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J Biol Chem, Vol. 273, Issue 46, 30175-30182, November 13, 1998
From the Thyroid Unit, Department of Medicine, Beth Israel
Deaconess Medical Center and Harvard Medical School,
Boston, Massachusetts 02215
On positive thyroid hormone response elements
(pTREs), thyroid hormone receptor (TR) binding to DNA in the absence of
ligand (thyroid hormone, T3) decreases transcription
(silencing). Silencing is due to a family of recently described nuclear
corepressor proteins (NCoR and SMRT) which bind to the CoR box in the
hinge region of TR. Ligand-dependent activation of TR is
associated with displacement of corepressors and recruitment of
coactivating proteins. Resistance to thyroid hormone (RTH) is due to
mutations in the Thyroid hormone receptor
(TR)1 is a member of the
steroid/thyroid hormone receptor superfamily and regulates the
expression of many target genes through its ability to bind to thyroid
hormone response elements (TREs) (1, 2). TR contains a variety of protein domains involved in DNA binding, hormone binding, receptor dimerization, and interaction with the basic transcription machinery. On positive response elements (pTREs), TR binding to DNA in the absence
of ligand (thyroid hormone, T3) decreases transcription (silencing) (3). Silencing is due to binding of the TR to a family of
recently described nuclear corepressor proteins (TRACs, NCoR (RIP 13),
SMRT, SUN-CoR) (4-15). Corepressors are thought to silence
transcription by promoting a closed chromatin configuration through
histone deacetylation. The CoR box (amino acids 211-240 of human
TR- Resistance to thyroid hormone (RTH) is the result of mutations in the
carboxyl terminus of the To date, six naturally occurring RTH mutations have been described in
or near the hinge region (amino acids 174-237 (44) of TR: A234T,
R243Q, R243W, V264D, T277A, and R282S (40, 42, 43). The former three
mutations are located in or near the CoR box. Based on the location of
corepressor binding, RTH mutations in or near the CoR box might be
expected to alter corepressor binding and affect TR function. While the
A234T mutation is reported to have mildly decreased T3
binding, R243Q and R243W are reported to bind T3 normally
in solution (40, 42, 43). Because A234T, R243Q, and R243W bind
T3 normally or near normally in solution, we decided to
investigate the mechanism by which they cause RTH.
Site-directed mutagenesis (CLONTECH, Palo
Alto, CA) was used to create hinge mutations in the context of the
human TR- The SRC-1 nuclear binding domain (NBD-1) fragment was generated by
polymerase chain reaction of amino acids 594-780 of an F-SRC-1 clone
generously provided by William W. Chin. The generation of the binding
domain of NCoR, NCoR-I, has been previously described (9).
Reporter constructs included two copies of idealized pTREs, direct
repeat with a four-base pair interval (DR+4) (41) and inverted
palindrome (chicken lysozyme F2) (46). Constructs contained the pTRE
element fused upstream of a Transfections were performed in CV-1 cells which are relatively
TR-deficient (47, 48). Each transfection included a mutant or wt TR- Gel-shift studies were performed using constructs of the wt and mutant
TRs placed in a pGEM vector. To study corepressor binding, a construct
was fashioned inserting the TR interacting domains of NCoR (9) into the
pKCR2 expression vector. TRs and the binding domain of NCoR were
in vitro translated using rabbit reticulocyte lysate
(Promega, Madison, WI). The nuclear binding domain (NBD-1) of SRC-1
(21, 49) was inserted into a procaryote glutationine S-transferase (GST) expression vector resulting in a GST
fusion protein. TR-SRC interactions were evaluated accordingly. A
polyclonal anti-GST antibody (Santa Cruz Biotechnology, Santa Cruz, CA)
which specifically disrupted the TR/SRC·GST complex was used to more clearly identify TR-SRC-1 binding.
The binding affinities for wt and mutant TRs in solution were assessed
with a filter binding assay utilizing 125I-labeled
T3 (50). Ka values were assigned after
Scatchard analysis. T3 binding on DNA was also assessed
following a standard gel-shift protocol except that
125I-labeled T3 replaced
32P-labeled DR+4 for visualization.
TR- Ligand-dependent Dissociation of Corepressor from
Mutant TRs Is Impaired--
We first evaluated mutant TR binding to
the nuclear corepressor, NCoR, using a gel-shift assay. The three hinge
mutants noted above (A234T, R243Q, and R243W), a severe GRTH mutant
(
In contrast, as shown in Fig. 3,
T3 dissociation of NCoR-I from certain mutant TRs was much
different than from wt TR on the DR+4 element. With wt TR, 5 nM T3 resulted in an almost complete dissociation of the TR/NCoR-I complex (panel A). The
artificial hinge mutant with normal T3 binding, E220R
(panel B), had a similar NCoR-I dissociation pattern. The
A234T mutant (panel C), however, required about 2-fold
higher concentration of T3 to dissociate NCoR-I than did wt
TR. The R243Q and R243W mutants (panels D and E)
required about 20-fold greater T3 concentration relative to wt TR to dissociate this complex. Finally the Recruitment of SRC-1 by Mutant TRs Is Mildly Impaired Relative to
wt TR--
To test whether hinge mutants could normally recruit SRC-1,
the NBD-1 of SRC-1 (21, 49) was fused to the GST protein and expressed
in bacteria. Purified GST and GST-SRC-1(NBD-1) were then employed in a
gel-shift study of wt and mutant TR binding to a DR+4 probe (Fig.
5). In the absence of T3,
SRC-1 did not form a complex with wt TR (panel A). A wt
TR·SRC-1 complex was first noted at 2.5 nM and was
maximal at 100 nM T3. These results were
unexpected since T3 is known to dissociate the TR homodimer and prevent its binding to DNA. The presence of a TR·SRC-1 complex indicates that the SRC-1(NBD-1) must, in some way, stabilize the binding of TR on DNA in the presence of T3. The three hinge
mutants also recruited the NBD-1 of SRC-1 with increasing
T3 concentrations. The R243Q mutant (panel C)
appeared qualitatively similar to wt TR in its ability to recruit SRC-1
over the T3 concentrations tested. The A234T (panel
B) and R243W (panel D) mutants required about 2-fold
greater T3 concentrations, relative to wt TR, to begin to
recruit SRC-1(NBD-1). In Fig. 6, these
experiments were repeated in the presence of RXR. SRC-1(NBD-1) was able
to bind to both wt TR homodimers and wt TR/RXR heterodimers
(panel A) as evidenced by the decrease in intensity of both
complexes and the presence of two closely spaced more slowly migrating
complexes on a lighter exposure of this autoradiogram (data not shown). Qualitatively, however, the defect in SRC-1 association caused by the
hinge mutants was similar to the data obtained without RXR. Thus, hinge
mutants display a significantly greater impairment in
T3-mediated dissociation from NCoR-I than
T3-mediated association to the NBD-1 of SRC-1.
TR Binding to NCoR Prevents SRC-1 Recruitment--
To determine
whether NCoR binding interfered with SRC-1 recruitment, the gel-shift
assays were repeated using TR interacting domains from both cofactors.
In Fig. 7 gel-shifts obtained using the
wt TR and two hinge mutants, R243Q and R243W are shown. Although the
TR·NCoR-I and TR·SRC-1(NBD-1) complexes migrated at a similar position, closer inspection revealed that the TR·SRC-1(NBD-1) complex
migrated slightly faster than the TR·NCoR-I complex. We used this
difference in migration position and an antibody directed against the
GST protein to distinguish the two complexes. In the absence of
T3 where only NCoR-I would be expected to bind to the wt
TR, one distinct complex was formed and the location and intensity of
the complex was not affected by addition of the GST antibody (panel A). Conversely at high T3 concentration
(100 nM) where only SRC-1(NBD-1) should bind to TR, a
faster migrating complex was formed which was completely eliminated by
the addition of the GST antibody. A gradual shift from the TR·NCoR-I
to the TR·SRC-1(NBD-1) complex was noted over the T3
concentrations tested (panel A).
Under no condition was a complex formed consistent with binding both
NCoR-I and SRC-1(NBD-1) to wt TR. Rather, as the T3
concentration rose, the shifted band migrated from the higher
TR·NCoR-I position to the lower TR·SRC-1(NBD-1) position. This was
particularly well seen at a T3 concentration of 2.5 nM, where both NCoR-I and SRC-1(NBD-1) were capable of
binding wt TR when tested individually. At this concentration and in
the absence of the GST antibody, wt TR bound in a faster migrating
complex consistent with its binding to SRC-1(NBD-1). Addition of the
GST antibody shifted the position of the complex slightly to a location
predicted for TR·NCoR-I binding. Since the GST antibody interferes
with formation of the TR·SRC-1(NBD-1) complex, these data suggest
that removal of SRC-1(NBD-1) from the TR allowed NCoR-I in solution to
bind to the wt TR.
The net result of the impaired NCoR-I dissociation from hinge mutants
is also shown in Fig. 7 (panels B and C). Only at
100 nM T3 was NCoR-I sufficiently dissociated
from the hinge mutants to permit significant SRC-1(NBD-1) recruitment,
based on the position of the protein-DNA complex. The GST antibody
blocks formation of this complex, confirming that at this
T3 concentration, the hinge mutants bound exclusively to
SRC-1(NBD-1). At 10 nM T3, SRC-1(NBD-1) bound
somewhat to the hinge mutants based on partial dissociation of the
shifted complex by the GST antibody (R243Q > R243W). This result
is consistent with the other data showing that the R243Q mutant
recruited SRC-1(NBD-1) at lower T3 concentrations than the
R243W mutant. Similar data was obtained with the A234T mutant (not
shown). Thus, an impairment in NCoR dissociation by T3, as
observed with the hinge mutants, prevents SRC-1 recruitment.
Solution and DNA Complex Binding of Mutant TR Do Not
Correlate--
It is unclear why the hinge mutants displayed such
significant impairment in dissociation from NCoR-I in the absence of
significant defects in solution binding to T3. Moreover,
the R243Q and R243W mutants required significantly greater
T3 concentrations to dissociate NCoR-I than did the A234T
mutant despite the fact that these receptors had very similar
T3 binding profiles. To investigate the reason for this
discrepancy, gel-shift assays were performed in parallel using either a
32P-labeled DR+4 probe or 125I-T3
to identify the shifted complexes. Fig. 8
is such an experiment comparing wt and mutant TRs. In vitro
translated RXR-
As found by others (52), the wt TR/RXR heterodimer was clearly labeled
by 125I T3 (lane 9) and the
homodimer was not labeled, despite equal amount of these complexes on
the 32P- labeled gel-shift. We interpret this result as
indicating that occupancy of one TR molecule by radiolabeled
T3 marks the heterodimer which is not dissociated by
T3, while T3 bound to one TR molecule is
sufficient to dissociate the homodimer and eliminate a labeled band at
that position. Because of the migration position of free 125I T3 on the gel, we could not determine if
the wt TR·NCoR-I complex was labeled. The heterodimeric complex
containing the A234T mutant was labeled 3-fold less well by
T3 (as determined by densitometry) and heterodimers
containing the R243Q, R243W, and Mutant TR Function Is Diminished at T3
Concentrations Where NCoR Remains Bound and Is Not Reversed by
Transfection of an NCoR Inhibitor (NCoR-I)--
As a model for thyroid
hormone action, two copies of a positive thyroid hormone response
element were fused upstream of a heterologous promoter luciferase
construct (pTK109-Luc) for use in transient transfection assays of CV-1
cells. On the DR+4 element (Fig. 9,
panel A), activation by the wt TR was first noted at 1 nM T3 and reached a maximum at 100 nM T3 Activation of this element by the
artificial hinge mutant (E220R) was similar to wt TR (data not shown).
The natural hinge mutants were clearly defective in activation compared
with wt TR. Activation by the A234T mutant was first achieved at 5 nM T3, while transactivation by the two 243 mutant TRs was first noted at 10 nM T3. At very high T3 doses, however, transactivation by all mutants
except
Since transfection of an NCoR inhibitor (NCoR-I) expression vector can
reverse TR-mediated repression on pTREs (9), we wanted to determine if
cotransfection of this plasmid could correct the defect in
transactivation observed with the hinge mutants. In Fig.
10A, cotransfection of
NCoR-I completely reversed ligand-independent repression by the wt TR
on the DR+4 element and yielded similar levels of reporter gene
activation in the presence of T3. Since endogenous
corepressors (NCoR and SMRT) and NCoR-I are released from wt TR at
physiological T3 concentrations, transcriptional repression
by endogenous corepressors is selectively removed by NCoR-I without
affecting T3-mediated coactivator recruitment and transactivation. In contrast, in Fig. 10B are data obtained
with one of the hinge mutants, R243W. Note that NCoR-I cotransfection completely eliminated TR-mediated repression but did not allow for
normal T3-mediated transactivation except at 100 nM T3. Importantly, these transfection studies
are consistent with gel-shift studies showing that TR hinge mutants are
markedly defective in their ability to dissociate NCoR-I and permit
SRC-1 (NBD-1) recruitment.
Our study of hinge mutations of the TR has revealed several
important properties of TR-mediated transactivation and shed new light
on the RTH syndrome. First, hinge mutants of the TR display a defective
release of NCoR in response to treatment with T3. Gel-shift
studies demonstrate that both 243 mutants, and to a lesser extent the
234 mutant, remain bound to NCoR at concentrations of T3
that completely dissociate NCoR from wt TR. Yoh et al. (13) first suggested that RTH mutants displayed aberrant interaction with
nuclear corepressors. As expected, mutations which bound T3
poorly failed to dissociate from corepressor except at very high
T3 concentrations. Our results with the We initially favored this hypothesis to explain our findings with the
hinge mutants. These mutants displayed good solution T3
binding (less than a 2-fold decrease), yet failed to dissociate from
NCoR in gel-shift experiments and continued to silence gene transcription in transfection studies except at very high
T3 concentrations. Importantly, however, we determined the
ability of these mutants to bind to T3 when complexed to
DNA. To our surprise, these mutants as RXR heterodimers bound
radiolabeled T3 poorly, if at all, in gel shift studies.
Our results are limited to the heterodimer complex since TR homodimers
were not labeled in this assay, perhaps due to the fact they would be
dissociated when bound by T3, and free radiolabeled
T3 obscured the TR·NCoR-I complex, Regardless these data
suggest that binding of T3 in solution and on DNA do not
always correlate as highlighted by mutations in the hinge region of TR.
Based on the crystal structure of TR- In evaluating TR cofactors and their role in RTH, the question of the
relative importance of corepressors and coactivators in the genesis of
RTH has arisen. For example, impaired ligand-dependent activity could be associated either with impaired (13) corepressor release or with defective coactivator recruitment (52, 57). Our data
suggest that impaired corepressor release is the primary defect in RTH
patients with hinge mutations and probably for most other patients with
RTH. This is based on two lines of evidence. 1) Most RTH mutations
affect T3 binding to TR in solution or on DNA, as is the
case with hinge mutants. This would result in impaired release of
corepressor at physiological T3 concentrations and continued gene silencing. 2) Both NCoR and SRC-1 do not bind
simultaneously to TR, suggesting at least two discreet steps are
necessary for transactivation. Addition of T3 must first
cause dissociation of NCoR before recruitment of SRC-1 can take place.
Either the binding locations of the two cofactors must overlap such
that both cannot be present on TR or the TR conformation when NCoR is
bound is such that SRC-1 binding is not favorable. This result is most
clearly seen using the NCoR inhibitor in transfection studies.
Transfection of NCoR-I competes with endogenous corepressors, resulting
in a loss of ligand-independent repression on pTREs caused by the R243W
mutation. Remarkably the mutant TR is still unable to activate
transcription except at very high T3 concentration. Since
mutant TR binding to NCoR-I is also resistant to T3
dissociation, SRC-1 or other coactivators are not recruited to the TR
due to lack of binding to the corepressor occupied TR, yielding a lack of transactivation.
We propose the following model (Fig.
11) based on our results. wt TR, in the
absence of ligand, binds corepressor and transcription is repressed.
Addition of T3 results in dissociation of corepressor, permitting recruitment of coactivators and transcriptional activation. Although, the hinge mutant TRs bind corepressor normally in the absence
of ligand resulting in normal silencing activity, T3 fails to dissociate the corepressor and thus coactivator recruitment is
prevented. While T3 binding by the hinge mutant TRs is
normal or near-normal in solution, their T3 binding is
clearly defective when complexed to DNA. Our data indicate that
DNA-binding modifies the ability of hinge mutants to bind
T3, yielding this novel mechanism of resistance by this
class of RTH mutants.
*
This work was supported by National Instiutes of Health
Grants DK-02423 (to J. D. S.) and DK-43653 and DK-49126 (to F. E. W.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
TR, thyroid hormone
receptor; TRE, thyroid hormone response element; pTRE, positive thyroid
hormone response element; TR-
Defective Release of Corepressor by Hinge Mutants of the
Thyroid Hormone Receptor Found in Patients with Resistance to
Thyroid Hormone*
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
isoform of the thyroid hormone receptor (TR-).
To date, three RTH mutations reportedly with near-normal T3
binding (A234T, R243Q, and R243W) have been described in or near the
CoR box. To determine the mechanism of RTH caused by these mutants, the
interaction of wild type (wt) and mutant TRs with the corepressor,
NCoR, and the coactivator, SRC-1, was tested in gel-shift assays. As
expected, NCoR bound wt TR in the absence of T3 and
dissociated from TR with increasing T3 concentration. SRC-1
failed to bind wt TR in the absence of T3, but bound to TR
with increasing avidity as T3 concentrations rose. At no
T3 concentration did both NCoR and SRC-1 bind to wt TR,
indicating that their binding to TR was mutually exclusive. Hinge
mutants bound NCoR normally in the absence of T3; however, dissociation of NCoR and recruitment of SRC-1 was markedly impaired except at very high T3 concentrations. Importantly, hinge
mutant TRs when complexed to DNA bound T3 poorly despite
their near-normal T3 binding in solution. These binding
studies correlated with functional assays showing defective
transactivation of pTREs by hinge mutants except at high T3
concentrations. Thus, we describe a novel mechanism of RTH whereby TR
hinge mutants selectively affect T3 binding when complexed
to DNA, and prevent NCoR dissociation from TR. Our data also suggest
that solution T3 binding by RTH mutants may not accurately
reflect physiologically relevant T3 binding by TR when
bound to DNA.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1), located in the hinge region of TR, binds corepressor proteins
(16). Addition of T3 results in displacement of
corepressors and a return of transcription to a basal rate (4, 5). In the presence of ligand, proteins termed coactivators are recruited to
mediate the ligand-dependent response (10, 12). A large number of nuclear receptor-interacting or coactivating proteins have
been isolated including SRC-1 (ERAP 160, p160), RIP 160, ERAP 140 (p140), RIP140, TIF1, TIF2 (GRIP1), TRIP1 (SUG-1), RAP 46, hRPF 1, ARA
70 (RFG), CBP (p300), p/CAF, P120, ACTR (AIB1, TRAM-1, pCIP), and GRIP
170 (10, 12, 17-29).
thyroid hormone receptor (TR-)
(30-33). Individuals with the disorder require greater thyroid hormone
(T3) concentrations in order to achieve
T3-dependent actions in tissues. RTH is a
dominant disorder in which most individuals are heterozygous for a
mutant TR- allele. In a phenomenon called dominant negative
activity, the mutant allele interferes with the activity of the normal
allele (34-37). RTH mutations congregate in two major "hot spots"
in the ligand binding domain of TR- (38, 39). Recently, mutations
have also been reported in the hinge region, suggesting a third hot
spot for mutations within the TR- locus (41-43).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1 isoform (45). All mutations were confirmed by DNA
sequencing of the TR-1 C terminus. The expression vector used for
transfections in this study was pSG5, containing either wild type (wt)
or mutant TR- cDNAs as EcoRI fragments. The human
TR-1 cDNA was used as the wt TR-1. Expression vector plasmid
preparations used in this study were carefully quantitated by agarose
gel electrophoresis. To confirm the integrity and quality of each
expression vector plasmid DNA preparation, in vitro
translation with [35S]methionine was performed using T7
polymerase and the products analyzed by SDS-polyacrylamide gel electrophoresis.
109 thymidine kinase promoter and the
luciferase gene to measure activity. The luciferase reporter gene was
derived from pA3 and contained two transcriptional stop sequences
upstream of the promoter to prevent read-through transcription. The
reporter was documented to have no positive thyroid hormone response in
the absence of response elements.
expression vector (500 ng) and a response element reporter construct
(10 µg). Cell cultures were transfected using a calcium-phosphate precipitation method and precipitate applied for 16 h in
Dulbecco's modified Eagle's medium containing 10% fetal bovine
serum, glutamine, and appropriate antimicrobials. The next day, media
was removed and Dulbecco's modified Eagle's medium containing both
anion exchange and charcoal stripped fetal bovine serum (10%) was
added ± T3. Concentrations of T3 were
used as noted. Data were pooled from at least three independent
experiments and displayed as mean ± S.E.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Mutations Used in This Study--
As shown in Fig.
1, three TR- mutations were evaluated
in this study (A234T, R243Q, and R243W). These mutations were
introduced into a TR-1 cDNA in pGEM 3, using site-directed
mutagenesis and transferred to the expression vector pSG5 for use in
transient transfection studies. Mutant TR binding to T3 in
solution (Table I) is not significantly
impaired with each mutant displaying only a 20-40% reduction in
T3 binding. These results confirm those of other
laboratories studying the T3 binding properties of these mutant receptors (40, 42, 43).
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Fig. 1.
Schematic representation of the location of
TR- mutations used in this study. Mutations lie in the hinge
region between the DNA and ligand binding domains.
Relative T3 binding to hinge mutant TRs in solution
337T) (51), and an artificial hinge mutant known to not interfere with corepressor binding (E220R) (7) were tested in this assay. A
portion of NCoR, containing the TR interacting domains but lacking the
repressing domains, termed NCoR-I, was employed in the assay (9).
In vitro translated TRs formed a dimer (D) in the absence of
NCoR-I on a DR+4 radiolabeled element. Addition of in vitro translated NCoR-I caused the dimer to be shifted to a new position and
serial dilution of the amount of NCoR-I added to the binding reaction
reduced the intensity of this complex. Ligand-independent binding of
NCoR-I to each of the mutants was similar to wt TR (Fig.
2). Addition of RXR-
or RXR- to the
binding reaction resulted in the formation of a strong heterodimeric
complex, and a significant reduction in the TR·NCoR-I complex (data
not shown).
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Fig. 2.
wt and mutant TRs bind NCoR equally in the
absence of T3. Gel-shift studies were performed with
32P-labeled DR+4 probe, the indicated TR and a range of
relative quantities of NCoR-TR binding domain. For each TR shown, the
first lane contains TR and probe alone. The subsequent lanes contain
NCoR-I in a reticulocyte lysate volume four times that of TR, equal to
that of TR, one quarter that of TR, and 1/16th that of TR. The TRs
shown are A, wt; B, E220R; C, A234T;
D, R243Q; and E, R243W; F,
337T.
337 mutant, which cannot bind T3, could not be dissociated from NCoR-I even
at the 100 nM T3 concentration. Similar
observations were made on the chicken lysozyme TRE (Lys, Fig.
4) and palindromic TRE (data not shown).
On the Lys element, a higher T3 concentration was necessary to completely dissociate NCoR-I from wt TR (100 nM).
Dissociation of NCoR-I from E220R and 234T was similar to wt, while
the R243Q and R243W mutant TRs required a 10-fold higher T3
concentration (1000 nM) than did wt TR for dissociation. As
expected, NCoR-I could not be dissociated from the 337T mutant even
at this very high T3 concentration.
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Fig. 3.
TR hinge mutants require more T3
to dissociate NCoR than does wt TR. A 32P-labeled DR+4
gel shift as in Fig. 2 was done with reticulocyte lysate generated TRs
incubated with NCoR binding domain across a range of T3
concentrations as noted: A, wt; B, E220R;
C, A234T; D, R243Q; E, R243W; and
F, 337T. Note that NCoR-I cannot be dissociated from the
337T mutant at any of the T3 concentrations tested
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Fig. 4.
Shown is a 32P-labeled Lys gel
shift as in Fig. 2 of TRs incubated with NCoR binding domain across a
range of T3 concentrations as noted. Hinge mutant TRs
required more than 10-fold more T3 to achieve the same
degree of dissociation as wt. TRs are A, wt; B,
E220R; C, A234T; D, R243Q; E, R243W;
and F, 337T.
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Fig. 5.
Hinge mutant recruitment of SRC-1 is only
mildly impaired relative to wt. A 32P-labeled DR+4 gel
shift as in Fig. 2 is demonstrated. Reticulocyte lysate generated TRs
were incubated with GST fusion protein generated SRC-1 (NBD-1) across a
range of T3 concentrations as noted: A, wt; B,
A234T; C, R243Q; and D, R243W. The R243Q mutant
is nearly normal in SRC-1 (NBD-1) recruitment; the A234T and R243W
mutants require a T3 concentration twice that for wt to
recruit SRC-1.
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Fig. 6.
The relative avidity of TRs for SRC-1 is not
altered by the presence of RXR. The TRs were incubated with RXR
and SRC-1 across the range of T3 concentrations noted. The
hinge mutants demonstrate only minimal defect in recruitment of SRC-1
with approximately twice the T3 concentration as that
required by wt TR. Pictured are A, wt; B, E220R;
C, A234T; D, R243Q; E, R243W; and
F, 337T.
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Fig. 7.
NCoR and SRC-1 cannot bind to TR
simultaneously. A 32P-labeled DR+4 gel shift was
performed with reticulocyte lysate generated TRs incubated with TR
binding domains of NCoR and SRC-1 across a range of T3
concentrations: A, wt; B, R243Q; and
C, R243W. An anti-GST antibody which specifically
dissociates the TR·SRC-1 complex was added at each T3
concentration. In the absence of ligand, the slower moving TR·NCoR
complex is evident. At high T3 concentrations, the faster
moving TR·SRC-1 complex appears. Although there are T3
concentrations at which both NCoR and SRC-1 bind TR, at no point is a
band representing both cofactors bound to TR present. With the two
hinge mutants shown, the TR·SRC-1 complex only appears at the highest
T3 concentrations when the TR·NCoR complex is
dissociated.
and the indicated TR- were added to each column
except columns 1 and 8 where an equal volume of unprogrammed lysate was
utilized (UP). In columns 2-7, reticulocyte
lysate generated TRs (wt, E220R, A234T, R243Q, R243W, and 337T,
respectively) were incubated with RXR- and 32P-labeled
DR+4, resulting in heterodimeric and homodimeric complexes of equal
intensity. In columns 9-14 the same paradigm was followed except that the 32P-labeled DR+4 pTRE was replaced by a
nonradioactive DR+4 pTRE and 125I-T3 was added
(10 nM final concentration).
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Fig. 8.
While the A243T mutant suffers only mildly
impaired T3 binding on DNA, the two 243 mutants suffer
significant impairment. The 337T mutant does not bind
T3 on DNA. Columns 1-7 show a
32P-labeled DR+4 gel shift of TRs incubated with
reticulocyte lysate generated RXR. Columns 8-14 show an
125I-labeled T3 and nonradioactive DR+4 gel
shift of the same TRs incubated with RXR. Columns 1 and
8 are unprogrammed control lanes. Other lanes contain TRs as
follows: 2 and 9, wt; 3 and
10, E220R; 4 and 11, A234T;
5 and 12, R243Q; 6 and 13,
R243W; 7 and 14, 337T. D,
homodimer; HD, heterodimer.
337T mutants were not labeled.
Clearly the 337T mutant would not be expected to bind
T3; however, results with the hinge mutants were unexpected given their solution T3 binding. These results indicate
that solution and DNA-complex binding of T3 by hinge
mutants of the TR do not correlate.
337T equaled that of wt TR. Each hinge mutant had strong
dominant negative activity against wt TR on the DR+4 element (data not shown). Qualitatively similar results were noted with the everted palindrome element derived from the chicken lysozyme gene promoter, Lys
(Fig. 9, panel B).
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Fig. 9.
A, on the DR+4 reporter, the R243
mutants suffer dimished ligand dependent stimulation at low and
intermediate doses of T3. The A234T mutant suffers more
mildly decreased activity. At high T3 doses, all three
hinge mutants tested have activity similar to wt. B, there
is diminished relative activity of the TR mutants across T3
concentrations on the Lys probe. As with DR+4, the degree of impairment
is greater for the 243 mutants than for the A234T mutant. The 337T
mutant has no significant ligand-dependent activity at any
T3 concentration tested.
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Fig. 10.
A, an NCoR inhibitor (NCoR-I) reverses
ligand-independent repression by wt TR. Shown are the results of
transient transfection of CV-1 cells with plasmids containing wt TR,
NCoR-I, and the luciferase gene preceded by DR+4. The T3
concentrations are noted on the x-axis. B, NCoR-I reverses
ligand-independent repression but does not permit normal T3
transactivation by the R243W mutant. Recruitment of coactivators is
prevented and ligand dependent transcription is retarded. Shown are the
results of transient transfection of CV-1 cells with plasmids
containing R243W TR, NCoRI, and the DR+4/luciferase reporter
gene.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
337T mutation
are in keeping with their findings. However, two mutants located in the
AF-2 domain (P453A and P453H) appeared to have a much greater defect in
T3-mediated dissociation from corepressor than would be
expected due to their mild decrease (4-5-fold) in T3
affinity. The authors suggested that a conformation change in the
extreme C terminus of TR caused by the mutation might explain this
apparent discrepancy by uncoupling T3 binding from
corepressor dissociation.
, hinge mutants might affect
T3 binding (53). These mutations are near helix two, which
is believed to stabilize the polar pocket which binds T3. Since regions of the hinge domain are known to contact DNA (54, 55) and
to mediate ligand binding (56) it is possible that DNA-binding induces
a conformation change in the mutant hinge domain so as either to
prevent ligand access to the heterodimeric bound TR or prevent
stabilization of ligand-binding on the TR. The net result is that
corepressor continues to bind to TR and prevent transactivation despite
the presence of T3 in solution. It is unclear whether this
discrepancy between solution and DNA-complex binding of T3
is specific for hinge mutants or will also be observed with RTH mutants
in other locations on the TR-.
View larger version (33K):
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Fig. 11.
Model of resistance caused by hinge mutants
of the TR. When wt TR is bound to DNA, addition of
T3 results in dissociation of corepressor, permitting
recruitment of coactivator and transcriptional activation. When hinge
mutant TR is bound to DNA, T3 cannot bind and NCoR is not
dissociated. SRC-1 recruitment is prevented and DNA transcription
remains repressed.
FOOTNOTES
To whom correspondence should be addressed: Thyroid Unit, Beth
Israel Deaconess Medical Center, Research North, Rm. 330C, 99 Brookline
Ave., Boston, MA 02215. Tel.: 617-667-4490; Fax: 617-667-2927.
, thyroid hormone receptor beta
isoform; T3, triiodothyronine; NCoR, nuclear corepressor; NCoR-I, nuclear corepressor inhibitor; SRC-1, steroid receptor
coactivator-1; RTH, resistance to thyroid hormone; wt, wild type; DR+4, direct repeat response element with 4-base pair separation; Lys, chicken lysozyme response element; NBD-1, nuclear binding domain-1; GST, glutathione S-transferase; RXR, retinoid X receptor
isoform; RXR, retinoid X receptor isoform.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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