EXPRESSION FROM THE TYROSINE AMINOTRANSFERASE PROMOTER (NT -350 TO -SPECIFIC AND DEPENDENT ON THE BINDING OF BOTH LIVER-ENRICHED AND UBIQUITOUS TRANS-ACTING FACTORS(1) IS LIVER)

The rat tyrosine aminotransferase (TAT) gene promoter (nucleotides - 3 50 to + 1; TAT(0.35)) was able to sustain liver-specific expression bo th ex vivo in transient transfection (TAT-expressing H4IIEC3 hepatoma cells vs. TAT non-expressing CCL1.2 fibroblasts) and in in vitro trans cription (rat river vs. spleen crude nuclear extracts). In either case , the index of tissue specificity (6.2 and 6.7 in ex vivo and in vitro experiments, respectively) was close to that obtained with 10 Kb of T AT gene 5'-flanking sequences in transient transfection. Using compute r-assisted search of homologies, DNase I footprinting, gel retardation and methylation interference assays, we showed that TAT(0.35) sequenc es spanning nt -156 to -175 and nt -268 to -281 interacted with the ri ver enriched NF-1(Liver) (a member of the NF1 gene family) and HNF1 re spectively, whereas those encompassing nt -57 to -85 and nt -283 to -2 88 interacted with the ubiquitous NF-Y and with ubiquitous 'CCAAT'-box binding factor(s), respectively. Competition studies in in vitro tran scription carried out with wild type and mutated oligonucleotides, dem onstrated that NF-Y cis-elements were crucial for basal TAT promoter a ctivity, both in liver and spleen whereas NF1(Liver) and HNF1 were onl y efficient in the liver (supported similar to 60% and 30% of basal TA T(0.35) activity respectively. Altogether, these results support the c onclusion that TAT(0.35) was able to sustain at least part of the live r specificity of TAT gene expression.