Changes in keratin gene expression are closely associated with changes in the differentiation state of the keratinocyte. For instance, the induction of the differentiation-specific keratins, K1 and K10 can already be observed in basal cells that have lost their proliferative capacity and are about to migrate to the spinous layer. The induction of K6 on the other hand, occurs rapidly in interfollicular epidermis that has suffered trauma and remains induced as long as the traumatizing stimulus persists. In both cases the induction of new keratin types is associated with a down regulation of the previous keratin genes with the newly induced proteins replacing the pre-existing network. As the central rod domains of these keratins are remarkably similar, the significance of new keratin expression probably lies in the expression of a new set of end-domains. However, the exact role of the end-domains in keratin filament biology and their impact on the function of the epidermis remains to be determined. An understanding of the molecular mechanism regulating expression of these keratin genes together with information on the biological roles of the various end-domains will provide insights into keratinocyte function in both normal and abnormal states. Both in vivo transgenic and in vitro tissue culture techniques will be used to explore the regulation of K1. The transgenic experimental system will be utilized to identify those elements required for correct tissue, developmental and differentiation specific expression. The micro analysis of these elements will be examined by insertion of putative regulatory regions in reporter constructs, followed by transfection into primary epidermal keratinocytes. Proteins that interact with these elements will be characterized and those that are unique to keratinocytes will be cloned. To explore the role of the unique end-domains on keratinocyte function, various mutations will be engineered into the N and C termini of K6 and introduced into the germline of transgenic mice. These end-domain mutations, as well as constructs which swap the end-domains between keratins, are designed not only to gain insights into end-domain function, but also to create mouse models of human skin disorders. This reverse genetics approach may potentially identify other keratin disorder in man. Finally, the animal models, produced as a results of these studies, can be exploited for the assessment of gene therapy protocols in the amelioration of these skin diseases.
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