Binding Specificity and Modulation of the Human ApoCIII Promoter Activity by Heterodimers of Ligand-Dependent Nuclear Receptors

Human apolipoprotein CIII (apoCIII) is a major determinant of plasma triglyceride metabolism. The regulatory elements that control both hepatic and intestinal transcription of the human apoCIII gene are localized between nucleotides -792 and -25 of the apoCIII promoter. Elements important for apoCIII promoter activity are three hormone response elements (HREs) and three SP1-binding sites. Orphan members of the nuclear hormone receptor superfamily can bind the HREs and strongly enhance or repress apoCIII promoter activity. In the present study we have investigated the ability of ligand-dependent nuclear hormone receptors to bind and modulate the human apoCIII promoter activity. Experiments using DNA binding and competition assays showed that the proximal element B (-87/-72) binds strongly, in addition to HNF-4, ARP-1, EAR-2, and EAR-3, heterodimers of RXRalpha with RARalpha, and less efficiently, homodimers of RARalpha and heterodimers of RXRalpha with T3Rbeta or PPARalpha. Element G (-669/-648), which was shown previously to bind ARP-1 and EAR-3 but not HNF-4, binds strongly heterodimers of RXRalpha with either RARalpha or T3Rbeta. Finally element I4 (-732/-712), which was shown to bind HNF-4, also binds strongly ARP-1 and EAR-3, as well as RXRalpha/RARalpha heterodimers and less efficiently, RXRalpha/T3Rbeta heterodimers. Methylation interference experiments have identified the protein-DNA interactions between different nuclear receptors and the respective HREs on the apoCIII promoter. RXRalpha/RARalpha heterodimers and HNF-4 homodimers bind to DR-1 motifs on elements B and I4, respectively. RXRalpha/T3Rbeta heterodimers and ARP-1 bind to DR-5 and DR-0 motifs respectively on element G. Cotransfection experiments in HepG2 cells showed that RXRalpha or a combination of RXRalpha and RARalpha increased the apoCIII promoter activity approximately 2-fold in the presence of the ligands 9-cis or all-trans RA. In contrast, a combination of RXRalpha and T3Rbeta transactivated the apoCIII promoter 1.5-fold in the presence of 9-cis RA but it repressed the apoCIII promoter activity in the presence of T3. Mutations in the HREs of elements B, G, or I4 or in the SP1-binding site of element H, which abolished the binding of nuclear hormone receptors or SP1 to their cognate site, reduced the promoter strength and exhibited different responses to the ligand-dependent nuclear receptors. The findings suggest that modulation of the apoCIII promoter activity by orphan and ligand-dependent nuclear receptors involves complex interactions among nuclear receptors, SP1 and possibly other factors bound to the enhancer and the proximal promoter region.