Upstream region of rat serum albumin gene promoter contributes to promoteractivity: presence of functional binding site for hepatocyte nuclear factor-3

Transcription of the serum albumin gene occurs almost exclusively in the li ver and is controlled in part by a strong liver-specific promoter. The upst ream region of the serum albumin gene promoter is highly conserved among sp ecies and is foot-printed in vitro by a number of nuclear proteins. However , the role of the upstream promoter region in regulating transcription and the identity of the transcription factors that bind to this region have not been established. In the present study, deletion analysis of the rat serum albumin promoter in transiently transfected HepG2 cells demonstrated that elimination of the region between -207 and -153 bp caused a two-fold decrea se in promoter activity (P < 0.05). Additional analysis of the -207 to -124 bp promoter interval led to the identification of two potential binding si tes for hepatocyte nuclear factor-3 (HNF-3) located at -168 to -157 bp (sit e X) and -145 to -134 bp (site Y). Electrophoretic mobility-shift assays pe rformed with the HNF-3 X and Y sites demonstrated that both sites are capab le of binding HNF-3 alpha and HNF-3 beta. Placement of a single copy of the HNF-3 X site upstream from a minimal promoter increased promoter activity by about four-fold in HepG2 cells, and the reporter construct containing th is site could be transactivated if co-transfected with an HNF-3 expression construct. Furthermore, inactivation of the HNF-3 X site by site-directed m utagenesis within the context of the -261 bp albumin promoter construct res ulted in a 40% decrease in transcription (P < 0.05). These results indicate that the positive effect of the -207 to -153 bp promoter interval is attri butable to the presence of the HNF-3 X site within this interval. Additiona l results obtained with transfected HepG2 cells suggest that the HNF-3 Y si te plays a lesser role in activation of transcription than the X site.