Cellular differentiation results in transcriptional induction of distinct sets of tissue-specific genes whose expression is required for organ function. Deciphering transcriptional control mechanisms is thus critical to understanding differentiation and aberrations of this process that lead to neoplastic transformation. We have utilized the transthyretin (TTR) DNA regulatory regions as a model in seeking to understand hepatocyte-specific gene transcription. Studies of TTR suggest that hepatocyte-specific gene regulation relies on combinatorial interaction of multiple DNA binding sites by several distinct families of liver- enriched transcription factors. One of these regulatory families is composed of hepatocyte nuclear factor-3 (HNF-3alpha, beta, and gamma) proteins, which mediate the liver-specific transcription of numerous genes important for liver function and are important for hepatocyte differentiation. The HNF-3 proteins bind DNA via a homologous winged helix motif and comprise an extensive family of tissue-specific transcription factors sharing homology in their winged helix DNA-binding motif. The long-term objective of this proposed research is to further our understanding of hepatocyte-specific gene regulation through the characterization of proteins involved in HNF-3 mediated transcriptional activation and determination of mechanisms which restrict expression of the HNF-3 genes. We have found that a novel liver-enriched nuclear protein, HNF-6, recognizes sequences in the TTR and HNF-3beta promoter regions. We will perform promoter mutagenesis studies to determine the contribution of HNF-6 recognition to TTR and HNF-3beta promoter activity. We propose to clone HNF-6 and use its cDNA to determine HNF-6's transcriptional activity and embryonic expression pattern. We will examine whether the -4 Kb HNF-3alpha upstream region and the -4 Kb plus its intron sequences are sufficient to elicit correct developmental and tissue expression patterns in transgenic mice. We have shown that in acute phase livers, HNF-3alpha expression is dramatically reduced as is expression of its target gene, TTR. We will use the transgenic lines to explore whether these HNF-3alpha regulatory sequences are sufficient to confer decreased expression in response to acute phase cytokines. We have identified HNF-3beta transcriptional activation domains at the N-terminus (amino acids 14 to 93) and C-terminus (amino acids 361 to 458) of the protein. We will use the yeast two hybrid system to isolate cDNA clones encoding proteins which interact with the HNF-3beta activation domains. The isolation of such clones will allow us to address fundamental questions regarding HNF-3 mediated activation of liver-specific genes.
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