Dual DNA-binding specificity of peroxisome-proliferator-activated receptor γ controlled by heterodimer formation with retinoid X receptor α

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Abstract
The peroxisome-proliferator-activated receptor γ (PPARγ) is a member of the steroid/thyroid nuclear receptor superfamily of ligand-activated transcription factors. PPARγ forms a heterodimer with the retinoid X receptor α (RXRα) and binds to a common consensus response element consisting of a direct repeat of two hexanucleotides spaced by one nucleotide (DR1 motif). However, other hexamer configurations for binding of PPARγ have not been considered. By using PCR-mediated random site selection, the DNA sequence preferences for PPARγ binding were examined. In this study, we have demonstrated that PPARγ has dual DNA-binding specificity; binding to both the DR1 motif and a palindromic sequence with three bases as spacers (Pal3 motif). The consensus sequence selected by equimolar amounts of PPARγ and RXRα was a perfect DR1 motif, whereas a relatively large population of Pal3 was observed when a 30-fold molar excess of PPARγ over RXRα was used. Gel-shift analysis revealed that the PPARγ homodimer could bind to Pal3 and that the affinity constant of the PPARγ homodimer for Pal3 was nearly the same as that of the PPARγ/RXRα heterodimer for DR1. The addition of RXRα decreased the binding affinity of PPARγ for Pal3, indicating that the DNA-binding specificity of PPARγ could be altered by heterodimer formation with RXRα.