With the discovery that mutations in fourteen different keratin genes can cause eight distinct epithelial fragility disorders, comes the realization that the keratin filament cytoskeleton is crucial to the structural integrity of epithelial tissues exposed to mechanical stress. The identification of mutations causing these disorders has improved our understanding of keratin structure and function, particularly with regard to the highly conserved regions of the central rod domain. However, two central questions in keratin biology still remain. The first question concerns the regulation of individual keratin genes, since the tightly regulated synthesis of these proteins is a pre-requisite for the normal development of the epidermis. To address this question, we will focus on two keratin genes that are expressed at different stages of epidermal development, keratins K14 and K1. K14 is one of the first genes expressed in the surface ectoderm after commitment to stratification. We have recently demonstrated that the transcription factor p63 is the molecular switch for initiation of the epidermal stratification program. This discovery has provided us with novel insights into the molecular mechanisms responsible for the induction of K14 expression during epidermal development. We have also discovered that a switch in p63 isoform expression is required to allow basal keratinocytes to withdraw from the cell cycle and commit to terminal differentiation. Since K1 is one of the first genes expressed in the epidermis after keratinocytes have committed to terminal differentiation, we will exploit this discovery to gain insight into the molecular mechanisms regulating K1 expression during maturation of the epidermis. The second question concerns the roles of the keratin N- and C-terminal end domains. These domains are distinctive for each keratin protein but are remarkably well conserved across species and presumably have functional significance. In vitro studies have suggested that these domains interact with desmosomes and/or the cell envelope, however very little is known regarding cell type-specific functions of these domains in vivo. Two transgenic approaches are proposed to address this question. Finally, epidermolytic hyperkeratosis (EHK), the dominantly inherited skin disorder caused by mutations in the post-mitotically expressed keratins, K1 or K10, presents a challenge for gene therapy. During the last funding period of this grant, we generated a mouse model that mimics EHK at both the genetic and phenotypic level. This mouse model has provided new insights into the molecular and cellular basis of EHK and we propose to use this model to test new gene therapy strategies. The results of this research will yield valuable information that can lead to novel therapeutic approaches for other diseases affecting post-mitotic keratinocytes.