A novel role for helix 12 of retinoid X receptor in regulating repression

Abstract

Nutrients, drugs, and hormones influence transcription during differentiation and metabolism by binding to high-affinity nuclear receptors. In the absence of ligand, some but not all nuclear receptors repress transcription as a heterodimer with retinoid X receptor (RXR). Here we define a novel role for helix 12 (H12) in sterically masking the corepressor (CoR) binding site in apo-RXR. Removing H12 converts RXR to a potent transcriptional repressor. The length but not the specific sequence of H12 is critical for masking RXR's intrinsic repression function. This contrasts with the amphipathic character required for mediating ligand-dependent activation and coactivator recruitment. Physiologically, we show that heterodimerization of RXR with apo-thyroid hormone receptor (TR) unmasks the CoR binding site in RXR and allows the TR-RXR heterodimer to repress. A molecular mechanism that involves sequence-specific interaction between RXR H12 and the coactivator-binding surface of the nuclear receptor is proposed for this heterodimerization-mediated unmasking. Peroxisome proliferator-activated receptor gamma does not interact as well with RXR H12, thus explaining its inability to repress transcription as an RXR heterodimer. The requirement to unmask RXR's latent repression function explains why only certain RXR partners repress transcription.

FIG. 1

RXR H12 masks an intrinsic repression function. (A) Deletion of the RXR C terminus converts RXR LBD into a potent repression domain. Full-length human RXRα LBD or RXR LBDΔ443 was fused to Gal4 DBD, and 1 μg was transfected into 293T cells along with a (Gal4 × 5)-simian virus 40-luciferase reporter gene. (B) Enhanced interaction between RXRΔ443 and N-CoR in vivo. Mammalian two-hybrid assay with Gal4-RXR or Gal4-RXRΔ443 as bait and VP16-N-CoR as prey. (C) Enhanced interaction between RXRΔ443 and N-CoR in vitro: GST pull-down of RXR or RXRΔ443 with GST alone or GST-N-CoR interaction domain. The input lane shows 10% of input. (D) CoR box sequence comparison. Rat TRα sequence is shown from amino acid 160. Human RARα sequence is shown from amino acid 180. Human RXRα sequence is shown from amino acid 224. Amino acids critical for interaction of CoR with TR and RAR are underlined, as are the homologous amino acids in RXR. (E and F) CoR box is required for repression (E) and interaction (F) between RXRΔ443 and CoR: mammalian two-hybrid assay with Gal4-RXRΔ443 or Gal4-RXRΔ443(AEV) as bait and VP16-SMRT as prey.

FIG. 2

Length is the critical determinant of the RXR H12 mask. (A) RXRα mutants used in the study. Sequences shown begin at amino acid 437. The positions of H11 and H12 are indicated. (B and C) Seven alanine residues in place of H12 are sufficient for interference with repression (B) and CoR interaction (C) with RXR. Fold repression values for Gal4-RXRΔ449 or Gal4-RXRΔ449-A7 are shown. (D) Schematic of conclusion from panels B and C. (E and F) Four but not three alanines after amino acid 443 are sufficient to block repression and CoR interaction. Three, four, and seven alanine residues were attached to the Gal4-RXRΔ449 (designated RXRΔ449-A3, -A4, and -A7), and these were assayed for repression (E) and CoR interaction activities (F). (G) Potential structural basis of the H12 mask. The apo-RXR structure is from Bourguet et al. (5). Note that the Ω loop flips outward, and the side chain of the fourth but not the third amino acid after 449 contacts the Ω loop, especially N262. The RXRΔ449 structure was modeled using Swiss-Pdb Viewer software. The lowest energy (force field score) conformation without amino acid clashes is shown. Note that the Ω loop is flipped down.

FIG. 3

Apo-RXR exists as an intermediate between CoR- and CoA-interacting conformations. (A) Apo-RXR and apo-TR are differentially sensitive to proteolysis. Arrows point to protected fragments. Proteins were translated in reticulocyte lysate and treated with trypsin (300 μg/ml) for various times (5, 10, and 20 min). The T3 concentration was 1 μM and SR11237 was used at a concentration of 50 μM. (B) Differential protease sensitivity of apo-RXRΔ443 and RXR-F313A.

FIG. 4

RXR provides a CoR interaction surface in the RXR-TR heterodimer. (A) Gal4-RXR, but not the RXR CoR box mutant, rescues Gal4-TR(AHT) for repression. (B and C) Gal4-RXR, but not the RXR CoR box mutant, rescues Gal4-TR(AHT) for interaction with VP16-SMRT (B) and VP16-N-CoR (C) in the mammalian two-hybrid assay.

FIG. 5

Unmasking the CoR interaction domain of RXR by “H12 docking” with a heterodimer partner. (A) RXR H12 contributes to the affinity of RXR for TR: mammalian two-hybrid assay with Gal4-RXR or Gal4-RXRΔ449 and VP16-N-CoR. (B) RXR H12 interacts specifically with apo-TR and apo-RAR in the GST pull-down assay. (C) Heterodimerization with TR alters protease sensitivity of RXR. Labeled RXR was incubated with a fivefold excess of unlabeled TR (in the presence and absence of T3 [12.5 μM]), then exposed to trypsin (1 mg/ml) for 3 min. (D) Model of conclusions from panels A, B, and C. (E and F) Gal4-RXRΔ449A7 cannot rescue Gal4-TR(AHT) for repression (E) and CoR interaction (F). (G) H12-AF2 mutants of TR used in panel H. (H) Ligand binding by TR abolishes complementation of repression between Gal4-RXR and Gal4-TR(AHT)E403A but not Gal4-TR(AHT)ΔAF2. Gal4-TR(AHT)ΔAF2, Gal4-TR(AHT)E403A, and Gal4-RXR were transfected separately or together and assayed for repression of (Gal4 × 5)-simian virus 40-luciferase reporter in the presence and absence of T3 (1 μM).

FIG. 6

Sequences in TR and RXR required for the unmasking of repression. (A) Sequences of RXR AF2 and the ML-AA mutant. (B and C) The ML-AA mutant of RXR fails to rescue TR(AHT) for repression (B) or N-CoR interaction (C). (D) Sequences of H5 of TRα and the I248R mutant. (E and F) The I248R mutant, which does not interact with coactivators, cannot functionally interact with RXR for repression (E) and N-CoR interaction (F).

FIG. 7

PPARγ does not repress transcription because it is unable to dock with RXR H12 and unmask the RXR CoR interaction surface. (A) Gal4-RXR does not complement Gal4-PPARγ in repression. (B) Wild-type TR but not wild-type PPARγ complements Gal4-RXR in repression. (C) PPARγ does not interact with the GST-RXR H12 in the GST pull-down assay. GST was fused to amino acids 443 to 462 of hRXRα. (D) PPARγ-RXRΔ443 heterodimer interacts with N-CoR on DNA: gel shift assay using in vitro-translated PPARγ and RXR proteins and bacterially expressed GST or GST-N-CoR interaction domain, binding to PPRE from the acyl- CoA oxidase gene.

FIG. 8

Model of the role of RXR H12 in masking and unmasking CoR interaction. H12 of RXR sterically masks the CoR interaction domain. The intrinsic affinity of RXR for N-CoR and SMRT can be unmasked by deletion of RXR H12 (top) or by heterodimerization with TR (bottom). T3 binding to TR-RXR heterodimer alters the conformation of TR in a manner that interferes with H12 docking, thereby contributing to CoR dissociation and derepression. For simplicity, the CoR is depicted in isolation but is most likely in a complex including some combination of proteins known to associate with N-CoR or SMRT, including Sin3 (2, 23, 37), HDAC (2, 23, 37), SUN-CoR (62), and ETO (17, 33, 57).