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

Abstract

The identification of a novel coregulator for nuclear hormone receptors, designated NRIF3, was recently reported (D. Li et al., Mol. Cell. Biol. 19:7191-7202, 1999). Unlike most known coactivators, NRIF3 exhibits a distinct receptor specificity in interacting with and potentiating the activity of only TRs and RXRs but not other examined nuclear receptors. However, the molecular basis underlying such specificity is unclear. In this report, we extended our study of NRIF3-receptor interactions. Our results suggest a bivalent interaction model, where a single NRIF3 molecule utilizes both the C-terminal LXXIL (receptor-interacting domain 1 [RID1]) and the N-terminal LXXLL (RID2) modules to cooperatively interact with TR or RXR (presumably a receptor dimer), with the spacing between RID1 and RID2 playing an important role in influencing the affinity of the interactions. During the course of these 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 (AD1) 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 (RepD1) 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 alternative splicing. These two isoforms contain the same RepD1 region as NRIF3. Consistent with this, Gal4 fusions of these two isoforms were also found to repress transcription. Cotransfection of NRIF3 or its two isoforms did not relieve the transrepression function mediated by their corresponding Gal4 fusion proteins, suggesting that the repression involves a mechanism(s) other than the recruitment of a titratable corepressor. Interestingly, a single amino acid residue change of a potential phosphorylation site in RepD1 (Ser(28) to Ala) abolishes its transrepression function, suggesting that the coregulatory property of NRIF3 (or its isoforms) might be subjected to regulation by cellular signaling. Taken together, our results identify NRIF3 as an interesting coregulator that possesses both transactivation and transrepression domains and/or functions. Collectively, the NRIF3 family of coregulators (which includes NRIF3 and its other isoforms) may play dual roles in mediating both positive and negative regulatory effects on gene expression.

FIG. 1

Domain organization of NRIF3 and its isoforms. NRIF3 consists of 177 amino acids. EnS consists of 111 amino acids and is 100% identical to the corresponding region of NRIF3. The first 161 amino acids of EnL are identical to those of NRIF3. EnL and NRIF3 differ in their unique C termini (9 residues in EnL, filled box; 16 residues in NRIF3, hatched box). The unique C terminus of NRIF3 (hatched box) harbors RID1, which contains an LXXIL motif. Another RID, RID2, is located at the N terminus of NRIF3 and contains the canonical LXXLL motif. A coiled-coil (dimerization) domain is mapped to the center of the NRIF3 molecule (residues 84 to 112, waved box) and is also found in EnS and EnL. This region also contains a putative leucine zipper-like motif. A transactivation domain (AD1) is mapped to the unique C terminus of NRIF3 (hatched box), a region that also harbors RID1. A transrepression domain (RepD1) is mapped in the N-terminal portion of NRIF3 (residues 20 to 50, dotted box), a region also common to EnS and EnL.

FIG. 2

EnS and EnL are nucleus localized. HeLa cells were transfected with an expression vector for GFP-EnS or GFP-EnL. Cells were then incubated at 37°C for 24 h before being examined under a fluorescence microscope to determine the subcellular location of the fusion protein. GFP fusions of both EnS and EnL are localized in the nucleus. The control GFP alone was found to be distributed throughout the cell (data not shown). GFP-NRIF3 was used as another control, and its pattern was found to be similar to that of GFP-EnS or GFP-EnL (data not shown).

FIG. 3

Integrity of helix 12 (the AF2 helix) is essential for the NRIF3-TR interaction. (A) ³⁵S-labeled wild-type TR (WT) or the mutant TR (L398R) was generated by in vitro translation. The labeled receptors were then examined for binding to purified GST control or GST-NRIF3 in the presence (+) or absence (–) of T3 as described in Materials and Methods. (B) Yeast two-hybrid assay. LexA-NRIF3 was examined for interaction with the control B42 alone, B42-TR LBD(120–408), or B42-TR LBD(120–398) that deletes 10 amino acid residues from helix 12. β-Galactosidase activities were determined in the presence (shaded columns) or absence (open columns) of T3.

FIG. 4

Interactions of RID1 with liganded LBDs in yeast are fusion partner dependent. The indicated baits LexA-RID1, LexA-TR LBD, and LexA-RXR LBD were examined for interactions with the indicated preys (as B42 fusions) in a yeast two-hybrid assay as described in Materials and Methods. β-Galactosidase activities were determined in the presence (shaded columns) or absence (open columns) of cognate ligands: T3 for TR, 9-cis RA for RXR.

FIG. 5

Characterization of the NRIF3-NRIF3 interaction. LexA-NRIF3 was studied in a yeast two-hybrid assay for interactions with various B42 fusions as depicted in the figure. The region of the coiled-coil domain is indicated as a waved box.

FIG. 6

The central coiled-coil domain is essential for the NRIF3-NRIF3 interaction. (A) Amino acid sequence of the coiled-coil domain of NRIF3 (residues 84 to 112). The putative leucine zipper-like structure is shown in boldface and underlined, with the occurrence of Leu, Leu, Ile, and Ile at every seventh position between residues 89 and 110. Leu⁸⁹ and Leu⁹⁶ are marked with asterisks. (B) LexA-NRIF3 (WT) was examined in a yeast two-hybrid assay for interactions with the following preys (as B42 fusions), respectively: wild-type NRIF3 (WT), mutant NRIF3 with an internal deletion of the coiled-coil domain (Δ1), and mutant NRIF3s L89R, L96R, and DM.

FIG. 7

Dimerization of NRIF3 is not required for receptor interactions. The baits LexA-TR LBD and LexA-RXR LBD were examined in a yeast two-hybrid assay for interactions with the following preys (as B42 fusions), respectively: wild-type NRIF3 (WT), NRIF3 L89R, NRIF3 L96R, and the double mutant (DM). β-Galactosidase activities were determined in the presence (shaded columns) or absence (open columns) of cognate ligands: T3 for TR, 9-cis RA for RXR.

FIG. 8

NRIF3-receptor interaction requires regions of both RID1 and RID2 and is influenced by the spacing between RID1 and RID2. The baits LexA-TR LBD and LexA-RXR LBD were examined in a yeast two-hybrid assay for interactions with the following preys (as B42 fusions), respectively: full-length NRIF3 (WT), the N-terminal portion of NRIF3(1–111) that contains RID2, the C-terminal portion of NRIF3(112–177) that contains RID1, and mutant NRIF3s that delete residues 87 to 111 (Δ1), or residues 112 to 161 (Δ2). β-Galactosidase activities were determined in the presence (shaded columns) or absence (open columns) of cognate ligands: T3 for TR, 9-cis RA for RXR.

FIG. 9

The C-terminal region of NRIF3 contains an autonomous transactivation domain (AD1). HeLa cells were transfected with the G5-tk-CAT reporter either alone or together with one of the following constructs: Gal4, Gal4-NRIF3 (full-length, 1 to 177), Gal4-EnS (equivalent to residues 1 to 111 of NRIF3, see Fig. 1), Gal4-EnL (full length; Fig. 1), and Gal4-AD1 (residues 162 to 177 of NRIF3, see Fig. 1). Cells were harvested 42 h after transfection, and CAT activities were then determined as described in Materials and Methods.

FIG. 10

The N-terminal portion of NRIF3 harbors a transrepression function. HeLa cells were transfected with the G5-SVB-CAT reporter either alone or together with one of the following constructs: Gal4, Gal4-NRIF3 (full length, 1 to 177), Gal4-EnS (equivalent to residues 1 to 111 of NRIF3; Fig. 1), Gal4-EnL (full length; Fig. 1), and Gal4-(112–177) (residues 112 to 177 of NRIF3). Cells were harvested 42 h after transfection, and CAT activities were then determined as described in Materials and Methods.

FIG. 11

The coiled-coil domain is not required for the transrepression function of NRIF3. HeLa cells were transfected with the G5-SVB-CAT reporter, together with one of the following constructs: Gal4, Gal4-NRIF3 (wild type), Gal4-(Δ87–111) (a mutant NRIF3 that deletes the coiled-coil domain), and Gal4-DM (a mutant NRIF3 containing double mutations L89R and L96R). Cells were harvested 42 h after transfection, and CAT activities were then determined as described in Materials and Methods.

FIG. 12

Gal4 fusions of NRIF3, EnL, and EnS function as potent repressors in GH4C1 cells. Constructs expressing Gal4-NRIF3, Gal4-EnL, or Gal4-EnS were transfected into GH4C1 cells, together with the G5-tk-CAT reporter, to evaluate the potential repression function by the Gal4 fusion proteins. Gal4-(112–177) (which shows no repression and was included as a control), Gal4-(Δ87–111 (a mutant NRIF3 that deletes the coiled-coil domain), and Gal4-DM (a mutant NRIF3 containing double mutations L89R and L96R) were used to examine the potential role of the coiled-coil domain.

FIG. 13

An essential repression domain (RepD1) is mapped to residues 20 to 50 of NRIF3. Constructs expressing Gal4 fusions of various regions of NRIF3 as illustrated were transfected into GH4C1 cells, together with the G5-tk-CAT reporter to evaluate repression. Gal4-(112–177), which shows no repression, was included as a control (construct 1 [not drawn]). The fold repression was calculated as described in Materials and Methods. The mapped RepD1 region spanning residues 20 to 50 is shown as a dotted box.

FIG. 14

A single amino acid substitution (Ser28 to Ala) abolishes RepD1-mediated repression. Gal4 fusions of the wild-type RepD1 [Gal4-(20–50) WT] or the mutant RepD1 [Gal4-(20–50) S28A] were examined for the ability to repress the G5-tk-CAT reporter in transfected GH4C1 cells. The fold repression was calculated as described in Materials and Methods.