SUMOylation of NRs has recently been established to play important roles in transrepression (Pascual et al., 2005). Since it is known that Nurr1 interacts with the protein inhibitor of activated STAT (PIAS) 4 (Galleguillos et al., 2004), which is a SUMO E3 ligase, we examined whether SUMOylation is also involved in Nurr1-mediated repression. As shown in Figure 3C, knockdown of Ubc9, an essential E2 enzyme for SUMOylation (Hay, 2005), reversed Nurr1-mediated repression of iNOS, suggesting that SUMOylation is required. Next, we confirmed that Nurr1 could be SUMOylated with SUMO2 and SUMO3 using PIAS4 as an E3 ligase (Fig. S6E and F) and found that IL1β stimulation could induce SUMOylation of Nurr1 in the absence of overexpression of PIAS4 (Fig. S6F). Mutational studies demonstrated that lysine 558 and, to a lesser extent lysine 576, are essential SUMO sites of Nurr1 (Fig. 3D). Since both K558R and K576R mutants are located in the ligand binding domain and close to the I-box and RXR is not required for repression activity (Fig. 3B and Fig. S6D), we hypothesize that SUMOylation is required for monomerization
