Tyrosine aminotransferase gene expression is confined to parenchymal cells of the liver, is inducible by glucocorticoids and glucagon, and is repressed by insulin. Three enhancers control this tissue-specific and hormone-dependent activity, one of which, located at -11 kb, is implicated in establishing an active expression domain. We have studied in detail this important regulatory element and have identified a 221-bp fragment containing critical enhancer sequences which stimulated the heterologous thymidine kinase promoter more than 100-fold in hepatoma cells. Within this region, we have characterized two essential liver-specific enhancer domains, one of which was bound by proteins of the hepatocyte nuclear factor 3 (HNF3) family. Analyses with the dedifferentiated hepatoma cell line HTC suggested that HNF3a and/or -y, but not HNF31, are involved in activating the tyrosine aminotransferase gene via the -11-kb enhancer. Genomic footprinting and in vitro protein-DNA binding studies documented cell-type-specific binding of ubiquitous factors to the second essential enhancer domain, which by itself stimulated the thymidine kinase promoter preferentially in hepatoma cells.These results will allow further characterization of the role of these enhancer sequences in developmental activation of the tyrosine aminotransferase gene.Differential gene expression is the basis of cellular diversity in complex organisms. Work from many laboratories has established that gene activity is regulated by the combinatorial action of sequence-specific DNA-binding proteins which, together with the basic transcription apparatus, control the frequency of transcription initiation at the promoter. Binding sites for transcriptional activator proteins, which mediate cell-type-specific, hormone-dependent, and developmentally regulated gene expression, can be found in the vicinity of the transcription start site and in enhancer sequences located several kilobases upstream or downstream (33, 43). More recently, cofactors or adaptors which couple activator proteins to the basal transcription factors by protein-protein interaction have been described (reference 42 and references therein).Studies on liver-specific genes have led to the identification of several liver-enriched transcriptional activators, the majority of which can be grouped into four families (see references 14 and 61 for reviews): hepatocyte nuclear factor 1 (HNF1) hierarchical order of liver-specific transcription factors (37,65).Tyrosine aminotransferase (TAT) is a gluconeogenic enzyme which has been studied as a classical system for more than 20 years (reviewed in reference 51). The TAT gene is switched on in the parenchymal cells of the liver shortly after birth, when hormone levels change dramatically (26, 28). Gene activity is induced by glucocorticoids and by glucagon, acting via its intracellular mediator cyclic AMP (cAMP) (25,29,58), and is repressed by insulin (60). Our analyses have defined three enhancers which are responsible for the liverspecific and hormone-inducibl...