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Molecular and Cellular Biology, September 1999, p. 6448-6457, Vol. 19, No. 9
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A Novel Role for Helix 12 of Retinoid X
Receptor in Regulating Repression
Jinsong
Zhang,1,2
Xiao
Hu,1 and
Mitchell A.
Lazar1,3,4,*
Departments of
Medicine,1
Biochemistry,2 and
Genetics3 and The Penn Diabetes
Center,4 University of Pennsylvania School
of Medicine, Philadelphia, Pennsylvania 19104
Received 7 May 1999/Returned for modification 21 June 1999/Accepted 24 June 1999
 |
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
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.
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
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 PPAR
2 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 RXR
449 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).
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RESULTS |
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 RXR
443 abrogated repression (Fig. 1E) as well
as CoR interaction with RXR (Fig. 1F). This result indicated that the
CoR interaction surface in RXR
443 is similar to that in TR and RAR,
although in the case of RXR it is obscured by H12.

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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 LBD 443 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 RXR 443 and N-CoR in vivo. Mammalian
two-hybrid assay with Gal4-RXR or Gal4-RXR 443 as bait and VP16-N-CoR
as prey. (C) Enhanced interaction between RXR 443 and N-CoR in vitro:
GST pull-down of RXR or RXR 443 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 RXR 443 and CoR: mammalian
two-hybrid assay with Gal4-RXR 443 or Gal4-RXR 443(AEV) as bait and
VP16-SMRT as prey.
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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 RXR
449 (designated RXR
449-A1 through -A7). An extension of one to three alanine residues did not
block potent repression by RXR
449 (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.

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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-RXR 449 or Gal4-RXR 449-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-RXR 449 (designated RXR 449-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 RXR 449
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.
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In the solved apo-RXR crystal structure, H12 makes multiple tight
contacts with the

loop between H1 and H3 (
5). Since
the
CoR box is located in H1, we reasoned that unmasking of the
CoR
interaction surface by deletion of H12 might be due to a conformational
change in the

loop, which is flipped outward in apo-RXR (Fig.
2G,
left). As previously noted (
5), this position of the
loop is constrained by amino acid clashes between the

loop and
H12,
especially the fourth amino acid, whose side chain sticks
out toward
the

loop and makes van der Waals and hydrogen bonding
contacts with
N262 (
d = 2.91 Å). By contrast, modeling studies
predict that the

loop would be flipped down in the conformation
of
lowest free energy for RXR

449 (Fig.
2G, right). This analysis,
although containing numerous assumptions, is based upon the solved
apo-RXR structure and provides a plausible explanation for why
RXR

449A3 but not RXR

449A4 is able to interact with
CoR.
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-RXR
443 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).

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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-RXR 443 and
RXR-F313A.
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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).

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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.
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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 RXR
443. 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).

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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-RXR 449 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-RXR 449A7 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).
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Since the protease sensitivities of apo-RXR with and without H12 are
distinguishable, we next used the protease assay to probe
the
conformational change in RXR induced by TR heterodimerization.
The
presence of apo-TR reduced the proteolytic stability of the
C-terminal
RXR fragment characteristic of the aporeceptor conformation
that does
not bind CoR (Fig.
5C; compare lanes 2 and 3). This
result indicates
that interaction with TR repositions RXR H12
to unmask the CoR
interaction surface (Fig.
5D). This predicts
that the ability of TR to
reposition H12 should be sequence specific.
Thus, we tested for
complementation of TR repression by RXR

449-A7.
This H12 sequence
mutant differed from wild-type RXR in that it
failed to rescue TR(AHT)
either for repression (Fig.
5E) or for
CoR interaction (Fig.
5F). Thus,
the specific sequence of H12
is an important determinant of CoR binding
to the TR-RXR
heterodimer.
T3 binding releases CoR from TR-RXR heterodimers. The strong TR-RXR
interaction mediated by H10 and H11 (the "ninth heptad")
is ligand
independent, but we hypothesized that the weak interaction
of RXR H12
with TR might be sensitive to ligand-induced changes
in the
conformation of TR. This was tested in the protease sensitivity
assay.
Indeed, the ability of TR heterodimerization to allosterically
promote
the "unmasked" conformation of RXR was reversed by T3
(Fig.
5C,
compare lanes 3 and 4), indicating that H12 does not
interact with
holo-TR. We next used an H12 deletion mutant (

AF2,
missing the last
nine amino acids) and an H12 point (E403A) mutant
of Gal4-TR(AHT) to
test the hypothesis that repositioning of TR
H12 in the presence of T3
is involved in the undocking of RXR
H12 and thus the release of CoR
from the TR-RXR heterodimer (Fig.
5G). T3 does not activate either TR
mutant because of the alterations
in H12 that prevent CoA binding.
Because of their CoR box mutations,
these TR mutants only weakly
repress transcription. However, Gal4-RXR
can rescue this defect (Fig.
5H). Addition of T3 to the Gal4-RXR-Gal4-TR(AHT)

AF2
dimer has no
effect on repression. The concentration of T3 used
(1 µM) was well
above the
Kd of the mutant
Gal4-TR

1(AHT)

AF2
(the measured
Kd was 27 nM). By contrast, addition of T3 to the
Gal4-RXR-Gal4-TR(AHT)E403A
dimer relieves repression. This suggests
that the T3-dependent switch
in position of TR H12 (which is deleted
in the

AF2 mutant) prevents
RXR H12 from interacting with TR,
thereby remasking the RXR repression
function. Thus, RXR H12 differentially
recognizes the liganded and
unliganded conformations of TR and
this switch regulates the repression
of the TR-RXR
heterodimer.
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).

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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).
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RXR H12 contains a sequence similar to the LXXLL motif (LXXML in RXR)
that is important for CoA interaction with a hydrophobic
cleft,
comprised of key amino acids in H3, H4, H5, and H12 in
TR
(
15). We therefore reasoned that this surface might also
be
the TR interaction site of RXR H12. To test this, we created
a mutation
in H5 [TR(AHT)I248R; Fig.
6D], corresponding to the
I302R mutation in
TR

that has been shown to bind T3 normally
but to be defective in
CoA binding and activation (
16). As predicted,
wild-type RXR
could not rescue this mutant in either repression
(Fig.
6E) or CoR
interaction (Fig.
6F). These data indicate that
a sequence-specific
interaction between H12 of RXR and the hydrophobic
cleft of apo-TR is
responsible for unmasking the CoR interaction
domain of RXR and thus
allows the TR-RXR heterodimer to repress
transcription.
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).

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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 -RXR 443 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.
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The PPAR

-RXR

443 heterodimer, unlike wild-type
PPAR

-RXR heterodimers, was able to interact with N-CoR on a PPAR
response
element (PPRE) (Fig.
7D). Interestingly, deleting AF2 from
both
PPAR

and RXR increased CoR binding on DNA even further.
Consistent
with this, deletion of PPAR

H12 increased repression,
especially
in the context of Gal4 fusion proteins (data not shown).
This
is not surprising in light of the apo-PPAR

structure, in which
the position of H12 appears to be similar to that in the
holoreceptor.
 |
DISCUSSION |
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).

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|
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.
The complexity of NHR function allows ligand, receptor, and target gene
specificity while retaining generally successful strategies
for
DNA-binding and transcriptional regulation. Numerous NHRs
utilize RXR
heterodimerization as a mechanism for enhancing DNA
binding affinity.
Here we have shown that repression by RXR heterodimers
is restricted to
partners like TR and RAR that can dock with RXR
H12 and unmask a latent
repression function in apo-RXR. Thus,
RXR governs partner-specific
repression, in addition to target
gene recognition (
40,
53,
65) and the capacity of the partner's
ligand to synergize with
RXR ligands (
11,
45,
46,
58,
59). The ability of RXR to
dictate receptor-specific functions
has enabled NHRs to share a
successful strategy for target gene
recognition while permitting
diverse ligands to have quantitatively
and qualitatively different
effects on gene
transcription.
 |
ACKNOWLEDGMENTS |
We thank Clarice Chen and Myles Brown for reading the manuscript
and for valuable discussions.
This work was supported by NIH grants DK43806 and DK45586 (to M.A.L.).
DNA sequencing was performed by the University of Pennsylvania sequencing facility, supported in part by the University of
Pennsylvania Center for the Molecular Study of Digestive Diseases (P30
DK50306.)
J.Z. and X.H. contributed equally to this work.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: University of
Pennsylvania School of Medicine, 611 CRB, 415 Curie Blvd.,
Philadelphia, PA 19104-6149. Phone: (215) 898-0198. Fax: (215)
898-5408. E-mail: lazar{at}mail.med.upenn.edu.
 |
REFERENCES |
| 1.
|
Allan, G. F.,
X. Leng,
S. Y. Tsai,
N. L. Weigel,
D. P. Edwards,
M. J. Tsai, and B. W. O'Malley.
1992.
Hormone and antihormone induce distinct conformational changes which are central to steroid receptor activation.
J. Biol. Chem.
267:19513-19520[Abstract/Free Full Text].
|
| 2.
|
Alland, L.,
R. Muhle,
H. Hou,
J. Potes,
L. Chin,
N. Schreiber-Agus, and R. A. DePinho.
1997.
Role for N-CoR and histone deacetylase in Sin3-mediated transcriptional repression.
Nature
387:49-55[Medline].
|
| 3.
|
Baniahmad, A.,
A. C. Kohne, and R. Renkawitz.
1992.
A transferable silencing domain is present in the thyroid hormone receptor, in the v-erbA onco-gene product and in the retinoic acid receptor.
EMBO J.
11:1015-1023[Medline].
|
| 4.
|
Baniahmad, A.,
X. Leng,
T. P. Burris,
S. Y. Tsai,
M.-J. Tsai, and B. W. O'Malley.
1995.
The 4 activation domain of the thyroid hormone receptor is required for release of a putative corepressor(s) necessary for transcriptional silencing.
Mol. Cell. Biol.
15:76-86[Abstract].
|
| 5.
|
Bourguet, W.,
M. Ruff,
P. Chambon,
H. Gronemeyer, and D. Moras.
1995.
Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-alpha.
Nature
375:377-382[Medline].
|
| 6.
|
Brent, G. A.,
M. K. Dunn,
J. W. Harney,
T. Gulick,
P. R. Larsen, and D. D. Moore.
1989.
Thyroid hormone aporeceptor represses T3-inducible promoters and blocks activity of the retinoic acid receptor.
New Biol.
1:329-336[Medline].
|
| 7.
|
Cavailles, V.,
S. Dauvois,
P. S. Danielian, and M. G. Parker.
1994.
Interaction of proteins with transcriptionally active estrogen receptors.
Proc. Natl. Acad. Sci. USA
91:10009-10013[Abstract/Free Full Text].
|
| 8.
|
Chen, J. D., and R. M. Evans.
1995.
A transcriptional co-repressor that interacts with nuclear hormone receptors.
Nature
377:454-457[Medline].
|
| 9.
|
Chen, J. D.,
K. Umesono, and R. M. Evans.
1996.
SMRT isoforms mediate repression and anti-repression of nuclear receptor heterodimers.
Proc. Natl. Acad. Sci. USA
93:7567-7571[Abstract/Free Full Text].
|
| 10.
|
Chen, J.-Y.,
S. Penco,
J. Ostrowski,
P. Balaguer,
M. Pons,
J. E. Starrett,
P. Reczek,
P. Chambon, and H. Gronmeyer.
1995.
RAR-specific agonist/antagonists which dissociate transactivation and AP1 transrepression inhibit anchorage-independent cell proliferation.
EMBO J.
14:1187-1197[Medline].
|
| 11.
|
Chen, J. Y.,
J. Clifford,
C. Zusi,
J. Starrett,
D. Tortolani,
J. Ostrowski,
P. R. Reczek,
P. Chambon, and H. Gronemeyer.
1996.
Two distinct actions of retinoid-receptor ligands.
Nature
382:819-822[Medline].
|
| 12.
|
Collingwood, T. N.,
A. Butler,
Y. Tone,
R. J. Clifton-Bligh,
M. G. Parker, and V. K. Chatterjee.
1997.
Thyroid hormone-mediated enhancement of heterodimer formation between thyroid hormone receptor and retinoid X receptor.
J. Biol. Chem.
272:13060-13065[Abstract/Free Full Text].
|
| 13.
|
Damm, K.,
C. C. Thompson, and R. M. Evans.
1989.
Protein encoded by v-erbA functions as a thyroid-hormone receptor antagonist.
Nature
339:593-597[Medline].
|
| 14.
|
Danielian, P. S.,
R. White,
J. A. Lees, and M. G. Parker.
1992.
Identification of a conserved region required for hormone dependent transcriptional activation by steroid hormone receptors.
EMBO J.
11:1025-1033[Medline].
|
| 15.
|
Darimont, B. D.,
R. L. Wagner,
J. W. Apriletti,
M. R. Stallcup,
P. J. Kushner,
J. D. Baxter,
R. J. Fletterick, and K. R. Yamamoto.
1998.
Structure and specificity of nuclear receptor-coactivator interactions.
Genes Dev.
12:3343-3356[Abstract/Free Full Text].
|
| 16.
|
Feng, W.,
R. C. J. Ribeiro,
R. L. Wagner,
H. Nguyen,
J. W. Apriletti,
R. J. Fletterick,
J. D. Baxter,
P. J. Kushner, and B. L. West.
1998.
Hormone-dependent coactivator binding to a hydrophobic cleft on nuclear receptors.
Science
280:1747-1749[Abstract/Free Full Text].
|
| 17.
|
Gelmetti, V.,
J. Zhang,
M. Fanelli,
S. Minucci,
P. G. Pelicci, and M. A. Lazar.
1998.
Aberrant recruitment of the nuclear receptor corepressor-histone deacetylase complex by the acute myeloid leukemia fusion partner ETO.
Mol. Cell. Biol.
18:7185-7191[Abstract/Free Full Text].
|
| 18.
|
Glass, C. K.,
D. W. Rose, and M. G. Rosenfeld.
1997.
Nuclear receptor coactivators.
Curr. Opin. Cell Biol.
9:222-232[Medline].
|
| 19.
|
Graupner, G.,
K. N. Wills,
M. Tzukerman,
X.-K. Zhang, and M. Pfahl.
1989.
Dual regulatory role for thyroid-hormone receptors allows control of retinoic-acid receptor activity.
Nature
340:653-656[Medline].
|
| 20.
|
Guex, N., and M. C. Peitsch.
1997.
SWISS-MODEL and the Swiss-Pdb viewer: an environment for comparative protein modeling.
Electrophoresis
18:2714-2723[Medline].
|
| 21.
|
Halachmi, S.,
E. Marden,
G. Martin,
H. MacKay,
C. Abbondanza, and M. Brown.
1994.
Estrogen receptor-associated proteins: possible mediators of hormone-induced transcription.
Science
264:1455-1458[Abstract/Free Full Text].
|
| 22.
|
Hassig, C. A., and S. L. Schreiber.
1998.
Nuclear histone acetylases and deacetylases and transcriptional regulation: HATs off to HDACs.
Curr. Opin. Chem. Biol.
1:300-308.
|
| 23.
|
Heinzel, T.,
R. M. Lavinsky,
T.-M. Mullen,
M. Soderstrom,
C. D. Laherty,
J. Torchia,
W.-M. Yuang,
G. Brard,
S. D. Ngo,
J. R. Davie,
E. Seto,
R. N. Eisenman,
D. W. Rose,
C. K. Glass, and M. G. Rosenfeld.
1997.
A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression.
Nature
387:43-48[Medline].
|
| 24.
|
Horlein, A. J.,
A. M. Naar,
T. Heinzel,
J. Torchia,
B. Gloss,
R. Kurokawa,
A. Ryan,
Y. Kamei,
M. Soderstrom,
C. K. Glass, and M. G. Rosenfeld.
1995.
Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor.
Nature
377:397-404[Medline].
|
| 24a.
| Hu, X., and M. A. Lazar. Unpublished results.
|
| 25.
|
Jeannin, E.,
D. Robyr, and B. Desvergne.
1998.
Transcriptional regulatory patterns of the myelin basic protein and malic enzyme genes by the thyroid hormone receptors 1 and 1.
J. Biol. Chem.
273:24239-24248[Abstract/Free Full Text].
|
| 26.
|
Kadosh, D., and K. Struhl.
1998.
Targeted recruitment of the Sin3-Rpd3 histone deacetylase complex generates a highly localized domain of repressed chromatin in vivo.
Mol. Cell. Biol.
18:5121-5127[Abstract/Free Full Text].
|
| 27.
|
Koradi, R.,
M. Billeter, and K. Wuthrich.
1996.
MOLMOL: a program for display and analysis of macromolecular structures.
J. Mol. Graphics
14:51-55[Medline].
|
| 28.
|
Kurokawa, R.,
M. Soderstrom,
A. Horlein,
S. Halachmi,
M. Brown,
M. G. Rosenfeld, and C. K. Glass.
1995.
Polarity-specific activities of retinoic acid receptors determined by a co-repressor.
Nature
377:451-454[Medline].
|
| 29.
|
Lavinsky, R. M.,
J. Kristen,
H. Thorsten,
J. Torchia,
T.-M. Mullen,
R. Schiff,
A. L. Del-Rio,
M. Ricote,
S. Ngo,
J. Gemsch,
S. G. Hilsenbeck,
C. K. Osborne,
C. K. Glass, and M. G. Rosenfeld.
1998.
Diverse signaling pathways modulate nuclear receptor recruitment of N-CoR and SMRT complexes.
Proc. Natl. Acad. Sci. USA
95:2920-2925[Abstract/Free Full Text].
|
| 30.
|
Leng, X.,
J. Blanco,
S. Y. Tsai,
K. Ozato,
B. W. O'Malley, and M.-J. Tsai.
1995.
Mouse retinoid X receptor contains a separable ligand-binding and transactivation domain in its E region.
Mol. Cell. Biol.
15:255-263[Abstract].
|
| 31.
|
Lin, B. C.,
S. H. Hong,
S. Krig,
S. M. Yoh, and M. L. Privalsky.
1997.
A conformational switch in nuclear hormone receptors is involved in coupling hormone binding to corepressor release.
Mol. Cell. Biol.
17:6131-6138[Abstract].
|
| 32.
|
Lin, R. J.,
L. Nagy,
S. Inoue,
W. Shao,
W. H. Miller, and R. M. Evans.
1998.
Role of the histone deacetylase complex in acute promyelocytic leukaemia.
Nature
391:811-814[Medline].
|
| 33.
|
Lutterbach, B.,
J. J. Westendorf,
B. Linggi,
A. Patten,
M. Moniwa,
J. R. Davie,
K. D. Huynh,
V. J. Bardwell,
R. M. Lavinsky,
M. G. Rosenfeld,
C. Glass,
E. Seto, and S. W. Hiebert.
1998.
ETO, a target of t(8;21) in acute leukemia, interacts with the N-CoR and mSin3 corepressors.
Mol. Cell. Biol.
18:7176-7184[Abstract/Free Full Text].
|
| 34.
|
Mangelsdorf, D. J., and R. M. Evans.
1995.
The RXR heterodimers and orphan receptors.
Cell
83:841-850[Medline].
|
| 35.
|
Marks, M. S.,
P. L. Hallenback,
T. Nagata,
J. H. Segars,
E. Appella,
V. M. Nikodem, and K. Ozato.
1992.
H-2RIIBP (RXR ) dimerization provides a mechanism for combinatorial diversity in the regulation of retinoic acid and thyroid hormone responsive genes.
EMBO J.
11:1419-1435[Medline].
|
| 36.
|
Martin, B.,
R. Renkawitz, and M. Muller.
1994.
Two silencing sub-domains of v-erbA synergize with each other, but not with RXR.
Nucleic Acids Res.
22:4899-4905.
|
| 37.
|
Nagy, L.,
H.-Y. Kao,
D. Chakvarkti,
R. J. Lin,
C. A. Hassig,
D. E. Ayer,
S. L. Schreiber, and R. M. Evans.
1997.
Nuclear receptor repression mediated by a complex containing SMRT, mSin3A, and histone deacetylase.
Cell
89:373-380[Medline].
|
| 38.
|
Nolte, R. T.,
G. B. Wisely,
S. Westin,
J. E. Cobb,
M. H. Lambert,
R. Kurokawa,
M. G. Rosenfeld,
T. M. Willson,
C. K. Glass, and M. V. Milburn.
1998.
Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor- .
Nature
395:137-143[Medline].
|
| 39.
|
Qi, J.-S.,
V. Desai-Yajnik,
M. E. Greene,
B. M. Raaka, and H. H. Samuels.
1995.
The ligand-binding domains of the thyroid hormone/retinoid receptor gene subfamily function in vivo to mediate heterodimerization, gene silencing, and transactivation.
Mol. Cell. Biol.
15:1817-1825[Abstract].
|
| 40.
|
Rastinejad, F.,
T. Perlmann,
R. M. Evans, and P. B. Sigler.
1995.
Structural determinants of nuclear receptor assembly on DNA direct repeats.
Nature
375:203-211[Medline].
|
| 41.
|
Reginato, M. J.,
S. T. Bailey,
S. L. Krakow,
C. Minami,
S. Ishii, and H. Takaka.
1998.
A potent antidiabetic thiazolidinedione with unusual PPAR -activating properties.
J. Biol. Chem.
273:32679-32684[Abstract/Free Full Text].
|
| 42.
|
Renaud, J.-P.,
N. Rochel,
M. Ruff,
V. Vivat,
P. Chambon,
H. Gronemeyer, and D. Moras.
1995.
Crystal structure of the RAR ligand-binding domain bound to all-trans retinoic acid.
Nature
378:681-689[Medline].
|
| 43.
|
Sande, S., and M. L. Privalsky.
1996.
Identification of TRACs, a family of co-factors that associate with and modulate the activity of nuclear hormone receptors.
Mol. Endocrinol.
10:813-825[Abstract/Free Full Text].
|
| 44.
|
Schulman, I. G.,
H. Juguilon, and R. M. Evans.
1996.
Activation and repression by nuclear hormone receptors: hormone modulates an equilibrium between active and repressive states.
Mol. Cell. Biol.
16:3807-3813[Abstract].
|
| 45.
|
Schulman, I. G.,
C. Li,
J. W. R. Schwabe, and R. M. Evans.
1997.
The phantom ligand effect: allosteric control of transcription by the retinoid X receptor.
Genes Dev.
11:299-308[Abstract/Free Full Text].
|
| 46.
|
Schulman, I. G.,
G. Shao, and R. A. Heyman.
1998.
Transactivation by retinoid X receptor-peroxisome proliferator-activated receptor (PPAR ) heterodimers: intermolecular synergy requires only the PPAR hormone-dependent activation function.
Mol. Cell. Biol.
18:3483-3494[Abstract/Free Full Text].
|
| 47.
|
Seol, W.,
H. S. Choi, and D. D. Moore.
1995.
Isolation of proteins that interact specifically with the retinoid X receptor: two novel orphan receptors.
Mol. Endocrinol.
9:72-85[Abstract/Free Full Text].
|
| 48.
|
Seol, W.,
M. J. Mahon,
Y.-K. Lee, and D. D. Moore.
1996.
Two receptor interacting domains in the nuclear hormone receptor corepressor RIP13/N-CoR.
Mol. Endocrinol.
10:1646-1655[Abstract/Free Full Text].
|
| 49.
|
Shao, D.,
S. M. Rangwala,
S. T. Bailey,
S. L. Krakow,
M. J. Reginato, and M. A. Lazar.
1998.
Interdomain communication regulating PPAR ligand binding.
Nature
396:377-380[Medline].
|
| 50.
|
Shibata, H.,
T. E. Spencer,
S. A. Onate,
G. Jenster,
S. Y. Tsai,
M. J. Tsai, and B. W. O'Malley.
1997.
Role of co-activators and co-repressors in the mechanism of steroid/thyroid receptor action.
Rec. Prog. Horm. Res.
52:141-164.
|
| 51.
|
Tagami, T., and J. L. Jameson.
1998.
Nuclear corepressors enhance the dominant negative activity of mutant receptors that cause resistance to thyroid hormone.
Endocrinology
139:640-650[Abstract/Free Full Text].
|
| 52.
|
Tanenbaum, D. M.,
Y. Wang,
S. P. Williams, and P. B. Sigler.
1998.
Crystallographic comparison of the estrogen and progesterone receptor's ligand binding domains.
Proc. Natl. Acad. Sci. USA
95:5998-6003[Abstract/Free Full Text].
|
| 53.
|
Umesono, K.,
K. K. Murakami,
C. C. Thompson, and R. M. Evans.
1991.
Direct repeats as selective response elements for the thyroid hormone, retinoic acid, and vitamin D3 receptors.
Cell
65:1255-1266[Medline].
|
| 54.
|
Uppenberg, J.,
S. Svensson,
M. Jaki,
G. Bertilsson,
L. Jendeberg, and A. Berkenstam.
1998.
Crystal structure of the ligand binding domain of the human nuclear receptor PPAR .
J. Biol. Chem.
273:31108-31112[Abstract/Free Full Text].
|
| 55.
|
Vivat, V.,
C. Zechel,
J. M. Wurtz,
W. Bourguet,
H. Kagechika,
H. Umemiya,
K. Shudo,
D. Moras,
H. Gronemeyer, and P. Chambon.
1997.
A mutation mimicking ligand-induced conformational change yields a constitutive RXR that senses allosteric effects in heterodimers.
EMBO J.
16:5697-5709[Medline].
|
| 56.
|
Wagner, R. L.,
J. W. Apriletti,
M. E. McGrath,
B. L. West,
J. D. Baxter, and R. J. Fletterick.
1995.
A structural role for hormone in the thyroid hormone receptor.
Nature
378:690-697[Medline].
|
| 57.
|
Wang, J.,
T. Hoshino,
R. L. Redner,
S. Kajigaya, and J. M. Liu.
1998.
ETO, fusion partner in t(8;21) acute myeloid leukemia, represses transcription by interaction with the human N-CoR/mSin3/HDAC1 complex.
Proc. Natl. Acad. Sci. USA
95:10860-10865[Abstract/Free Full Text].
|
| 58.
|
Westin, S.,
R. Kurokawa,
R. T. Nolte,
G. B. Wisely,
E. M. McInerney,
D. W. Rose,
M. V. Milburn,
M. G. Rosenfeld, and C. K. Glass.
1998.
Interactions controlling the assembly of nuclear receptor heterodimers and co-activators.
Nature
395:199-202[Medline].
|
| 59.
|
Willy, P. J.,
K. Umesono,
E. S. Ong,
R. M. Evans,
R. A. Heyman, and D. J. Mangelsdorf.
1995.
LXR, a nuclear receptor that defines a distinct retinoid response pathway.
Genes Dev.
9:1033-1045[Abstract/Free Full Text].
|
| 60.
|
Wong, C.-W., and M. L. Privalsky.
1998.
Transcriptional silencing is defined by isoform- and heterodimer-specific interactions between nuclear hormone receptors and corepressors.
Mol. Cell. Biol.
18:5724-5733[Abstract/Free Full Text].
|
| 61.
|
Wurtz, J. M.,
W. Bourguet,
J. P. Renaud,
V. Vivat,
P. Chambon,
D. Moras, and H. Gronemeyer.
1996.
A canonical structure for the ligand binding domain of nuclear receptors.
Nat. Struct. Biol.
3:87-94[Medline].
|
| 62.
|
Zamir, I.,
J. Dawson,
R. M. Lavinsky,
C. K. Glass,
M. G. Rosenfeld, and M. A. Lazar.
1997.
Cloning and characterization of a corepressor and potential component of the nuclear hormone receptor repression complex.
Proc. Natl. Acad. Sci. USA
94:14400-14405[Abstract/Free Full Text].
|
| 63.
|
Zamir, I.,
H. P. Harding,
G. B. Atkins,
A. Horlein,
C. K. Glass,
M. G. Rosenfeld, and M. A. Lazar.
1996.
A nuclear hormone receptor corepressor mediates transcriptional silencing by receptors with different repression domains.
Mol. Cell. Biol.
16:5458-5465[Abstract].
|
| 64.
|
Zamir, I.,
J. Zhang, and M. A. Lazar.
1997.
Stoichiometric and steric principles governing repression by nuclear hormone receptors.
Genes Dev.
11:835-846[Abstract/Free Full Text].
|
| 65.
|
Zechel, C.,
X. Q. Shen,
P. Chambon, and H. Gronemeyer.
1994.
Dimerization interfaces formed between the DNA binding domains determine the cooperative binding of RXR/RAR and RXR/TR heterodimers to DR5 and DR4 elements.
EMBO J.
13:1414-1424[Medline].
|
| 66.
|
Zhang, J.,
I. Zamir, and M. A. Lazar.
1997.
Differential recognition of liganded and unliganded thyroid hormone receptor by retinoid X receptor regulates transcriptional repression.
Mol. Cell. Biol.
17:6887-6897[Abstract].
|
Molecular and Cellular Biology, September 1999, p. 6448-6457, Vol. 19, No. 9
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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-
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-
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