DETERMINATION OF TISSUE-SPECIFICITY OF THE ENHANCER BY COMBINATORIAL OPERATION OF TISSUE-ENRICHED TRANSCRIPTION FACTORS - BOTH HNF-4 AND C EBP-BETA ARE REQUIRED FOR LIVER-SPECIFIC ACTIVITY OF THE ORNITHINE TRANSCARBAMYLASE ENHANCER/

The enhancer of the rat ornithine transcarbamylase gene is located 11 kilobases upstream from the transcription start site and has been show n to be hepatoma cell-specific. Using transgenic mice, we showed that this enhancer is capable of activating transcription in a liver-specif ic manner, inverting the tissue specificity of the homologous promoter that is by itself more active in the small intestine than in the live r. Transient transfection analysis with cultured hepatoma cells indica ted that the enhancer activity resides in the approximately 110-base p air region containing four protein-binding sites, two for hepatocyte n uclear factor-4 (HNF-4) and two for CCAAT/enhancer binding protein (C/ EBP), both of which are liver-selective transcription factors. Concate merization of a region containing one HNF-4 and one C/EBP site led to reconstitution of the hepatoma cell-specific enhancer, and intactness of these two sites was strictly required for the enhancer activity. Fu rthermore, cotransfection experiments showed that both HNF-4 and C/EBP beta are necessary, and neither alone sufficient, for activation of th e reconstituted enhancer in nonhepatic cells. Requirement of combinato rial operation of at least two liver-enriched transcription factors fo r transcriptional activation successfully explains why these liver-sel ective but not strictly liver-specific factors can confer more restric ted liver specificity on transcription of their target genes.