Chromatin immunoprecipitation (ChIP) experiments indicated that Nurr1 was recruited to LPS-responsive promoters following LPS treatment, exemplified by the TNFα promoter (Fig. 3A), suggesting that it was acting locally to repress transcription. Two different general mechanisms of NR-mediated repression have been described: active repression, involving sequence-specific DNA binding, and transrepression, involving tethering of NRs to negatively regulated target genes via protein-protein interactions (Glass and Ogawa, 2006). A mutant of Nurr1 (Nurr1C280A/E281A, CEAA), defective for sequence-specific DNA binding and unable to activate NGFI-B responsive element (NBRE)-luciferase, a reporter for Nurr1 monomer-binding, was fully able to repress iNOS induction by LPS (Fig. 3B and Fig. S6D). In addition, mutations directed at the heterodimerization (I-box) domain (Aarnisalo et al., 2002) of Nurr1 (Nurr1K555A/L556A/L557A, KLL) that prevented its ability to activate a Nurr1/RXR-dependent (DR5)-promoter did not interfere with Nurr1-mediated repression of iNOS (Fig. 3B). On the other hand, this I-box mutation increased transcriptional activation of Nurr1 monomers through the NBRE element, as previously reported (Aarnisalo et al., 2002) (Fig. S6D).
