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. 2009 Jun;23(6):794-808.
doi: 10.1210/me.2008-0236. Epub 2009 Mar 12.

The PPARgamma2 A/B-domain plays a gene-specific role in transactivation and cofactor recruitment

Anne Bugge  1 , Lars Grøntved, Mads M Aagaard, Rehannah Borup, Susanne Mandrup
Affiliations

The PPARgamma2 A/B-domain plays a gene-specific role in transactivation and cofactor recruitment

Anne Bugge et al. Mol Endocrinol. 2009 Jun.

Abstract

We have previously shown that adenoviral expression of peroxisome proliferator-activated receptors (PPARs) leads to rapid establishment of transcriptionally active complexes and activation of target gene expression within 5-8 h after transduction. Here we have used the adenoviral delivery system combined with expression array analysis to identify novel putative PPARgamma target genes in murine fibroblasts and to determine the role of the A/B-domain in PPARgamma-mediated transactivation of genomic target genes. Of the 257 genes found to be induced by PPARgamma2 expression, only 25 displayed A/B-domain dependency, i.e. significantly reduced induction in the cells expressing the truncated PPARgamma lacking the A/B-domain (PPARgammaCDE). Nine of the 25 A/B-domain-dependent genes were involved in lipid storage, and in line with this, triglyceride accumulation was considerably decreased in the cells expressing PPARgammaCDE compared with cells expressing full-length PPARgamma2. Using chromatin immunoprecipitation, we demonstrate that PPARgamma binding to genomic target sites and recruitment of the mediator component TRAP220/MED1/PBP/DRIP205 is not affected by the deletion of the A/B-domain. By contrast, the PPARgamma-mediated cAMP response element-binding protein (CREB)-binding protein (CBP) and p300 recruitment to A/B-domain-dependent target genes is compromised by deletion of the A/B-domain. These results indicate that the A/B-domain of PPARgamma2 is specifically involved in the recruitment or stabilization of CBP- and p300-containing cofactor complexes to a subset of target genes.

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Figures

Fig. 1.
Fig. 1.
Characterization of AdHA-PPARγ2 and AdHA-PPARγCDE. A, Schematic diagram of the HA-tagged full-length and A/B-domain-deleted PPARγ encoded by the recombinant adenoviruses. Numbers indicate amino acid positions of domain borders (Swiss-Prot; www.expasy.org). B, Western blot showing HA-PPARγ expression in NIH-CAR cells transduced for 8 h with AdEmpty (AdE), AdHA-PPARγ2 (AdHA-γ2), or AdHA-PPARγCDE (AdHA-γCDE) and treated with vehicle (DMSO) or PPARγ agonist rosiglitazone (Rosi) as indicated. Whole-cell extract from 3T3-L1 adipocytes (ADI) is used for comparison. Cell extracts were subjected to SDS-PAGE and immunoblotted using antibodies against PPARγ and TFIIB. C, Validation of HA-tags on PPARγ proteins. Whole-cell lysates were prepared as in B and immunoblotted using antibodies against HA, TFIIB, and PPARγ as indicated. D, Expression levels of adenoviral E4 (AdE4), total PPARγ, and endogenous PPARγ mRNA 8 h after transduction with the indicated adenoviral vector and treatment with vehicle (DMSO), rosiglitazone, or antagonist GW9662 (GW) as indicated. mRNA expression was determined by RT-qPCR and normalized to the corresponding TFIIB levels. E, Deletion of the A/B-domain does not affect the subcellular localization of HA-PPARγCDE. Cells were transduced as in B and fixed 8 h after transduction. Cells were stained with DAPI (blue), primary αPPARγ antibody, and Alexa flour 546-conjugated secondary antibody (red). Fluorescence was detected by confocal microscopy and images were merged. F, Lack of the A/B-domain does not affect in vitro DNA-binding activity of HA-PPARγ. Nuclear extracts were prepared 8 h after transduction of NIH-CAR cells as in B, subjected to SDS-PAGE, and immunoblotted using an antibody against PPARγ (left). EMSAs were performed using these nuclear extracts (right). All results are representative of at least three independent experiments.
Fig. 2.
Fig. 2.
Comparison of the transcriptional activity of HA-PPARγ2 and HA-PPARγCDE on genomic target genes. NIH-CAR cells were transduced with AdEmpty (AdE), AdHA-PPARγ2 (AdHA-γ2), or AdHA-PPARγCDE (AdHA-γCDE) and treated with vehicle (DMSO), rosiglitazone (Rosi), or antagonist GW9662 (GW) as indicated. RNA was isolated 8 h after transduction, and mRNA expression was determined by RT-qPCR and normalized to the corresponding TFIIB levels. Results are representative of at least three independent experiments.
Fig. 3.
Fig. 3.
Comparison of the transcriptional activity of HA-PPARγ2, HA-PPARγCDE, and HA-PPARαA/BγCDE on genomic target genes. A, Schematic diagram of the HA-tagged PPARα and -γ and the chimeric PPARαA/BγCDE encoded by the recombinant adenoviruses. Numbers indicate amino acid positions of domain borders (Swiss-Prot; www.expasy.org). B–D, NIH-CAR cells were transduced with AdEmpty (AdE), AdHA-PPARγ2 (AdHA-γ2), AdHA-PPARγCDE (AdHA-γCDE), or AdHA-PPARαA/BγCDE (AdHA-αA/BγCDE) and treated with vehicle (DMSO) or rosiglitazone (Rosi) as indicated. The cells were harvested 8 h after transduction and subjected to either SDS-PAGE and immunoblotted using antibodies against PPARγ and TFIIB or RNA purification. mRNA expression was determined by RT-qPCR and normalized to the corresponding TFIIB levels. Results are representative of at least three independent experiments.
Fig. 4.
Fig. 4.
The effect of A/B-domain deletion on the lipogenic potential of HA-PPARγ2. NIH-CAR cells were transduced with AdEmpty (AdE) and treated with vehicle (DMSO) or transduced with AdHA-PPARγ2 (AdHA-γ2) or AdHA-PPARγCDE (AdHA-γCDE) and treated with rosiglitazone (Rosi). A, Cells were harvested 48 h after transduction and subjected to SDS-PAGE and immunoblotted using antibodies against PPARγ and TFIIB. B, Lipid synthesis was assessed in triplicate by determining incorporation of [14C]acetate into total lipids 48 h after adenoviral transduction of which the last 24 h were in the presence of [14C]acetate. Extracted lipids were separated on silica thin-layer chromatography plates and radioactivity in the triglyceride fraction was quantified. Results are representative of two independent experiments.
Fig. 5.
Fig. 5.
HA-PPARγ2:RXR and HA-PPARγCDE:RXR heterodimers bind equally well to most genomic PPREs. Cross-linked chromatin was harvested from NIH-CAR cells 8 h after transduction with either AdEmpty (AdE) and treatment with vehicle (DMSO) or transduction with AdHA-PPARγ2 (AdHA-γ2) or AdHA-PPARγCDE (AdHA-γCDE) and treatment with rosiglitazone (Rosi). Relative occupancy at PPREs of interest was determined by ChIP-qPCR using antibodies against HA and RXR, respectively. Results are representative of at least three independent experiments. The figure shows means of two experiments with indication of range.
Fig. 6.
Fig. 6.
The HA-PPARγ2- and HA-PPARγCDE-induced RNAPII occupancy at target gene promoters reflects mRNA expression. Cross-linked chromatin was harvested from NIH-CAR cells 8 h after transduction with either AdEmpty (AdE) and treatment with vehicle (DMSO) or transduction with AdHA-PPARγ2 (AdHA-γ2) or AdHA-PPARγCDE (AdHA-γCDE) and treatment with rosiglitazone (Rosi). Relative occupancy at target gene promoters of interest was determined by ChIP-qPCR using an antibody against RNAPII. Results are representative of at least three independent experiments. The figure shows means of two experiments with indication of range.
Fig. 7.
Fig. 7.
HA-PPARγ2 and HA-PPARγCDE recruit TRAP220 equally well to genomic PPREs. Cross-linked chromatin was harvested from NIH-CAR cells 8 h after transduction with either AdEmpty (AdE) and treatment with vehicle (DMSO) or transduction with AdHA-PPARγ2 (AdHA-γ2) or AdHA-PPARγCDE (AdHA-γCDE) and treatment with rosiglitazone (Rosi). Relative occupancy at PPREs of interest was determined by ChIP-qPCR using an antibody against TRAP220. Results are representative of at least three independent experiments. The figure shows means of two experiments with indication of range.
Fig. 8.
Fig. 8.
HA-PPARγ2 and HA-PPARγCDE differ in their ability to recruit CBP and p300 occupancy to genomic PPREs. Cross-linked chromatin was harvested from NIH-CAR cells 8 h after transduction with either AdEmpty (AdE) and treatment with vehicle (DMSO) or transduction with AdHA-PPARγ2 (AdHA-γ2) or AdHA-PPARγCDE (AdHA-γCDE) and treatment with rosiglitazone (Rosi). Relative occupancy at PPREs of interest was determined by ChIP-qPCR using antibodies against CBP or p300, respectively. Results are representative of at least three independent experiments. The figure shows means of two experiments with indication of range.
Fig. 9.
Fig. 9.
CBP and p300 knockdown differentially affects the expression of PPARγ target genes. NIH-CAR cells were transfected with siRNA against CBP, p300, or both 24 h before transduction with either AdEmpty (AdE) and treatment with vehicle (DMSO) or transduction with AdHA-PPARγ2 (AdHA-γ2) and treatment with rosiglitazone (Rosi). The siRNA against luciferase (Luc) was used as a control. The cells were harvested 8 h after transduction. A, Protein expression was determined by SDS-PAGE and immunoblotting using antibodies against PPARγ, CBP, p300, or Sp1. B, mRNA expression was determined by RT-qPCR and normalized to the corresponding TFIIB levels. Results are representative of three independent experiments.
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