Several evidence suggest that the transcription factor Tcf3 might play a key role in maintaining skin stem cell properties. An important step in exploring Tcf3's functions in skins will be to identify and characterize its target genes. I will first generate inducible Tcf3 transgenic mice using the tetracycline inducible system. This will allow me to characterize Tcf3's effect on postnatal animals and assess the dose dependent effect of Tcf3 on keratinocytes proliferative characteristics in vitro. With the inducible system, I will culture keratinocytes from the transgenic mice to perform microarray analyses to identify genes that are affected by Tcf3 induction. I will use Northern and in situ hybridization to confirm for genes that are affected by Tcf3 expression, and analyze promoters of the candidate genes for binding to Tcf3. By time course analyses of Tcf3 induction and the candidate genes' expression, I should be able to further narrow down the list of the candidate genes. In parallel, I will perform chromatin IP coupled with CpG microarray analyses to identify Tcf3 direct target genes. The combined approaches will allow me to select a few target genes to test for their relevance in skin stem cell maintenance by manipulating their expression in the skin, by either transgenic technology using K 14/5 promoter or knockout technology.
