Domain structure of the NRIF3 family of coregulators suggests potential dual roles in transcriptional regulation

The identification of a novel coregulator for nuclear hormone receptors, de signated NRIF3, was recently reported (D. Li et al., Mol. Cell. Biol. 19:71 91-7202, 1999). Unlike most known: coactivators, NRIF3 exhibits a distinct receptor specificity in interacting with and potentiating the activity of o nly TRs and RXRs but not other examined nuclear receptors. However, the mol ecular basis underlying such specificity is unclear. In this report, we ext ended our study of NRIF3-receptor interactions. Our results suggest a bival ent interaction model, where a single NRIF3 molecule utilizes both the C-te rminal LXXIL (receptor-interacting domain 1 [RID1]) and the N-terminal LXXL L (RID2) modules to cooperatively interact with TR or RXR (presumably a rec eptor dimer), with the spacing between RID1 and RID2 playing an important r ole in influencing the affinity of the interactions. During the course of t hese studies, we also uncovered an NRIF3-NRIF3 interaction domain. Deletion and mutagenesis analyses mapped the dimerization domain to a region in the middle of NRIF3 (residues 84 to 112), which is predicted to form a coiled- coil structure and contains a putative leucine zipper-like motif. By using Gal4 fusion constructs, we identified an autonomous transactivation domain (ADI) at the C terminus of NRIF3. Somewhat surprisingly, full-length NRIF3 fused to the DNA-binding domain of Gal4 was found to repress transcription of a Gal4 reporter. Further analyses mapped a novel repression domain (RepD 1) to a small region at the N-terminal portion of NRIF3 (residues 20 to 50) . The NRIF3 gene encodes at least two additional isoforms due to alternativ e splicing. These two isoforms contain the same RepD1 region as NRIF3. Cons istent with this, Gal4 fusions of these two isoforms were also found to rep ress transcription. Cotransfection of NRIF3 or its two isoforms did not rel ieve the transrepression function mediated by their corresponding Ga14 fusi on proteins, suggesting that the repression involves a mechanism(s) other t han the recruitment of a titratable corepressor. Interestingly, a single am ino acid residue change of a potential phosphorylation site in RepD1 (Ser(2 8) to Ala) abolishes its transrepression function, suggesting that the core gulatory property of NRIF3 (or its isoforms) might be subjected to regulati on by cellular signaling. Taken together, our results identify NRIF3 as an interesting coregulator that possesses both transactivation and transrepres sion domains and/or functions. Collectively, the NRIF3 family of coregulato rs (which includes NRIF3 and its other isoforms) may play dual roles in med iating both positives and negative regulatory effects on gene expression.