INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Nuclear hormone receptors (NHRs) allow cells to regulate gene transcription in response to nutritional, metabolic, and hormonal signals. Many NHRs repress transcription in the unliganded state (3, 6, 13, 19, 39). This silences target genes and amplifies the ligand signal. Repression is a function of the ligand-binding domain (LBD) of unliganded (apo-) NHR, which recruits corepressor (CoR) molecules to target genes. The main NHR CoRs are N-CoR (for nuclear receptor corepressor) (24, 47, 63) and SMRT (for silencing mediator for retinoid and thyroid hormone receptors) (8, 43). The CoRs are components of multiprotein repression complexes that also include mSin3 and histone deacetylase (2, 23, 37), the latter providing an enzymatic link to the nucleosome (22, 26). The biological importance of repression by NHRs is increasingly clear from studies of human diseases, including thyroid hormone resistance (51) and acute promyelocytic leukemia (32).

Ligand binding to the NHR leads to dissociation of the CoR complex. This depression step accounts for a portion of the increase in gene activity associated with hormone binding. In addition, the hormone-bound (holo-) NHR LBD recruits a coactivator (CoA) complex containing multiple histone acetyltransferases (HATs), including CREB-binding protein (CBP), p300/CBP-associated factor, and a member of the steroid receptor coactivator class of CoAs (reviewed in references 18 and 50). The localized HAT activity is thought to counteract the repressive effects of deacetylated histone and thus lead to enhanced transcription of the hormonally responsive gene.

Remarkably, the stability of the huge CoR and CoA complexes with NHR on DNA is dependent upon the absence or presence of a small lipophilic ligand. NHR apo- and holoreceptor conformations are similar overall, with 12 highly ordered  helices (H1 to H12) (reviewed in reference 61). Numerous differences in the position of these helices between the apo- and holoreceptors have been inferred by comparing the apo-retinoid X receptor (RXR) (5) with holo-retinoic acid receptor (RAR) (42) and holo-thyroid hormone receptor (TR) (56) structures. The most striking difference is in the positioning of H12, which is rotated back toward the ligand-binding pocket in the holoreceptor structure. The change in position of H12 is much less marked in apo- versus holo-peroxisome proliferator-activated receptor  (PPAR) (38, 54). H12 contains an amphipathic region whose sequence is critical to the ligand-dependent recruitment of the CoA complex (14, 21). This amphipathic helix also plays a less-well-defined role in CoR release by NHRs (31, 66).

The structures of apo- and holoreceptors have provided important insights into the molecular underpinnings of hormone action. However, information is limited because NHRs have not been cocrystallized as heterodimers. Indeed, the majority of NHRs, including the TR, RAR, and PPAR, require heterodimerization with RXR for high-affinity binding to hormonally responsive elements (HREs) in target genes (reviewed in reference 34). The RXR-NHR interface also determines target gene specificity by restricting the spacing between directly repeated half-sites that constitute the HRE, thus contributing to the precision of hormone action (40, 53). RXR-TR and RXR-RAR heterodimers interact with corepressors off and on DNA, in vitro as well as in vivo (9, 28, 60, 64). The observation that two repression domains are required for CoR complex formation (25, 64) suggests an active role of RXR. Consistent with this, TR mutants that cannot heterodimerize with RXR are defective in repression (4, 39), and interaction with RXR via a heterologous dimerization interface rescues this defect (66).

RXR by itself weakly interacts with CoRs (48) and only weakly represses transcription (36, 44, 66). Here we show that this is because H12 of RXR sterically prevents CoR binding. Deletion of H12 reveals the potential of RXR to bind CoR and repress transcription. Remarkably, unlike other functions of H12, such as CoA recruitment (7, 21, 55), it is the length and not the sequence of H12 that is critical to masking this intrinsic repression function. Moreover, heterodimerization with receptors that reposition H12, such as TR, unmasks RXR's latent repressive potential. Not all RXR-heterodimerizing NHRs are capable of the latter interaction, thereby providing an explanation of variations in their repression function.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Receptor and CoR expression plasmids. Most plasmids have been described previously (66). Others were created by using PCR, endonuclease restriction digestion, or a combination of these techniques followed by ligation. Gal4-PPAR contains amino acids 204 to 505 of mouse PPAR2 cDNA. Details of other constructs are described in the text and/or figure legends. All constructs were directly sequenced.

Interaction assays. Mammalian two-hybrid and glutathione S-transferase (GST) pull-down assays were performed as previously described (66). Gel shift assays have been previously described (64).

Transcription assays. Transient transfection of 293T cells and luciferase reporter assays were performed as previously described (66). Cells were transfected either with calcium phosphate as previously described (66) or by Lipofectamine reagent (Gibco/BRL) in accordance with the manufacturer's instructions. In each case, -galactosidase expression vector was cotransfected as a control for transfection efficiency. Results shown are normalized to -galactosidase expression. Fold activation in the mammalian two-hybrid assay was normalized to luciferase activity in the absence of the VP16-prey vector. For fold repression, results were normalized to the Gal4 DNA-binding domain (DBD) or receptor alone and the reciprocal was plotted. In every case, experiments were repeated two to six times, and duplicates and the range of the results of a representative experiment are shown.

Limited proteolytic digestion assays. Protease digestions and analyses were performed as previously described (49). Trypsin concentrations and times of incubation are provided in the figure legends.

Loop building. The  loop of RXR449 was built using the coordinates of apo-RXR (5) and Swiss-Pdb Viewer software (20). Free energies (force field scores) were calculated for all conformations during loop building, and the lowest energy conformation without amino acid clashes is shown in Fig. 2G. All structures were displayed using MOLMOL software (27).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

RXR H12 masks the intrinsic repression function of apo-RXR by preventing CoR interaction. Unlike TR, the RXR LBD only minimally represses transcription when fused to the Gal4 DBD (Fig. 1A and references 32 and 62). Deletion of the RXR C terminus at amino acid 443 converts RXR LBD into a potent repression domain (Fig. 1A), consistent with the results of others (30, 44). Here, we show that the increased repression by the C-terminal deletion correlates with a dramatic increase in the ability of RXR to interact with N-CoR in vivo (Fig. 1B) and in vitro (Fig. 1C). Figure 1D aligns H1 of RXR with the "CoR box" that is required for CoR interaction with TR and RAR; repression by TR is abolished by mutation of the underlined A, H, and T residues to G, G, and A, respectively (24). Mutation of the homologous amino acids (AEV to GGA) in RXR in the context of RXR443 abrogated repression (Fig. 1E) as well as CoR interaction with RXR (Fig. 1F). This result indicated that the CoR interaction surface in RXR443 is similar to that in TR and RAR, although in the case of RXR it is obscured by H12.

View larger version (28K): [in this window] [in a new window]   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 RXR LBD or RXR LBD443 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 RXR443 and N-CoR in vivo. Mammalian two-hybrid assay with Gal4-RXR or Gal4-RXR443 as bait and VP16-N-CoR as prey. (C) Enhanced interaction between RXR443 and N-CoR in vitro: GST pull-down of RXR or RXR443 with GST alone or GST-N-CoR interaction domain. The input lane shows 10% of input. (D) CoR box sequence comparison. Rat TR sequence is shown from amino acid 160. Human RAR sequence is shown from amino acid 180. Human RXR 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 RXR443 and CoR: mammalian two-hybrid assay with Gal4-RXR443 or Gal4-RXR443(AEV) as bait and VP16-SMRT as prey.

Length is the critical determinant of the H12 mask. We next tested the role of RXR H12 using the C-terminal mutants summarized in Fig. 2A. Deletion at amino acid 449 was sufficient to unmask RXR repression (Fig. 2B). The core amphipathic region of RXR H12 is comprised of amino acids 450 to 456. Remarkably, complete replacement of these amino acids with alanines was sufficient to mask the repression function of RXR (Fig. 2B) as well as the ability of RXR to interact with N-CoR (Fig. 2C). Our results suggest that the presence of H12, rather than its primary amino acid sequence or amphipathicity, was critical for masking repression (Fig. 2D). To further test this hypothesis, one to seven alanine residues were attached to the C terminus of RXR449 (designated RXR449-A1 through -A7). An extension of one to three alanine residues did not block potent repression by RXR449 (Fig. 2E and data not shown). Strikingly, substitution of a single additional alanine (four total), as well as a total of five to seven alanines, was sufficient to block repression (Fig. 2E and data not shown). N-CoR interaction was similarly permitted by the tri-alanine extension but prevented by addition of a fourth alanine residue (Fig. 2F). These results show that the H12 mask is steric and that the length of H12 is the critical factor in RXR's inability to interact with CoR and repress transcription.

View larger version (29K): [in this window] [in a new window]   FIG. 2.   Length is the critical determinant of the RXR H12 mask. (A) RXR 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-RXR449 or Gal4-RXR449-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-RXR449 (designated RXR449-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  loop flips outward, and the side chain of the fourth but not the third amino acid after 449 contacts the  loop, especially N262. The RXR449 structure was modeled using Swiss-Pdb Viewer software. The lowest energy (force field score) conformation without amino acid clashes is shown. Note that the  loop is flipped down.

Apo-RXR exists as an intermediate between CoR- and CoA-interacting conformations. The ability of RXR H12 to sterically prevent corepressor interaction contrasts with apo-TR and apo-RAR, where H12 does not have this function. This suggests that the conformation of apo-RXR is fundamentally different from that of other NHRs, perhaps more akin to the liganded conformation that excludes CoR binding. The conformation of NHR LBDs can be probed by limited protease digestion, wherein the C terminus of the aporeceptor is more prone to proteolytic cleavage than that of the holo-NHR (1). As predicted by the functional studies, RXR LBD was partially protected under conditions that led to complete proteolysis of apo-TR LBD (Fig. 3A). Like apo-TR, apo-RXR443 was completely proteolyzed (Fig. 3B). Unlike RXR, however, deletion of H12 from apo-TR did not affect its proteolytic stability (data not shown). Although the conformation of apo-RXR was different from that of the H12 deletion, it was also distinct from that of holo-RXR, which was protected from proteolysis to a far greater extent (Fig. 3A). This protection was due to the holoreceptor conformation, and not interference by ligand, because a constitutively active mutant (F313A) (55) was similarly protected from proteolytic digestion even in the absence of ligand (Fig. 3B).

View larger version (52K): [in this window] [in a new window]   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-RXR443 and RXR-F313A.

The CoR interaction surface of RXR is critical for repression by the RXR-TR heterodimer. We next explored the ability of RXR to rescue repression-defective TR. The TR CoR box mutant, Gal4-TR(AHT), is inactive in repression assays (references 24 and 66) (Fig. 5). As previously shown (66), Gal4-RXR rescued the repression-defective Gal4-TR(AHT) by restoring repression (Fig. 4A) as well as CoR interaction (Fig. 4B and C). The contribution of the CoR interaction surface of RXR to repression by the TR(AHT)-RXR heterodimer was confirmed by using the Gal4-RXR(AEV) CoR box mutant, which was unable to rescue the TR CoR box mutant in terms of either repression (Fig. 4A) or CoR interaction (Fig. 4B and C).

View larger version (15K): [in this window] [in a new window]   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.

Interaction of RXR H12 with TR unmasks the CoR interaction domain of RXR. The requirement for the RXR CoR box indicated that the masking effect of H12 is relieved by heterodimerization with TR. We hypothesized that H12 of RXR interacts with apo-TR, thereby repositioning H12 and unmasking the CoR interaction surface of RXR. The major RXR heterodimer interface involves H10 and H11 and, as previously shown (35), RXR H12 is not required for interaction with TR (Fig. 5A). However, mammalian two-hybrid experiments showed that while it is not required for heterodimerization, RXR H12 contributes to the interaction of RXR with apo-TR (Fig. 5A). The ability of RXR H12 to interact with TR was directly tested in a GST pull-down experiment, comparing GST alone with GST fused to the 20 C-terminal amino acids (443 to 462) that are deleted in RXR443. Indeed, Fig. 5B shows that RXR H12 interacted specifically with apo-TR. TR(AHT) also interacted with RXR H12 (data not shown). The interaction between RXR H12 and TR was relatively weak but reproducible, similar to that between RAR and RXR H12 (Fig. 5B and reference 58).

View larger version (37K): [in this window] [in a new window]   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-RXR449 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-RXR449A7 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)AF2. Gal4-TR(AHT)AF2, 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).

The sequence specificity of the interaction between apo-TR and RXR H12 is similar to that of receptor-CoA interactions. Recent crystallographic data have indicated that the AF2 helix of apo-PPAR interacts with the CoA binding pocket of another PPAR molecule (38). A similar interaction has been observed in the estrogen receptor crystal structure (52). This led us to hypothesize that RXR AF2 mutations that prevented CoA binding would similarly fail to rescue repression by TR(AHT). To test this idea, two amino acids in the RXR AF2 amphipathic helix were mutated to alanine (ML454AA) (Fig. 6A). This mutation has previously been shown to abolish ligand-dependent activation and CoA recruitment by RXR (55). In the TR(AHT) complementation assay, this mutant displayed markedly reduced repression (Fig. 6B) as well as interaction with CoR (Fig. 6C).

View larger version (29K): [in this window] [in a new window]   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 TR 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).

PPAR is unable to interact with RXR H12 and unmask the RXR CoR interaction surface. Although PPAR binds to N-CoR and SMRT in GST pull-down assays, the PPAR-RXR heterodimer fails to interact with CoRs on DNA and, thus, full-length PPAR does not repress transcription (64). Gal4-PPAR is a weak repressor, consistent with the detectable but weak ability of PPAR to bind N-CoR and SMRT in vitro (41, 64). However, unlike its effects on Gal4-TR(AHT), Gal4-RXR was unable to complement Gal4-PPAR for repression (Fig. 7A). Furthermore, full-length TR but not full-length PPAR complemented Gal4-RXR for repression (Fig. 7B). This suggested that RXR H12 would not dock with PPAR. Indeed, this was confirmed by GST pull-down assay (Fig. 7C and reference 58).

View larger version (26K): [in this window] [in a new window]   FIG. 7.   PPAR 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-PPAR in repression. (B) Wild-type TR but not wild-type PPAR complements Gal4-RXR in repression. (C) PPAR does not interact with the GST-RXR H12 in the GST pull-down assay. GST was fused to amino acids 443 to 462 of hRXR. (D) PPAR-RXR443 heterodimer interacts with N-CoR on DNA: gel shift assay using in vitro-translated PPAR and RXR proteins and bacterially expressed GST or GST-N-CoR interaction domain, binding to PPRE from the acyl- CoA oxidase gene.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Apo-RXR has a conformation distinct from repressive and active states. We have shown that apo-RXR exists in a neutral conformation intermediate between that of CoR and CoA binding conformations. This conformation is characterized by a novel protease sensitivity and by the inability to interact with CoA or CoR. In this conformation, H12 restricts CoR interaction as a function of the length and not the specific sequence of H12. This is in sharp contrast to H12 of other receptors, such as TR and RAR, where H12 does not restrict CoR interaction (8). Thus, not all apo-NHR conformations are the same. Moreover, the crystal structure of apo-RXR cannot be used to reliably predict the structure of other apo-NHRs.

Deletion of H12 unmasks RXR's repression function. The repression function of RXR is similar to that of other NHRs, in that an intact CoR box is required. Deletion of H12 alters the conformation of the receptor in such a way as to provide access of CoRs to the appropriate interaction surfaces in apo-RXR. Unlike other functions of H12, which depend upon the sequence of the amphipathic  helix, the ability of RXR H12 to mask the repression surface is primarily determined by the length of H12. Furthermore, introducing RXR H12 into TR does not block repression (24a). These data clearly indicate that regulation of repression by H12 is primarily steric, rather than allosteric. This argues against the allosteric model suggested by Schulman et al. (44), in which the activation function of RXR H12 neutralizes its repression function. The modeling analysis shown in Fig. 2G provides a plausible explanation for the steric basis of the H12 mask.

Molecular mechanism for heterodimerization-mediated unmasking of RXR's repression function. The more physiological mechanism that we have identified for repositioning H12 in RXR is heterodimerization with TR. Interaction of RXR H12 with the CoA binding surface of TR unmasks the CoR interaction surface of RXR by removing steric hindrance (Fig. 8). This mechanism requires the sequence of the RXR H12 to be specifically recognized by the heterodimer partner. It is likely that RAR behaves similarly, since biochemical studies and molecular modeling suggest that H12 of RXR can interact with the CoA binding pocket of RAR (58). The interaction between RXR H12 and TR (or RAR) is weaker than that between the main heterodimerization interfaces. This may allow reversible interaction between RXR H12 and TR to occur without disrupting the DNA binding of the heterodimer, which mediates activation as well as repression. The concept that RXR differentially interacts with unliganded and liganded TR is supported by the observation that the TR CoR box is required for RXR interaction in the absence but not in the presence of T3 (12, 66). Here we have shown that T3 binding releases RXR H12 by a TR H12-dependent mechanism, thereby contributing to the release of CoR from the heterodimer (Fig. 8).

View larger version (28K): [in this window] [in a new window]   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).

Regulation of repression by RXR. PPAR interacts with both N-CoR and SMRT in solution, albeit weakly (41). This is consistent with the observation that apo-PPAR itself is in an intermediate conformation, as suggested by structural studies (38, 54) as well as protease assays (49). PPAR-RXR heterodimers bind CoRs extremely weakly when bound to a PPRE (64), and here we show that deletion of H12 from RXR (or both RXR and PPAR) dramatically enhances the CoR binding affinity of the PPAR-RXR heterodimer. This correlates with the lack of detectable interaction between RXR H12 and PPAR (Fig. 7 and reference 58). The failure of RXR H12 to interact with PPAR explains the inability of the wild-type PPAR-RXR heterodimer to recruit CoRs to the PPRE. Consistent with this, the PPAR-RXR heterodimer does not repress transcription from a PPRE (64). A recent report suggests that microinjection of antibody to SMRT reverses the inhibitory effect of activated Ras on ligand-dependent PPAR activation (29). However, the effect of apo-PPAR on basal expression of a PPRE-containing reporter gene was not analyzed in that study. Indeed, to our knowledge, there is no published report of PPAR significantly repressing basal transcription when bound to a heterodimer binding site. The present results suggest that PPAR's lack of repressive function is due to the inability of PPAR to interact with RXR H12, coupled with the reduced inherent CoR binding affinity relative to TR and RAR.