An Inhibitory Region of the DNA-Binding Domain of Thyroid Hormone Receptor Blocks Hormone-Dependent Transactivation
Ying Liu1,
Akira Takeshita,
Takashi Nagaya,
Aria Baniahmad,
William W. Chin and
Paul M. Yen1
Division of Genetics, Department of Medicine Brigham and
Womens Hospital and Harvard Medical School Boston,
Massachusetts 02115
Department of Endocrinology and
Metabolism (T.N.) Research Institute of Environmental
Medicine Nagoya University Nagoya, Japan
Genetisches
Institut (A.B.) Justus-Liebig-Universitat D-35392, Giessen,
Germany
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ABSTRACT
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We have employed a chimeric receptor system in
which we cotransfected yeast GAL4 DNA-binding domain/retinoid X
receptor ß ligand-binding domain chimeric receptor (GAL4RXR), thyroid
hormone receptor-ß (TRß), and upstream activating sequence-reporter
plasmids into CV-1 cells to study repression, derepression, and
transcriptional activation. In the absence of
T3, unliganded TR repressed transcription to
20% of basal level, and in the presence of T3,
liganded TRß derepressed transcription to basal level. Using this
system and a battery of TRß mutants, we found that TRß/RXR
heterodimer formation is necessary and sufficient for basal repression
and derepression in this system. Additionally, an AF-2 domain mutant
(E457A) mediated basal repression but not derepression, suggesting that
interaction with a putative coactivator at this site may be critical
for derepression. Interestingly, a mutant containing only the TRß
ligand binding domain (LBD) not only mediated derepression, but also
stimulated transcriptional activation 10-fold higher than basal level.
Studies using deletion and domain swap mutants localized an inhibitory
region to the TRß DNA-binding domain. Titration studies further
suggested that allosteric changes promoting interaction with
coactivators may account for enhanced transcriptional activity by LBD.
In summary, our findings suggest that TR heterodimer formation with RXR
is important for repression and derepression, and coactivator
interaction with the AF-2 domain may be needed for derepression in this
chimeric system. Additionally, there may be an inhibitory region in the
DNA-binding domain, which reduces TR interaction with coactivators, and
prevents full-length wild-type TRß from achieving transcriptional
activation above basal level in this chimeric receptor system.
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INTRODUCTION
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Thyroid hormone receptors (TRs) are nuclear hormone receptors that
regulate transcription of target genes by binding to thyroid hormone
response elements (TREs) in their promoter regions. As such, they
function as ligand-regulatable transcription factors which can activate
transcription of positively-regulated target genes in the presence of
T3. TRs can bind to TREs as homodimers and heterodimers,
particularly with retinoid X receptors (RXRs). On the basis of DNA
binding in the presence of T3, cotransfection studies with
TR mutants, in vitro transciption studies, and yeast systems
that do not contain endogenous TR and RXR, TR/RXR heterodimers likely
are the transcriptionally active complexes involved in
T3-mediated transcriptional activation for most TREs
(1, 2, 3).
Interestingly, for several positively regulated target genes, TRs also
can repress basal transcription in the absence of ligand. Addition of
T3 relieves this basal repression and activates
transcription above basal level. We and others previously have shown
that binding of unliganded TRs to TREs, several groups have isolated
and cloned the cDNAs of proteins (corepressors) that interact with TRs
in a ligand-dependent manner such as N-CoR (nuclear receptor
corepressor) and SMRT (8, 9, 10, 11). In particular, these proteins interact
with unliganded, but not liganded, TRs. The subregion of TR that
interacts with corepressors appears to be located in the hinge region
between the DNA- and ligand-binding domains (LBDs) (8, 9). Functional
studies suggest that these putative corepressors can mediate basal
repression of transcription when associated with TRs.
In addition to corepressors, there are several putative coactivators
such as steroid receptor-coactivator-1 (SRC-1), transcriptional
intermediary factor II (TIFII)/GRIP1, P300/CBP
cointegrator-associated protein (p/CIP), and the recently described
receptor-associated coactivator-RAC3/ACTR/AIB1/thyroid hormone receptor
activator molecule-1 (TRAM-1) that interact with TRs or other members
of the nuclear hormone receptor family that may be important in
mediating ligand-dependent transcriptional activation for these
receptors (1219a). In contrast to corepressors, these proteins
selectively interact with the liganded, rather than unliganded, nuclear
hormone receptors. Mutations and deletions of a highly conserved
section of the extreme carboxy-terminal region of TRs and other nuclear
hormone receptors [activation function (AF)-2 domain] have shown that
this region is critical for mediating ligand-dependent transcription
and in some cases, interactions with putative coactivators (20, 21, 22, 23). It
is possible that this region may interact directly with coactivators or
may exert allosteric effects on TR conformation that may influence TR
interaction with coactivators. The role of the AF-2 domain in
modulating basal repression and derepression is currently not well
characterized.
Presently, little is known about the potential role of the DNA-binding
region on transcriptional activity by TRs. It appears that steric
effects mediated by DNA binding may affect retinoic acid receptor/RXR
and TR/RXR interactions with corepressors and/or coactivators (24).
Additionally, mutations in the first zinc finger of TRß still can
allow DNA binding by abrogating transcriptional activity (25). However,
the intrinsic role of the DNA-binding domain (DBD) on transcription has
not been investigated fully.
The GAL4 chimeric receptor system has been successfully employed
recently to study the specific role(s) of TR subregions in
transcriptional activation and protein-protein interactions (23, 26, 27). Using this system, and a battery of TRß mutants (Fig. 1
, A and B), we have found that TRß/RXR
heterodimers may have different transcriptional activity depending upon
which heterodimer partner binds DNA. We also found that TRß/RXR
heterodimer formation is necessary and sufficient for basal repression
and derepression in this system. Additionally, a mutation in the TR
AF-2 region did not alter basal repression but was unable to mediate
derepression, suggesting that interactions with coactivators may be
important for derepression. Surprisingly, the TRß LBD not only could
repress basal transcription but also could transactivate above basal
level in the presence of T3. Deletion and domain swap of
different regions of TRß suggest that there is a region in the second
zinc finger of TRß that may inhibit ligand-dependent transcriptional
activation by the full-length receptor by an allosteric mechanism.

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Figure 1. Chimeric Receptor System and TRß Mutants
A, Model depicting chimeric receptor system. B, Summary of TRß and
TRß mutants. Receptors were translated in vitro
according to manufacturers instructions (Promega) and then analyzed
for [125I]T3 binding or DNA binding to the F2
TRE as previously described (22, 40, 42, 50). Data are summary of
previously published and new results (40, 42, 50).
T3-dependent transactivation by these receptors was
measured via a luciferase reporter plasmid containing F2 TRE in
cotransfection experiments (40).
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RESULTS
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We first examined the transcriptional activity of GAL4RXR and
GAL4TRß in the absence or presence of their cognate ligands (Fig. 2
). 9-cis-RA stimulated
GAL4RXR-mediated transcriptional activity 25-fold, and T3
stimulated GAL4TRß transcriptional activity greater than 15-fold.
Interestingly, 9-cis RA also could stimulate
GAL4TRß-mediated transcription presumably via heterodimerization with
endogenous RXR. We previously have observed that RXRß is
predominantly expressed in CV-1 cells using isoform-specific antibodies
(Ref. 28 and P. M. Yen, unpublished results). Cotransfection of RXRß
did not further augment this 9-cis-RA effect on
GAL4-TR-mediated transcripton. In contrast, T3 did not
affect GAL4RXR-mediated transcription presumably because CV-1 cells
contain little or no endogenous TRs.

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Figure 2. Transcriptional Transactivation by GAL4 RXR and
GAL4TRß in the Presence or Absence of Ligand and/or Heterodimer
Partners
hTRß or mRXRß expression vector (0.1 µg) was cotransfected in
CV-1 cells in the absence or presence of 10-6 M
T3 or 9-cis-RA for 24 h as indicated.
In these experiments, treated cells were then harvested and luciferase
was measured. Luciferase activity was normalized to ß-galactosidase
activity and then calculated as fold basal luciferase activity with
1-fold basal activity defined as the luciferase activity with control
GAL4 and pcDNA vector alone in the absence of ligand. pcDNA vector was
added to some samples so that each sample had the same amount of total
expression vector. Each value represents the mean of four samples, and
bars denote SD of the mean.
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In the absence of ligand, GAL4TRß, but not GAL4RXR, repressed
transcription to less than 20% of basal level. When TRß was
coexpressed with GAL4RXR, similar basal repression occurred in the
absence of ligand. Thus, GAL4TRß/RXR and GAL4RXR/TRß complexes are
both able to mediate basal repression. However, in contrast to
GAL4TRß alone and GAL4TRß and RXRß, which activated transcription
in the presence of T3, GAL4RXR and TRß derepressed basal
repression in a T3-dependent manner, but did not activate
transcription significantly above basal level. Additionally, GAL4RXR
and TRß blocked 9-cis-RA-mediated transcription in the
presence or absence of T3 similar to previously reported
findings (29, 30). Thus, in this chimeric system, TRß had different
effects on RXR-mediated transcription depending on the absence or
presence of cognate ligand for either TR or RXR.
It has been difficult to separate derepression from transcriptional
activation in previous studies using conventional cotransfection
studies employing full-length receptors and TRE-containing reporters.
The previous findings thus prompted us to study in greater detail the
mechanisms of GAL4RXR- and TRß-mediated repression and
derepression. We first examined the role of heterodimerization on
repression and derepression by using TRß mutants in the ninth heptad
regions, which selectively formed homo- or heterodimers on
electrophoretic mobility shift assays (Fig. 1
). In these studies the
mutants were co-transfected with GAL4 vector as a control or GAL4RXR
vector with the upstream activating sequence (UAS)-reporter. As seen in
Fig. 3
, the hTRß and GAL4 samples had
weak basal repression and derepression in the presence of
T3, suggesting there may be a weak TRE present in the
expression vector. When cotransfected with GAL4RXR, hTRß repressed
basal transcription to 10% basal level and derepressed in the presence
of T3. Heterodimer-specific mutant R429Q behaved similar to
wild-type TRß, but homodimer-preferential mutant L428R was unable to
mediate basal repression and derepression. G345R, a natural mutant from
a patient with resistance to thyroid homone, exhibited constitutive
basal repression. TRAHTm, a TRß that contains three amino
acid substitutions in the hinge region and had decreased affinity with
the putative corepressor, N-CoR (nuclear receptor corepressor), showed
reduced basal represssion in the absence of ligand (8). These findings
suggest that heterodimerization and an intact hinge region are
important for basal repression in this system, and derepression depends
on T3 binding to TR.

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Figure 3. Repression and Derepression by GAL4RXR and hTRß
or TRß Mutants for Homodimerization (L428R), Heterodimerization
(R429Q), T3 Binding (G345R), and Basal Repression
(TRAHTM)
hTRß or hTRß mutant vectors (0.1 µg), GAL4 or GAL4RXR expression
vectors (0.1 µg), UAS-containing reporter plasmid (1.7 µg), and
ß-galactosidase control vector (1.0 µg) were cotransfected in CV-1
cells in the absence or presence of 106 M
T3 for 24 h as indicated. In these experiments,
treated cells were then harvested and luciferase was measured.
Luciferase activity was normalized to ß-galactosidase activity and
then calculated as fold basal luciferase activity with control GAL4 and
pcDNA vector alone in the absence of ligand. pcDNA vector was added to
some samples so that each sample had same amount of total expression
vector. Each value represents the mean of three to six samples, and
bars denote SD of the mean.
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We next examined the effect of point mutations in the AF-2 region
located in the extreme carboxy-terminal region of TRß on repression
and derepression (Fig. 4
). Previously,
Chatterjee and co-workers (22) showed that these mutations markedly
reduced transcriptional activation. As seen in Fig. 2
, both E457A and
E457D were able to mediate basal repression in the absence of ligand.
However, in the presence of T3, E457D, which contains a
more conservative amino acid substitution, derepressed to basal
transcription level, but E457A did not. These findings suggest that
mutations in the AF-2 region not only affect transcriptional activation
but also may affect derepression and thus lead to constitutive basal
repression.

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Figure 4. Repression, Derepression, and Transcriptional
Activation by GAL4RXR and rTRß, AF-2 Domain rTRß Mutants (E457A,
E457D), or LBD Mutants
rTRß, AF-2 domain rTRß mutants, or rTRß LBD vectors (0.1 µg),
GAL4 or GAL4RXR expression vectors (0.1 µg), UAS-containing reporter
plasmid (1.7 µg), and ß-galactosidase control vector (1.0 µg)
were cotransfected in CV-1 cells in the absence or presence of
10-6 M T3 for 24 h as
indicated. In these experiments, treated cells were then harvested and
luciferase was measured. Luciferase activity was normalized to
ß-galactosidase activity and then calculated as fold basal luciferase
activity with 1-fold basal activity defined as the luciferase activity
with control GAL4 and pcDNA vector alone in the absence of ligand.
pcDNA vector was added to some samples so that each sample had same
amount of total expression vector. Each value represents the mean of
four samples, and bars denote SD of the
mean.
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We also examined the effects of the TR ligand binding domain in this
system (Fig. 4
). TR-LBD (which also contains the hinge region for
nuclear localization and putative corepressor interaction sites) was
able to mediate basal repression in the absence of ligand.
Surprisingly, LBD not only derepressed basal repression, but also
stimulated transcription greater than 10-fold over basal level. The
LBDs that contain AF-2 mutations were able to repress basal
transcription and had markedly impaired transcriptional activation.
These findings with full-length TRß and TR-LBD suggested that there
may be an inhibitory region for T3-mediated transcriptional
activation that was located in either the amino-terminal region or DBD
of TRß. We thus studied the effects on basal repression and
transcriptional activation using a mutant TRß in which the
amino-terminal region has been deleted, TRß-
N (Fig. 5
). The receptor had decreased basal
repression but exhibited no transcriptional activation in the presence
of T3. Similar effects by TRß-
N on basal repression
recently have been reported by Hollenberg et al. (31).
Additionally, TR
, which contains a 40-amino acid amino-terminal
region that does not have homology with TRß was unable to activate
transcription in the presence of T3. Taken together, these
results suggest that the amino-terminal region is unlikely to be
mediating inhibition of transcriptional activation by full-length
TRß.

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Figure 5. Repression, Derepression, and Transcriptional
Activation by GAL4RXR and rTR , TRß, and TRß N
rRT , rTRß, and TRß N vectors (0.1 µg), GAL4 or GAL4RXR
expression vectors (0.1 µg), UAS-containing reporter plasmid (1.7
µg), and ß-galactosidase control vector (0.1 µg) were
cotransfected in CV-1 cells in the absence or presence of
10-6 M T3 for 24 h as
indicated. In these experiments, treated cells were then harvested and
luciferase measured. Luciferase activity was normalized to
ß-galactosidase activity and then calculated as fold basal luciferase
activity with 1-fold basal activity defined as the luciferase activity
with control of GAL4 and pcDNA vector alone in the absence of ligand.
pcDNA vector was added to some samples so that each sample had same
amount of total expression vector. Each value represents
the mean of four samples, and bars denote SD
of the mean.
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We next used a chimeric TR, TRß-TGT, in which the DBD has been
swapped with the corresponding domain from the human glucocorticoid
receptor (GR) and observed a 4-fold activation above basal
transcription level in the presence of T3 (Fig. 6A
). These findings suggested that there
may be a subregion within the DBD that may be involved in inhibiting
transcriptional activation. Accordingly, we used TRß mutants in which
each zinc finger was swapped with the corresponding zinc finger of GR
(TRß-TG and TRß-GT) and studied their abilities to activate
transcription (Fig. 6B
). TRß-TG, but not TRß-GT, was able to able
to stimulate transcription greater than 10-fold in the presence of
T3 suggesting that the inhibitory region was located in or
near the second zinc finger.

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Figure 6. Transcriptional Activation by Zinc Finger Swap
Mutants (TRß-TGT, TRß-TG, and TRß-GT)
TRß-TGT in pRSV, TRß-TG and TRß vectors (0.1 µg), GAL4 or GAL
4RXR expression vectors (0.1 µg), UAS-containing vector plasmid (1.7
µg), and ß-galactosidase control vector (1.0 µg) were
cotransfected in CV-1 cells in the absence or presence of
10-6 M T3 for 24 h as
indicated. In these experiments, treated cells were then harvested and
luciferase was measured. Luciferase activity was normalized to
ß-galactosidase activity and defined as the luciferase activity with
control GAL4 and pcDNA or pRSV vector alone in the absence of ligand.
pcDNA or pRSV vector was added to some samples so that each sample had
the same amount of total expression vector. Each value represents the
mean of four samples, and bars denote SD of
the mean. A, TRß-TGT; B, TRß-TG and TRß-GT.
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The mechanism of this inhibition could be due to allosteric changes in
the TR DBD that modulate the conformation of TR and reduce its affinity
for coactivators. Alternatively, it could be due to cellular inhibitors
that interact with this region of TR and reduce full-length TRs
affinity for coactivators. To determine whether there may be a cellular
inhibitor blocking the transcriptional activity of full-length TRß,
we cotransfected a 3-fold excess of L428R expression vector with TRß
and GAL4RXR expression vectors to examine whether it would titrate out
a putative inhibitor(s) (Fig. 7
). This
mutant forms heterodimers poorly and did not repress basal
transcription or transactivate (Figs. 1
and 3
). As seen in Fig. 7
, L428R was unable to enhance transcriptional activation by TRß,
suggesting that it was not titrating out an inhibitor. L428R also did
not affect the transcriptional activation by LBD. Even addition of a
20-fold excess of L428R had no significant effect on transcriptional
activity by TRß or LBD (data not shown). Similar results also were
obtained when we used a mutant TR containing only the TRß DBD and
hinge region in this system (data not shown). These results argue
against a titratable inhibitor interfering with the transcriptional
activation of TRß.

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Figure 7. Titration of a Putative Inhibitor by L428R Mutant
TRß vector (0.1 µg), L428R mutant (0.3 µg), GAL4, or GAL4RXR
expression vectors (0.1 µg), UAS-containing reporter plasmid (1.7
µg), and ß-galactosidase control vector (1.0 µg) were
cotransfected in CV-1 cells in the absence or presence of
10-6 M T3 for 24 h as
indicated. In these experiments, treated cells were then harvested and
luciferase measured. Luciferase activity was normalized to
ß-galactosidase activity and then calculated as fold basal luciferase
activity with 1-fold basal activity defined as the luciferase activity
with control GAL4 and pcDNA vector alone in the absence of ligand.
pcDNA vector was added to some samples so that each sample had same
amount of total expression vector. Each value represents the mean of
three samples, and bars denote SD of the
mean.
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DISCUSSION
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We have used a GAL4 chimeric receptor system to study the
mechanisms of repression, derepression, and transcriptional activation
for TRs. We observed some common features as well as differences
depending on whether GAL4TRß or GAL4RXR was used. GAL4TRß, but not
GAL4RXR, repressed basal transcription in the absence of ligand.
However, when these chimeric receptors were cotransfected with their
corresponding heterodimer partner, both repressed basal transcription.
GAL4RXR mediated 9-cis-retinoic acid-dependent
transcriptional activation; however, similar to previous studies, TRß
partially blocked the amount of 9-cis-RA-dependent
transcriptional activation by GAL4RXR (29, 30).
9-cis-RA also activated transcription by GAL4TRß
(presumably via endogenous RXR heterodimerized with GAL4TRß) and
GAL4TRß and RXR. The amount of transcriptional activation by
9-cis-RA was similar to that observed for the reciprocal
complex of GAL4RXR and TRß. GAL4TRß or GAL4TRß and RXRß
activated transcription in the presence of T3. In contrast,
GAL4RXR and TRß only derepressed transcription in the presence of
T3. These findings suggest that there may be different
effects on derepression and transcriptional activation depending on
which heterodimer partner is bound to DNA and/or conformational
differences between GAL4RXR/TRß and GAL4TRß/RXR heterodimer
complexes.
The observation that GAL4RXR and TRß repressed and derepressed in the
absence or presence of T3 suggested that this system would
enable us to study these two functions of the TR apart from
transcriptional activation. Studies using natural and artificial TRß
mutants showed that heterodimerization and an intact hinge region that
can interact with corepressors such as N-CoR are necessary for basal
repression. T3 binding is critical for derepression as a
natural TRß mutant that has minimal T3 binding exhibited
constitutive basal repression in the presence of T3.
Our studies with AF-2 mutants suggest that mutations in this region do
not significantly affect basal repression. However, one of the mutants
(E457A) was unable to derepress in the presence of T3. This
mutant has similar hormone binding affinity as wild-type TRß (22, 32). These findings suggest that mutations in the AF-2 region may
modulate derepression. It is possible that binding of coactivators may
be necessary for release of corepressors from TR, and the equilibrium
between corepressor- and coactivator-bound TR determines the amount of
basal repression, derepression, and transcriptonal activation. Recent
work by Baniahmad et al. (23) and Schulman et al.
(27) also support this possibility.
TR-LBD, which contains the hinge region to ensure nuclear
localization, was able to repress basal transcription in this system.
Surprisingly, it not only derepressed basal transcription but also was
able to activate transcription in the presence of T3. When
the AF-2 region LBDs were used, there was little or no trancriptional
activation above basal level, confirming the critical role of this
region for transcriptional activation. Our data suggest the difference
between the transcriptional activity of full-length TRß and TR-LBD is
due to inhibition of transcriptional activation by a subregion near or
within the second zinc finger of the DBD. It is not known whether this
subregion inhibits transcription by TR/RXR heterodimers bound to TREs.
Indeed, when LBD is cotransfected with TRE-containing reporters, we and
others did not observe either basal repression or transcriptional
activation. However, Forman et al. (33) showed that LBD
is unable to bind as a dimer with TR or RXR to TREs suggesting that it
may not be possible to detect this inhibitory function on conventional
TRE-containing reporters.
The mechanism of this inhibition may be due to cellular inhibitors that
interact with the TR DBD or to allosteric changes in the TR DBD that
modulate the conformation of TR and reduce its affinity for
coactivators. Casanova et al. (34) have provided evidence
for a cellular inhibitor that interacts with unliganded TR in a region
that involves the ninth heptad region of the cTR
LBD and is released
when T3 binds to TR. Burris et al. (35) have
shown that a TR-interacting protein identified by two-hybrid screening
inhibits TR-mediated transcription by binding to the TR hinge and
amino-terminal region of the LBD. In contrast, our titration
experiments suggest that allosteric changes induced by the DBD, rather
than cellular inhibitors, likely account for the difference in
transcriptional activation. Furthermore, cotransfection studies in P19
embryonal carcinoma cells exhibited similar differences among the
transcriptional activities of TRß, TR LBD, and TRß-TG, as observed
in CV-1 cells, and thus argue against a cell-specific inhibitor.
Finally, we have performed far-Western blots of nuclear extracts using
32P-labeled glutathione-S-transferase (GST) rat
TRß LBD and full-length rat TRß (32). Similar to recent work by
Fondell and Roeder using a coimmunoprecipitation assay, we observed
that the TRs can interact with several different nuclear proteins in a
T3-dependent manner (36); however, GST-rat TRß LBD
interacted much more strongly with this group of nuclear proteins than
GST full-length rat TRß (A. Takeshita, unpublished data). Taken
together, these findings strongly suggest that the DBD may modulate the
conformation of the AF-2 region of TR. Recently, the crystal structure
of ligand-bound TR
LBD was solved (37). Our data raise the
possiblity that the coordinates of certain subregions within the LBD
crystal structure, such as the AF-2 domain, may be different for the
full-length receptor TR than the LBD and should be interpreted with
caution. Crystal structures of TR containing both the DBD and LBD will
be helpful in resolving this issue.
In summary, allosteric changes by other regions of the receptor,
protein-protein interactions (as seen by the differences between
T3-mediated transactivation by GALRXR/TRß and
GALTRß/RXR complexes in Fig. 1
), DNA binding, and ligand binding may
all influence TR conformation in critical contact regions with
coactivators. Recent observations that TR can be phosphorylated further
raise the issue of whether additional factors may modulate TR
interactions with coactivators (38, 39). Nevertheless, it appears that
integration of all these contributions to the conformation of the
liganded TR/RXR complex likely is the first critical step that dictates
the interactions with corepressors, coactivators, and the basal
transcriptional machinery. The summation of these interactions, then,
may result in the repression, derepression, and activation of
transcription of target genes.
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MATERIALS AND METHODS
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Creation of TRß mutants
All expression vectors for TRs were expressed in pcDNA1/Amp
(pcDNA) (Invitrogen, San Diego, CA). hTRß, R429Q, G345,
TRß-
N.rTRß, and rTR
have been previously described (40, 41).
R428Q, in pBS (gift from Dr. L. Jameson, Northestern University,
Chicago, IL) was subcloned into pcDNA (42). TRAHTM in pcDNA
was created using an in vitro mutagenesis kit (Promega,
Madison, WI) and a mutagenesis primer that changed codons 223, 224, and
227 from A, H, and T to G, G, and A, respectively (8). TRß-TG and
TRß-GT, in which the sequences encoding first zinc and second zinc
fingers of human TRß have been swapped with hGR, were generated by
PCR amplification of the DNA sequences coding for each zinc finger from
TRßnx and GRnx plasmids in pRSV (43) (gifts from Dr. R. M.
Evans, Salk Institute, San Diego, CA) and then subcloned into
HindIII and KpnI sites of the pcDNA polylinker.
TRß-TGT contains the hTRß DBD substituted with the hGR DBD in pRSV
(43). AF-2 LBD mutants, 457A-LBD and 457D-LBD, were created by PCR
amplification using primers containing mutations of the codon 457 and
HindIII restriction site and a primer containing a
SmaI restriction site and TRß cDNA sequence starting from
codon 174 (encodes an internal methionine). Full-length AF-2 mutants,
E457A and E457D, were created by using the same primers containing the
mutations of codon 457 and a primer containing a SmaI
restriction and TRß cDNA containing the first translational start
site methionine. These PCR fragments were isolated, purified, and then
subcloned into pcDNA expression vectors. mRXRß in pcDNA has been
previously described.
GAL4RXR and GAL4TRß encode amino acids 1147 of GAL4 DNA-binding
sequence and of amino acids 157410 of the mRXRß (gift of Dr. K.
Ozato, NIH, Bethesda, MD) and 173161 of hTRß-1 LBDs (23, 44).
To generate the UAS-reporter, an oligonucleotide containing the
GAL4-binding site, UAS, a previously described 17-bp sequence was used
(45). This oligonucleotide contained BamHI and
EcoRI restriction sites on either end and was subcloned into
the reporter vector, PT109, which contains a viral thymidine kinase
minimal promoter and the firefly luciferase cDNA, as previously
described (46). Clones were isolated, sequenced, and maxi-prepped by
affinity chromatography (Qiagen, Chatsworth, CA) before used in
transfections.
Cotransfection Studies
cDNA clones encoding the TRs and TRß mutants in pcDNA
expression vector (Fig. 1
) as well as GAL4, GAL4TRß, and GAL4RXR were
used in the cotransfection experiments. Reporter plasmids containing
the UAS and the luciferase cDNA in PT109 described above were used (45, 46).
CV-1 cells were grown in DMEM/10% FCS. The serum was stripped of
T3 by constant mixing with 5% (wt/vol) AG1-X8 resin
(Bio-Rad, Richmond, CA) twice for 12 h at 4 C before
ultrafiltration. The cells were transfected with expression (0.1 µg)
and reporter (2 µg) plasmids as well as a RSV-TRß-galactosidase
control plasmid (1 µg) in 3.5-cm plates using the calcium-phosphate
precipitation method (47). Cells were grown for 24 h in the absence or
presence of 10-6 M T3 (Sigma, St.
Louis, MO) or 9-cis- retinoic acid (Biomol), and harvested.
Cell extracts then were analyzed for both luciferase and
ß-galactosidase activity to correct for transfection efficiency (48, 49). Except where indicated, the corrected luciferase activities of
untreated samples were normalized to the luciferase activities of
samples containing pcDNA (vector) and GAL4 expression vectors in the
absence of ligand (1-fold basal).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Paul M. Yen, Molecular and Cellular Endocrinology Branch, NIDDK/NIH, Building 10, Room 8D12, 9000 Rockville Pike, Bethesda, Maryland 20892.
We thank Dr. Ron Evans (Salk Institute, La Jolla, CA), Dr.
Larry Jameson (Northwestern University, Chicago, IL), Dr. Keiko Ozato
(NIH, Bethesda, MD), and Dr. Samuel Refetoff (University of Chicago,
Chicago, IL) for kind provision of plasmids.
Dr. Yen received support from the March of Dimes Foundation for this
study.
1 Current address: Molecular and Cellular Endocrinology Branch, NIDDK,
NIH, Bethesda, Maryland 02892. 
Received for publication May 8, 1997.
Revision received September 11, 1997.
Accepted for publication October 6, 1997.
 |
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