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We previously identified various upstream and downstream regulatory elements and factors important for hepatic expression of the human angiotensinogen (ANG) gene, the precursor of vasoactive octapeptide angiotensin II. In the present study, to further investigate the molecular mechanism of human ANG transcriptional regulation, we generated transgenic mice carrying the fusion gene composed of the 1. 3-kilobase promoter of the human ANG gene, its downstream enhancer, and the chloramphenicol acetyltransferase reporter gene. Because expression of the chloramphenicol acetyltransferase gene was observed strongly in the liver and weakly in the kidney, we suspected that hepatocyte nuclear factor (HNF) 4 with a tissue expression pattern similar to that of the reporter gene would regulate ANG transcription. In vitro assays indicated that HNF4 bound to the promoter elements and strongly activated the ANG transcription, but that chicken ovalbumin upstream promoter transcription factor (COUP-TF), a transcriptional repressor, dramatically repressed human ANG transcription through the promoter elements and the downstream enhancer core elements. Furthermore, COUP-TF dramatically decreased the human ANG transcription in the mouse liver by the Helios Gene Gun system in vivo. These results suggest that an interplay between HNF4 and COUP-TF could be important in hepatic human ANG transcription.

On the basis of cDNA cloning, primer extension, and transfection experiments, we identified a novel 5′ exon of the human activin βA subunit gene, and found its enhancer and promoter regions as well as multiple transcription start sites. A series of deletion and mutation analyses of the enhancer sequences defined the 45-base pair core region (DR-1 core) containing two short elements with similarity to AP-1 (12-O-tetradecanoylphorbol-13-acetate response element; TRE) and CREB/ATF (cyclic AMP response element; CRE) binding sites, both of which were necessary for full enhancer activity. Gel shift and antibody supershift assays using DR-1 core region revealed the formation of two specific DNA-protein complexes, one of which could be partially dissociated by a competing oligonucleotide containing a single copy of the consensus TRE, but the other of which contained neither CREB/ATF nor AP-1 as major components. Although 12-O-tetradecanoylphorbol-13-acetate and cAMP induced the activin enhancer/promoter-driven CAT activity, such drug induction was obscured when either the TRE- or CRE-like elements were mutated in the native promoter context. Our results demonstrate that the promoter and enhancer regions identified here are essential for maintaining the efficient promoter activity of the human activin βA subunit gene.