FIG. 6.

PPAR subtype-specific binding to endogenous PPREs in the presence of specific agonists. (A) HA-PPARs are expressed at equal levels. Whole-cell extracts from NIH-CAR cells transduced with 55 PFU/cell of AdHA-PPARα, -γ2, or -β/δ for 8 h show equal protein expression of the PPAR subtypes. Proteins were separated by SDS-PAGE and immunoblotted using antibodies against the HA tag and TFIIB, respectively. (B) The ability of the different PPAR subtypes to bind to target sites in the presence of their specific agonists (Fig. 5) was determined by ChIP. Chromatin was harvested 8 h following transduction, and ChIP was performed using antibodies against the HA epitope and RXR. Relative occupancy (recovery in the presence of PPAR expression over recovery in the nontransduced cells) was determined using primers positioned at the PPREs of the indicated genes (Table 2). (C) PPARγ2 fails to further activate the transcription of the ME gene and only slightly activates that of the ACOx1 gene although receptors and cofactors are significantly recruited to the corresponding PPREs. Expression of the ME and ACOx1 genes following adenoviral transduction was determined as described in the legend of Fig. 5A. Chromatin was harvested 8 h following transduction, and ChIP was performed using antibodies against the HA tag, RXR, CBP, TRAP220, and RNA Pol II. Recovery at the ME and ACOx1 PPREs is indicated. (D) The PPAR-induced recruitment of the cofactors TRAP220 and CBP to PPREs is correlated with binding of the respective subtype to the target sites. ChIP experiments were performed as described for panel B except that antibodies against TRAP220 and CBP were used. Relative occupancy is indicated. Results are representative of three independent experiments.