This is the firs application from a new investigator with the overall objective to investigate the regulation of caudal-related homeobox (CDX) proteins in normal colon and colorectal cancer. CDX1 and CDX2 are transcription factors that are involved in regulation of proliferation and differentiation of the intestinal epithelial cell. Additionally, expression of replacement of CDX gene expression in colon carcinoma cell lines dramatically inhibits cellular proliferation by cell cycle arrest in G1. Thus, altered regulation of CDX genes in colonic epithelial cells is involved in the development of the neoplastic phenotype. Therefore, this proposal is based on the hypothesis that an understanding of the regulation of CDX genes in both normal and neoplastic cells will elucidate pathways that control the phenotype in cancer cells. The goal of this project is to elucidate the normal regulation of CDX1 and CDX2 and to determine changes that occur in the colonic neoplasia. Preliminary are presented that demonstrate variable of CDX genes in colorectal cancer cell lines and that this differential expression is regulated the level of gene transcription. To accomplish this goal, four specific aims are proposed: 1) To elucidate the transcriptional regulation of the human CDX1 gene in normal and neoplastic colonocytes using DNase I hypersensitivity. mapping and transfection and DNA-protein interaction studies. Preliminary data shows that there is a specific hypersensitive site in a cell line that expresses CDX1 that is absent in non-intestinal cell lines and intestinal cell lines and intestinal cell lines that do not express CDX1. The function of this site will be explored and the remainder of the gene will be studied for additional sites. 2) To elucidate the transcriptional regulation of CDX2 in normal and neoplastic tissue. Similar to CDX1, we have mapped and characterized and characterized the CDX2 gene. Transfection studies suggest that elements for directing intestine-specific expression are outside the immediate upstream region of the gene. DNase I hypersensitive site analysis will also be employed to identify potential regulatory regions. 3) To study the regulation of CDX genes in transgenic mice. These studies will validate the function of regulatory elements identified in Aims 1 and 2 in the whole animals. 4) To examine cellular signaling pathways that regulate CDX genes in colon cancer cells. Once regulatory elements are defined, the cellular pathways that regulate expression in normal and neoplastic cells will be studied. Completion of these studies will provide a detailed picture of the transcriptional regulation of CDX genes in normal and neoplastic cells. This will provide the basis for understanding the network of cellular pathways that control these important cell specific transcription factors and how they are altered in neoplasia.