The role of the proximal promoter and the far-upstream enhancer in the hepatocyte-specific and hormonal regulation of the
carbamoyl-phosphate synthetase I (CPS) gene was investigated in transient transfection assays using primary rat hepatocytes,
hepatoma cells, and fibroblasts. These experiments revealed that the activity of the promoter is comparable in all cells tested and is, therefore, not responsible for tissue-specific expression. The 5'-untranslated region of the
mRNA is a major, non-tissue specific stimulator of expression in FTO-2B
hepatoma cells, acting at the post-transcriptional level. A 469-base pair
DNA fragment, 6 kilobase pairs upstream of the transcription start-site in the CPS gene, confers strong
hormone-dependent tissue specific expression, both in combination with the CPS promoter and a minimized viral
thymidine kinase promoter. Sequences similar to a
cyclic AMP-responsive
element and a glucocorticosteroid-responsive
element were found in the isolated enhancer. Substitutional mutations in these sites strongly affected
hormone-induced expression. Analysis of the interaction between the enhancer and parts of the CPS promoter revealed that, in addition to the TATA box, the GAG box, a motif similar to the GC box near the TATA motif, is instrumental in conferring the enhancer activity.