivo. Initially, we will identify the cis- and trans-acting factors which influence normal keratin genes. The gene for keratin K14, the acidic member of the basal cell-specific pair, will serve as a paradigm for our subsequent studies of the genes coding for the other keratins of normal and pathologic epidermis. The regulatory regions (promoters, enhancers and negative regulatory elements) will be identified by transfection into cells of constructs which contain the regulatory regions of the keratin genes linked to the chloramphenicol-acetyl-transferase (CAT) reporter gene. Sequences identified as involved in control will be amplified by the polymerase chain reaction and studied in more detail in CAT assays. Trans-acting factors will be studied in 4 cell lines which vary in their expression of the keratins under study (fibroblasts - none; HeLa - K18 and K8; SCC 12 - K14, K16, K5, K6; NM1 - K14, K16, K10, K5, K6, K1). The factors will be identified by gel retardation, footprinting and methylation interference assays. Mutations produced in the cis-active regulatory regions (through the polymerase chain reaction) will prove the functional significance of trans-acting factors. Parallel studies will then be done with cultured human epidermal cells, normal and psoriatic epidermis so that we begin to document the changes in transcriptional control associated with disease. Finally, we will study the interactions among the regulatory factors for the six human epidermal keratin genes. The experiments will require co- transfection studies and ultimately transgenic mice. Our goal will be to understand the coordinate expression which is so important in this large, clinically significant gene family.
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