COMPLEX INTERACTIONS BETWEEN SP1 BOUND TO MULTIPLE DISTAL REGULATORY SITES AND HNF-4 BOUND TO THE PROXIMAL PROMOTER LEAD TO TRANSCRIPTIONAL ACTIVATION OF LIVER-SPECIFIC HUMAN APOCIII GENE

Footprinting analysis of the human apoCIII promoter identified a set of four proximal (A-D) and six distal (E-J) regulatory elements between nucleotides -792 and -25 [Ogami, K., et al. (1990) J. Biol. Chem. 265, 9808-9815]. The distal regulatory elements of the apoCIII gene increase by 10-fold the strength of the homologous as well as of heterologous proximal promoters. Required for such transcriptional enhancement is the presence of an intact hormone response element (HRE) on the proximal promoter which binds a variety of nuclear hormone receptors. To understand the mechanism of this transcriptional activation, we identified the nature and the importance of the factors which bind to the upstream regulatory elements of the apoCIII promoter by DNA binding, competition, supershift, and transient transfection assays. These analyses showed that the upstream apoCIII promoter contains multiple binding sites for the ubiquitous transcription factor SP1, which recognizes the regulatory elements F, H, and I, The regulatory element G represents a specialized HRE which is recognized by the orphan receptors ARP-1 and EAR-3 but not by HNF-4. A single activity designated CIII J1 binds to the regulatory element J. The same or a similar activity binds as a minor component to the regulatory elements F and I where SP1 is the predominant binding activity. Finally, a minor activity designated CIII I5 binds to the regulatory element I. In vitro mutagenesis of the distal promoter showed that mutations in elements H and G which affect the binding of SP1 and ARP-1 and EAR-3, respectively, had the most severe effect and decreased the promoter strength in HepG2 cells to 14% and 26% of control, respectively. The promoter strength was similarly decreased in CaCo-2 cells to 25% of control, by mutations in element H, but either was slightly affected or increased 1.6-fold by mutations in element G. HNF-4 transactivated 8- and 6-fold hepatic and intestinal transcription, respectively, driven by the full-length apoCIII promoter. The transactivation patterns in HepG2, CaCo-2, and HeLa cells were different; however, in all cases the transactivation pattern was significantly affected by mutations in elements B, G, and H. The findings suggest the involvement of a complex mechanism in the regulation of transcription of the human apoCIII gene. A major role is exerted by HNF-4 or other nuclear hormone receptors that bind to the regulatory element B as well as by SP1 or by related factors that bind to upstream regulatory sites. It appears that the binding of multiple SP1 molecules to these sites directly or indirectly increases the binding or the activation potential of HNF-4, thus promoting the transcriptional activation of the apoCIII gene.