BINDING-SPECIFICITY AND MODULATION OF THE APOA-I PROMOTER ACTIVITY BYHOMODIMERS AND HETERODIMERS OF NUCLEAR RECEPTORS

Three proximal regulatory elements, AIB, AIC, and AID, of the apoA-I g ene are necessary and sufficient for its hepatic expression in vivo an d in vitro. DNA binding and competition assays showed that elements AI B and AID contain hormone response elements composed of imperfect dire ct repeats that support the binding of the hepatic nuclear factor-4, o ther nuclear orphan receptors, and the ligand-dependent nuclear recept ors retinoic X receptor (RXR alpha), RXR alpha/RAR alpha, and RXR alph a/T(3)R beta. Substitution mutations on repeats 1 and 2 in the hormone response sites of elements AIB and AID, respectively, abolished the b inding of all nuclear receptors and reduced promoter activity to backg round levels, indicating the importance of both hormone response eleme nts for the hepatic expression of the apoA-I gene. Cotransfection expe riments in HepG2 cells with normal and mutated promoter constructs and plasmids expressing nuclear hormone receptors showed that RXR alpha h omodimers transactivated the wild type promoter 150% of control, in th e presence of 9-cis-retinoic acid (RA), whereas RXR alpha/T(3)R beta h eterodimers repressed transcription to 60% of control, in the presence of T-3. RXR alpha/RAR alpha and hepatic nuclear factor-4 did not affe ct the transcription, driven by the proximal apoA-I promoter. Potassiu m permanganate and dimethyl sulfate interference experiments showed th at 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 bindin g is retained. This heterodimeric binding to the mutated element AID i s mediated solely by interactions with repeat 2 and one adjacent nucle otide and is confined to a heptameric core recognition motif. The inte ractions of the RXR alpha heterodimers with repeat 2 are associated wi th low levels of ligand independent transcriptional activity. The find ings suggest that the specific types of homo- and heterodimers of nucl ear 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.