Peroxisome Proliferator-Activated Receptor γ-Dependent Repression of the Inducible Nitric Oxide Synthase Gene

The peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily that activates target gene transcription in a ligand-dependent manner. In addition, liganded PPARγ can inhibit transcription of genes induced by gamma interferon (IFN-γ) and/or lipopolysaccharides (LPSs), including the inducible nitric oxide synthase (iNOS) gene. Inhibition of the iNOS promoter is achieved partially through antagonizing the activities of NF-κB, AP-1, and STAT1, which are known to mediate effects of LPS and IFN-γ. Previous studies have suggested that transrepression of these factors by nuclear receptors involves competition for limiting amounts of the general coactivators CREB-binding protein (CBP) and p300. CBP and p300 are thought to be recruited to nuclear receptors through bridging factors that include SRC-1, although CBP also interacts directly with PPARγ through its amino terminus. These observations have raised questions concerning the involvement of SRC-1-like factors in CBP recruitment and transrepression. We here provide evidence that PPARγ's ability to repress iNOS transcription requires the ligand-dependent charge clamp that mediates interactions with CBP and SRC-1. Single amino acid mutations in PPARγ that abolished ligand-dependent interactions with SRC-1 and CBP not only resulted in complete loss of transactivation activity but also abolished transrepression. Conversely, a CBP deletion mutant containing the SRC-1 interaction domain but lacking the N-terminal PPARγ interaction domain was inactive as a PPARγ coactivator and failed to rescue transrepression. Together, these findings are consistent with a model in which transrepression by PPARγ is achieved by targeting CBP through direct interaction with its N-terminal domain and via SRC-1-like bridge factors.