Abstract
Many genes of the cytochrome P450 3A (CYP3A) subfamily, including several human and rat isoforms, are inducible by glucocorticoids. In the rat CYP3A23 gene, a 110-base pair segment of the proximal 5'-flanking region mediates dexamethasone activation. Three binding sites (DexRE-1, DexRE-2, and Site A), identified by DNase I footprinting analysis, were characterized for their relative contribution to both basal activity and dexamethasone inducibility. Site-directed mutagenesis of DexRE-1 (-144 to -169) and DexRE-2 (-118 to -136) demonstrated that each contained a core imperfect AGGTCA direct repeat, which comprised a consensus nuclear receptor binding site, and was essential for dexamethasone responsiveness but was not required for basal activity. Competition gel shift and supershift analyses revealed that both sites can bind the orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor. Site A (-85 to -110) was shown to be important for both basal activity and dexamethasone responsiveness. Point mutants displayed a reduced (2-3-fold) induction response, compared with 15-fold for wild-type, which was accompanied by a 40-60% drop in basal activity. Site A was shown to bind the liver-enriched nuclear receptor hepatocyte nuclear factor 4. Our studies demonstrate that the mechanism mediating glucocorticoid-inducible transcriptional activity of CYP3A23 involves multiple binding sites for members of the nuclear receptor superfamily.
Highlights
Site A (؊85 to ؊110) was shown to be important for both basal activity and dexamethasone responsiveness
We demonstrated through gel shift analyses that the orphan receptor chicken ovalbumin upstream promoter transcription factor (COUP-TF) binds at DexRE-1 and DexRE-2 and, by cotransfection experiments, that Site A corresponds to a functional hepatocyte nuclear factor 4 (HNF-4) site
The heavy lines over each of the three regions indicate sequences protected in DNase I footprinting analysis [30], and the consensus sequences highlighted in boldface for DexRE-1 and DexRE-2 are imperfect AGGTCA direct repeats with four- and three-nucleotide spacing, respectively
Summary
Site A (؊85 to ؊110) was shown to be important for both basal activity and dexamethasone responsiveness. Analyses were performed using several DexRE-1 mutants that displayed either strongly decreased or enhanced dexamethasone responsiveness in transient transfection experiments in H4IIE rat hepatoma cells (Fig. 2).
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