Binding specificity and modulation of the ApoA-I promoter activity by homo- and heterodimers of nuclear receptors

Three proximal regulatory elements, AIB, AIC, and AID, of the apoA-I gene are necessary and sufficient for its hepatic expression in vivo and in vitro. DNA binding and competition assays showed that elements AIB and AID contain hormone response elements composed of imperfect direct repeats that support the binding of the hepatic nuclear factor-4, other nuclear orphan receptors, and the ligand-dependent nuclear receptors retinoic X receptor (RXR alpha), RXR alpha/RAR alpha, and RXR alpha/T(3)R beta. Substitution mutations on repeats 1 and 2 in the hormone response sites of elements AIB and AID, respectively, abolished the binding of all nuclear receptors and reduced promoter activity to background levels, indicating the importance of both hormone response elements for the hepatic expression of the apoA-I gene. Cotransfection experiments in HepG2 cells with normal and mutated promoter constructs and plasmids expressing nuclear hormone receptors showed that RXR alpha homodimers transactivated the wild type promoter 150% of control, in the presence of 9-cis-retinoic acid (RA), whereas RXR alpha/T(3)R beta heterodimers repressed transcription to 60% of control, in the presence of T-3. RXR alpha/RAR alpha and hepatic nuclear factor-4 did not affect the transcription, driven by the proximal apoA-I promoter. Potassium permanganate and dimethyl sulfate interference experiments showed that RXR alpha homodimers, RXR alpha/RAR alpha, and RXR alpha/T(3)R beta heterodimers participate in protein-DNA interactions with 12, 13, and 11 out of the 14 nucleotides, respectively, that span repeats 1 and 2 and the spacer region separating them on the hormone response element of element AID. The binding of RXR alpha homodimers and RXR alpha/T(3)R beta heterodimers is associated with ligand-dependent activation by 9-cis-RA or repression by T-3. Upon deletion or mutation of repeat 1, homodimeric binding of RXR alpha is lost whereas heterodimeric binding is retained. This heterodimeric binding to the mutated element AID is mediated solely by interactions with repeat 2 and one adjacent nucleotide and is confined to a heptameric core recognition motif. The interactions of the RXR alpha heterodimers with repeat 2 are associated with low levels of ligand independent transcriptional activity. The findings suggest that the specific types of homo- and heterodimers of nuclear hormone receptors occupying the hormone response elements of apoA-I and the availability of the ligand may play an important role in the transcriptional regulation of the human apoA-I gene.