In the mammalian fetus, the alpha-fetoprotein (AFP) gene is transcribed at high levels in the yolk sac, all hepatocytes, and most small intestinal epithelial cells. At birth the AFP gene is transcriptionally repressed in all of these cells, with the exception of a subset of enteroendocrine cells representing less than 1% of the epithelial cells in the adult small intestine. In the adult liver, AFP gene expression is reactivated in primary hepatocellular carcinomas, and serum AFP levels are monitored to aid in the diagnosis of this type of cancer. The cis and trans-acting factors responsible for AFP repression in the neonate, and reactivation of transcription in hepatomas have not yet been defined. Recent experiments in transgenic mice have resulted in the identification of a region of sequence lying between -250 basepairs (bp) and -838 bp of the AFP gene that is important for the repression of the AFP gene in the neonatal liver and gut. Deletion of these sequences results in high level AFP transgene transcription in pericentral hepatocytes and in goblet cells of the small intestine. We hypothesize that single or multiple cis-acting transcriptional repressor elements lie between -250 bp and -838 bp of the AFP gene, and that these elements bind protein factor(s) that mediate repression of AFP transcription. To test, we propose to map the position of the repressor elements by transfection of a series of AFP expression constructs containing systematic 5' and 3' deletions of the -250 bp to - 838 bp region, and a series of linker scanning mutations into two intestinal epithelial cell lines, and a differentiated nontransformed hepatocyte cell line. Preliminary studies indicate that Caco-2 cells, H2 cells, a subclone of the Ht-29 cell line, and the liver AML12 cell line, will be useful for mapping the repressor elements. Using the minimal region of sequence that is able to direct repression in these cell lines, DNA mobility shift assays will be performed to detect protein binding, and DNase I footprinting will be used to identify specific protein binding sites. Constructs containing mutations or deletions of protein binding sites will be generated and introduced into cell lines and transgenic mice to confirm that deletion of these sequences abrogates repression in vivo. To assess the biological role of the repressor elements during liver regeneration and hepatoma formation, we will determine if transcription of the repressor minus transgenes is influenced when animals are treated with chemical carcinogens and hepatotoxins. This will tell us if the repressor elements are targeted when gene expression is reinduced during development of hepatomas. If we demonstrate protein binding to novel cis-acting elements, we will attempt to clone and characterize the proteins mediating repression. During cell differentiation, specific sets of genes are activated and repressed in different cell types. The identification of novel proteins responsible for repression of the AFP gene may provide insight about mechanisms regulating maturation and cell differentiation in the liver and the intestine. In addition, we may be able to begin to dissect the molecular signals that lead to the inactivation of repression and the initiation of AFP expression in primary liver cancers.
