Mutations that cause aberrant activation of the Wnt Signaling Pathway are found in early stages of tumor development in colon carcinoma. The downstream components of the pathway are transcription factors that belong to the LEF/TCF family, and the focus of our lab is to study the regulation of the LEF1 gene. Northern blot analysis demonstrates that there are 3 LEF1 messages (3.6kb, 3.0kb, and 2.2kb) expressed from the locus. Two different promoters generate the 3.6 and 2.2 kb mRNA messages and have been characterized. The promoter for the 3.6kb message is a target of the Wnt pathway and is aberrantly expressed in colon cancer cell lines. The 3.0kb message is also aberrantly expressed in cancer, but its origin is not known. The focus of this proposal is to test the hypothesis that a region in exon 1 of the LEF1 gene harbors a third promoter which directs expression of the third LEF1 message (3.0kb) (Aim 1). Because the 3.6 and 3.0 kb messages are expressed together in colon cancer cell lines, we hypothesize that they are coordinately regulated. Due to the identification of a region in promoters 1 and 3 in exon 1 that contains putative Wnt and TGFbeta-response elements, experiments will be carried out to test if these elements individually play a role in the regulation of one or both promoters (Aim 2). If this putative regulatory region affects promoter activity, we plan to determine whether the Wnt and TGFbeta pathways cooperate to activate or act antagonistically to negatively regulate promoter 1 and 3 (Aim 3). Mutations in the TGFbeta Pathway are late events in the development of tumorigenesis, therefore, we hypothesize that the aberrant expression of LEF1 in colon carcinomas may be due to mutations that activate the Wnt pathway and mutations that inactivate the TGFbeta pathway.