An intact DNA-binding domain is not required for peroxisome proliferator-activated receptor γ (PPARγ) binding and activation on some PPAR response elements

Abstract

Peroxisome proliferator-activated receptor γ (PPARγ) interacts with retinoid X receptor (RXR) on PPAR response elements (PPREs) to regulate transcription of PPAR-responsive genes. To investigate the binding of PPARγ and RXR to PPREs, three mutations were constructed in the DNA-binding domains of PPARγ; two of the mutants maintained the structure of zinc finger I (PPARγ-GS and PPARγ-AA), and a third mutation disrupted the protein structure of zinc finger I (PPARγ-CS). Results indicated that the mutations of PPARγ that maintained intact zinc fingers were capable of binding to a variety of PPREs in the presence of RXR and could activate transcription on several PPREs. In parallel, a mutation was created in the DNA-binding domain of RXRα that maintained the structure of the zinc fingers (RXR-GS) but did not bind DNA and was transcriptionally inactive. Examination of the 3′ half-site of several PPREs revealed that variations from the consensus sequence reduced or abolished transcriptional activity, but conversion to consensus improved transcriptional activity with PPARγ-GS and PPARγ-AA. Examination of the 5′ half-site indicated that the upstream three nucleotides were more important for transcriptional activity than the downstream three nucleotides. Our data demonstrated that stringent binding of RXR to the 3′ half-site of a PPRE is more influential on the binding of the PPARγ/RXR heterodimer than the ability of PPARy to bind DNA. Thus, unlike RXR, PPARγ exhibits promiscuity in binding on a PPRE, suggesting that the definition of a PPRE for PPARγ may need to be expanded.