The long term objectives of this proposal is to understand the establishment and maintenance of liver-specific gene transcription during development. Evidence is accumulating that the establishment of the differential transcription of tissue-specific genes during development from the same genomic DNA is regulated by a cascade of transcription factors. Therefore, the establishment of tissue specific gene expression involves the execution of a network of temporally controlled transcription factors whose function are necessary to program the transcription of different genes in each cell-type in the body. The DNA regulatory regions of tissue- specific genes consist of multiple DNA elements that stimulate expression through the interaction with cell-specific transcription factors. This reinforces the importance of deciphering the mechanisms that restrict activity of cell-specific transcription factors and of the analyses of functional protein domains involved in transcriptional activation. To initiate experiments toward these goals, the characterization of two liver- specific transcription factors (termed hepatocyte nuclear factors; HNF) that regulate the expression of the transthyretin (TTR) gene will be performed. The two transcription factors, HNF 3 and HNF 4, that will be analyzed in this proposal also participate in coordinate expression of several other unrelated genes in the liver. Therefore they are involved in the general regulation of liver genes. they availability of the HNF 3 cDNA clone will allow initiation of these studies and the isolation of the clone encoding HNF 4 will expand this analysis. The expression of these HNF cDNA will allow the synthesis of specific antisera that is critical for the identification of protein-protein interactions and the investigation of the cellular distribution of these factors. The HNF cDNAs and antibodies will be used to determine whether transcriptional or translational mechanisms maintain the activity of these factors restricted to hepatocytes. The mechanism which limits their activity is important for the understanding of hepatocyte-specific expression, since this property prevents the expression of these liver genes in other cell-types. Future extension of these studies will involve the investigation of the cascade schemes that regulate the HNF cell-specific activity. The HNF clones are also used to construct deletion mutations to identify functional protein domains involved in DNA recognition and possible protein-protein interactions. In addition these HNF deletion mutations are used in a cotransfection assay with a test gene that requires HNF activity for expression to delineate protein domains involved in transcriptional activation. Since deletion within the HNF coding region may disrupt protein structure, the functional domains are confirmed by site-directed mutations which alter only several amine acid residues. These mutations will also provide insight into the type of amino acid sequence that is required for the functional activity. These studies will strengthen our understanding of the molecular basis controlling tissue-specific gene transcription.
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