Bugge, Anne Skovsø5; Grøntved, Lars5; M. Aagaard, Mads3; Borup, Rehannah4; Mandrup, Susanne5
1 Faculty of Science, SDU2 Department of Biochemistry and Molecular Biology, Faculty of Science, SDU3 Syddansk Universitet4 Rigshospitalet5 Department of Biochemistry and Molecular Biology, Faculty of Science, SDU
We have previously shown that adenoviral expression of PPARs leads to rapid establishment of transcriptionally active complexes and activation of target gene expression within 5-8 hours following transduction (Nielsen at al. 2006). Here we have used the adenoviral delivery system to identify novel putative PPARγ target genes in murine fibroblasts and to determine the role of the A/B-domain in PPARγ2 mediated transactivation of genomic target genes. RNA isolated from three biological replicates of fibroblasts transduced with adenovirus encoding full length PPARγ2 or A/B-domain deleted PPARγ (PPARγCDE) in the presence of agonist were evaluated by expression array analysis. In total, 218 genes were found to be significantly induced by acute PPARγ expression and agonist as compared to the empty adenovirus. Interestingly, only 22 of these genes displayed A/B-domain dependency, i.e. significantly reduced induction in the cells expressing PPARγCDE. Nine of the 22 A/B-domain dependent genes were involved in lipid storage and in line with this, triglyceride accumulation was considerably decreased in the cells expressing PPARγCDE compared to cells expressing full length PPARγ2. Using chromatin immunoprecipitation (ChIP) we demonstrate that the reduced transcriptional activity of PPARγCDE, on the A/B-domain dependent target genes, was not due to decreased binding of the RXR:PPARγ heterodimer or the recruitment of the mediator component TRAP220 to the genomic PPREs. By contrast we show that PPARγ mediated CBP and p300 recruitment to these genes is abolished by deletion of the A/B-domain, indicating that the A/B-domain of PPARγ2 is specifically involved in the recruitment of co-activator complexes to a subset of target genes. This work was supported by grants from the Danish Natural Science Research Foundation and from the EU FP6 STREP project X-TRA-NET.