My primary objective is to elucidate the mechanisms that control cell fate and differentiation in keratinocytes. The genes encoding keratins, K5 and K14 are abundantly and uniquely transcribed in dividing keratinocytes of all stratified tissues. Hence these genes are ideal to explore the role of cis-elements and transcription factors that confer epithelial-specificity. Some cis-acting elements of the minimal promoters of these genes have been identified, but regions sufficient for tissue specific and differentiation-specific gene expression include as yet unidentified upstream elements. Using DNAse I hypersensitivity assays, I have now uncovered novel upstream DNA sequences that display cell-type specific changes in chromatin. To test whether these regions are indeed important in keratinocyte-specific gene expression, I will a) determine whether keratinocyte-specific proteins account for cell type-specific DNAse hypersensitivity I observed, b) use mutagenesis, keratinocyte transfection and transgenic mice to assess whether the regions are functionally important, c) clone and characterize the transcription factors that govern K5 and K14 gene expression, and d) assess the relevance of these factors to determination of keratinocyte cell fate. This knowledge will also be extremely valuable in optimizing the keratinocyte as a vehicle for gene-therapy and drug delivery.
