From the Section of Endocrinology, Department of
Medicine, the University of Chicago, Chicago, Illinois 60637 and
§ Division of Endocrinology, Beth Israel Deaconess Medical
Center, Harvard Medical School, Boston, Massachusetts 02215
Received for publication, July 19, 2002, and in revised form, October 22, 2002
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ABSTRACT |
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The The synthesis and secretion of thyroid hormones are exquisitely
regulated by a negative feedback system that involves the hypothalamus,
pituitary, and thyroid gland (the hypothalamic-pituitary-thyroid axis:
H-P-T axis).1 The synthesis
of thyrotropin-releasing hormone (TRH), produced in the paraventricular
nucleus of the hypothalamus, and the In the traditional model of TR action, TR regulates gene expression by
binding to specific thyroid hormone-response elements (TRE) on target
genes. A number of artificial and natural positive thyroid
hormone-response elements (pTREs) have been described, and they can be
classified based on the half-site arrangement in the element: DR+4,
PAL, and LAP (LYS) (9-11). On target genes negatively regulated by
T3, response elements (nTREs) have been described in the
TRH and TSH- In contrast, some investigators have suggested that TR DNA binding is
not required for negative regulation. This mechanism has been proposed
to explain negative regulation of the TSH- TRs, like other members of the superfamily of nuclear hormone
receptors, have a conserved protein structure including a central DNA
binding domain (DBD) and a C-terminal ligand binding domain (27, 28).
The DBD is composed of two zinc finger motifs in which the zinc ion is
tetrahedrally coordinated by four cysteines (29, 30). C-terminal to
each zinc finger is an amphipathic isoform of thyroid hormone receptor
(TR-
) has a key role in the feedback regulation of the
hypothalamic-pituitary-thyroid (H-P-T) axis. The mechanism of
trans-repression of the hypothalamic thyrotropin-releasing hormone
(TRH) and pituitary thyroid-stimulating hormone (TSH) subunit genes,
however, remains poorly understood. A number of distinct mechanisms for
TR-
-mediated negative regulation by thyroid hormone have been
proposed, including those that require and do not require DNA binding.
To clarify the importance of DNA binding in negative regulation, we
constructed a DNA-binding mutant of TR-
in which two amino acids
within the P box were altered (GSG for EGG) to
resemble that found in the glucocorticoid receptor (GR). We termed this
mutant GS125, and as expected, it displayed low binding affinities for
positive and negative thyroid hormone-response element (pTRE and nTRE,
respectively) in gel-mobility shift assays. In transient transfection
assays, the GS125 mutant abolished transactivation on three classic
pTREs (DR+4, LAP, and PAL) and all negatively regulated promoters in
the H-P-T axis (TRH, TSH-
, and TSH-
). However, GS125 TR-
bound
to a composite TR/GR-response element and was fully functional on this
hybrid TR/GR-response element. Moreover, the GS125 TR-
mutant
displayed normal interactions with transcriptional cofactors in
mammalian two-hybrid assays. These data do not support a DNA-binding
independent mechanism for thyroid hormone negative regulation in the
H-P-T axis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and
subunits of
thyrotropin (thyroid-stimulating hormone; TSH) in the anterior lobe of
the pituitary is inhibited at the transcriptional level by thyroid
hormone (T3) (1-4). Negative regulation of gene expression
by T3 is an integral part of the function in the H-P-T axis, and these effects are mediated by the
isoform of the thyroid hormone receptors (TR-
). Of the
isoforms, TR-
2 is
quantitatively and functionally more important than TR-
1 in
regulating the hypothalamus and pituitary (5-8). The molecular
mechanisms responsible for TR-mediated negative regulation of target
genes are not well understood.
subunit genes (12-17). In these nTREs, TR bound as a
monomer to widely spaced half-sites or in the case of TRH with retinoid
X receptor (RXR) to an unusual element (13, 18-20). Mutagenesis
studies (21, 22) suggest that TR DNA binding to the half-site is
required for negative regulation. Activation of these nTRE in the
absence of T3 is mediated by the N terminus of TR-
2 via
a mechanism involving binding of coactivator proteins (23, 24).
promoter where an nTRE
has not been clearly identified (25, 26). In this model, corepressors
bind to TR in solution and activate gene expression; in the presence of
T3, corepressors dissociate from the TR and return to the
TSH-
promoter to repress gene expression. A major concern in this
model, however, is how specificity of negative regulation is achieved
in the absence of DNA binding. Thus, both on and off DNA binding
mechanisms have been proposed to explain negative regulation by thyroid hormone.
-helix. The
-helix of the
first finger motif is responsible for sequence-specific DNA
recognition, whereas the
-helix following the second finger motif
provides phosphate contacts to the DNA and stabilizes the structure
(31). The central two nucleotides of the hexameric half-site consensus
motif (AGNNCA) in a hormone-response element is determined by three
receptor-specific amino acids in the DNA recognition
-helix,
referred to as the P box (32). Nuclear receptors have been divided into
two subfamilies according to the identity of their P box amino acid
sequences (33). The subfamily of glucocorticoid receptor (GR) has a GSV
(glycine-serine-valine) P box sequence and is able to bind to DNA
elements containing the nucleotide sequence of AGAACA. The subfamily
that includes the TRs has a more diverse P box sequence. TRs contain an
EGG (glutamic acid-glycine-glycine) P box sequence and recognize DNA elements with the nucleotide sequence of AGGTCA (Fig.
1).
View larger version (33K):
[in a new window]
Fig. 1.
Schematic diagram of the zinc finger region
of the DBD of thyroid hormone receptor .
The shaded circles indicate the P box amino acids. The
boxed region outlines the DNA recognition
-helix. The GR
and thyroid hormone receptor
P box sequence and their consensus
DNA-binding half-sites are shown in the lower half of the
figure. The first two amino acids of the GS125 mutation correspond to
the P box of GR, whereas the third amino acid corresponds to that of
TR.
Within the P box, the first two amino acids are most important in
defining binding to a DNA-response element. Therefore, based on the
differences between the P box of GR and TR, we constructed a P box
mutant that substituted the first two amino acids in TR with the first
two amino acids of GR (EGG to GSG) (Fig. 1).
The GS125 TR- mutant had markedly impaired function on both positive and negative TRE due to disrupted DNA binding. However, this unique DBD
mutant was fully functional when tested on a composite TR/GR DNA
response element. We demonstrate, therefore, that DNA binding by TR-
is required on all nTREs known to regulate the H-P-T axis.
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MATERIALS AND METHODS |
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Plasmid Constructions--
Point mutations in the thyroid
hormone receptor were constructed by site-directed mutagenesis in
the context of the TR-
2 isoform. GS125 mutant TR-
2 cDNA was
inserted into the pKCR2 (pSG5) expression vector at an EcoRI
site; wild type TR-
1 and TR-
2 were constructed in a similar way
and have been described previously (34, 35). The construction of
thyroid hormone-responsive luciferase reporter genes (DR+4-TKLuc,
LAP-TKLuc, and PAL-TKLuc) has been reported previously (23). Negatively
regulated gene promoters in the identical luciferase reporter (pA3Luc)
have also been described: human TRH
900 to +55, human TSH-
846
to +44, and human TSH-
1192 to +37 bp (36). The hybrid TR/GRE 3, as two copies, was inserted upstream of TK 109 in pA3Luc. The GAL4
reporter plasmid UAS-TK-Luc contains two copies of the GAL4 recognition
sequences upstream of TK109 in pA3Luc. Construction of nuclear receptor corepressor (NCoR), steroid receptor coactivator-1 (SRC-1), and RXR-
Gal4BD fusion expression vectors was described previously (37). The
VP16AD-GS125 mutant TR-
2 was created by exchanging appropriate
restriction fragments within the VP16AD-TR-
2 construct, which has
been reported previously (38).
Electrophoretic Mobility Shift Assay--
The oligonucleotide
sequences of DR+4 and TRH site 4 probe were reported previously (13,
37). Hybrid TR/GR-response elements, TR/GRE 1, 2, and 3-DR+4 were
designed to contain an intact 5' half-site and a mutant 3' half-site
separated by a four-nucleotide gap. This strategy was based on polarity
studies of the DR+4 element where it has been shown that RXR binds to
the 5' and TR binds to the 3' half-sites in a DR+4 (39, 40). Wild type
or mutant TRs were transcribed/translated in vitro in
reticulocyte lysate (Promega Corp., Madison WI) using T7 polymerase. To
quantify protein production, 35S incorporation and direct
visualization on SDS-PAGE were used. Four µl of TR- and 2 µl of
in vitro translated RXR-
or an equivalent amount of
unprogrammed lysate were used in each reaction with 32P-radiolabeled probe. Binding reactions were performed in
20 µl of binding buffer containing 20% glycerol, 20 mM
HEPES, pH 7.6, 50 mM KCl, 1 mM dithiothreitol,
1 µg of poly(dI-dC), and 0.1 µg of salmon sperm DNA at room
temperature for 20 min. The protein-DNA complexes were resolved on 5%
nondenaturing polyacrylamide gels and visualized after autoradiography.
Western Blot Analysis--
Whole cell extracts were prepared
with RIPA Buffer (41) including 1 mM phenylmethylsulfonyl
fluoride, 1 mM dithiothreitol, 40 µl/ml Protease
Inhibitor Mixture (Roche Molecular Biochemicals). Proteins were
resolved by SDS-PAGE and transferred onto nitrocellulose transfer
membranes (Protran®; Schleicher & Schuell, Keene, NH). Immunodetection was performed using mouse monoclonal TR- antibody (MA1-215; Affinity Bioreagents, Golden, CO) and anti-mouse,
horseradish peroxidase-conjugated IgG (Amersham Biosciences). Protein
bands were visualized using ECL-Plus kit (Amersham Biosciences)
according to the manufacturer's instructions.
Avidin-Biotin Complex DNA Binding (ABCD) Assay--
Whole cell
extracts from GH3 cells or transfected 293T cells were
incubated with 3 µg of biotinylated double-stranded DNA at 4 °C
for 4 h with constant rotation. Sense and antisense TRE oligonucleotides were 5'-biotinylated as follows: rGH sense (5'-ttt cgg
tgg aaa ggt aag atc agg gac gtg acc gca gga gag caa a-) and antisense
(5'-ttt gct ctc ctg cgg tca cgt ccc tga tct tac ctt tcc acc gaa a-);
hTSH- sense (5'-ttt ttt ggg tca cca cag cat ctg ctc acc aat gca aag
taa gaa a-) and antisense (5'-ttt ctt act ttg cat tgg tga gca aga tgc
tgt ggt gac cca aaa aa-); and mutated hTSH-
sense (5'-ttt ttt
ttt tca cca cag cat ctg ctc acc aat gca aag taa gaa a-) and
antisense (5'-ttt ctt act ttg cat tgg tga gca aga tgc tgt ggt
gaa aaa aaa aa-). The DNA-protein suspensions
were precipitated with streptavidin-agarose (Invitrogen) for 1 h,
washed with RIPA Buffer, and re-precipitated by centrifugation. The
bound materials were eluted and separated with boiling and brief
centrifugation and then resolved by SDS-PAGE. Western blot analysis was
performed as described previously (41).
ChIP Assays--
ChIP assays were performed essentially as
described previously (42) with the following modifications. 293T cells
were transfected with 10 µg of hTSH- pA3Luc or TK 109 in pA3Luc
and 5 µg of either TR-
2 or GS125 mutant TR-
2 pKCR2 using the
calcium phosphate method. After no treatment or treatment with 10 nM T3, formaldehyde was added at 1% to culture
medium, and cells were incubated for 10 min at 37 °C to cross-link
proteins to the transfected promoter plasmids. Cells were collected and
resuspended in 200 µl of SDS lysis buffer (1% SDS, 10 mM
EDTA, 50 mM Tris-HCl, pH 8.1) with 1 mM
phenylmethylsulfonyl fluoride and protease inhibitors. Lysates were
cleared by centrifugation and diluted 10-fold with dilution buffer
(0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl). 10% of the
lysate was kept to quantitate the amount of DNA present in different
samples at the PCR analysis as an input. After pre-clearing with a
salmon sperm DNA/protein A-agarose/50% slurry (Upstate Biotechnology,
Lake Placid, NY), immunoprecipitation with a TR-
antibody (MA1-215;
Affinity Bioreagents) was performed at 4 °C overnight.
Immunoprecipitated complexes were collected by salmon sperm DNA/protein
A-agarose and followed by sequential washes in low salt wash buffer
(0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM
Tris-HCl, pH 8.1, 150 mM NaCl), high salt wash buffer
(0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM
Tris-HCl, pH 8.1, 500 mM NaCl), LiCl immune complex wash
buffer (0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1), and TE
buffer. Precipitates were eluted with elution buffer (1% SDS, 0.1 M NaHCO3), and 5 M NaCl was added
to reverse cross-links at 65 °C for 4 h. DNA was recovered by
phenol/chloroform extraction and ethanol precipitation. Pellets were
resuspended in TE buffer and subjected to PCR using specific 5' primers
for the hTSH-
pA3Luc (5'-caa agt gac agc gta ctc tct t) or TK 109 pA3Luc (5'-ccg ccc agc gtc ttg tca tt) and a common 3' luciferase primer (5'-cta gag gat aga atg gcg ccg ggc ctt tct).
Cell Culture and Transient Transfection Assay--
293T cells
were maintained in Dulbecco's modified Eagle's medium supplemented
with L-glutamine, 10% fetal calf serum, 100 µg/ml
penicillin, 0.25 µg/ml streptomycin, and amphotericin. Transient transfections were performed in subconfluent 6-well plates on subconfluent cells; 1.7 µg of reporter construct with 0.1 µg of receptor expression vector or 0.1 µg of pKCR2 vector alone were added
to each well using the calcium phosphate technique. 12-15 h after
transfection the cells were washed with PBS, and culture medium
containing fetal bovine serum treated with anion exchange resin (type
AGX-8, analytical grade; Bio-Rad) was added. Twenty four hours after
transfection, 10 nM T3 was added, and 42-48 h after transfection culture were harvested and assayed for luciferase activity. Mammalian two-hybrid assays were performed also using the
calcium phosphate technique in 6-well plates, with each well receiving
1.7 µg of upstream activating sequence (UAS)-TK luciferase reporter,
0.1 µg of Gal4DBD-SRC-1 NBD, Gal4DBD-NcoR, or Gal4DBD-RXR and 0.1 µg of VP16-TR-1, -TR-
2, or VP16-GS125 mutant TR-
2.
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RESULTS |
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The DNA Binding Affinity of the GS125 TR- Is Reduced on Both
Positive TRE and Negative TREs--
Recent cell culture and TR
knockout model studies suggest that the
2 isoform of the thyroid
hormone receptor (TR-
2) plays the predominant role in the feedback
regulation of the H-P-T axis (43-45). Therefore, we introduced the
GS125 mutation into the TR-
2 isoform and used it for the following
experiments. To assess the DNA binding property of the GS125 mutant,
in vitro translated protein was analyzed by gel-shift
studies using various radiolabeled TRE probes. A DR+4 probe was
employed as a pTRE and a TRH site 4 probe was utilized as a nTRE. The
site 4 sequence from human TRH promoter is a well established nTRE
(13). As shown in Fig. 2, wild type
TR-
1 and TR-
2 bound to either the DR+4 or TRH site 4 probe as
both a homodimer and as a heterodimer with RXR. In contrast the GS125
mutant was unable to bind to either element in the absence or presence
of RXR. In data not shown, weak binding of the GS125 mutant to a DR+4
probe was noted if the gel-shift study was performed at 4 °C, but
the corresponding wild type TRs also had markedly increasing binding.
Thus, the GS125 mutation displayed significantly reduced affinities for
both positive and negative TREs.
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To demonstrate direct interaction of TR- to the negative TRE
in vivo, a modified avidin-biotin complex DNA binding (ABCD) assay was employed with non-transfected GH3 cells (Fig.
3). 5'-Biotinylated double-stranded DNA
fragments containing either a rat GH pTRE, a human TSH-
nTRE, or
mutated human TSH-
TRE were incubated with whole cell extracts from
GH3 cells in the presence or absence of T3 (21,
47). Bound proteins were precipitated with streptavidin-agarose and
tested by Western blot analysis using a specific TR-
antibody. TR-
was equally recruited to naturally occurring pTRE and nTRE in
the absence or the presence of T3. In the control
experiment, a mutation in the human TSH-
nTRE abrogated TR-
binding (Fig. 3C). These data clearly demonstrated that DNA
binding of TR-
was DNA-dependent on negative as well as
on positive TREs. Next, the DNA-binding affinity of the GS125 TR-
2
mutant was evaluated using whole cell extracts from transfected 293T
cells. Results in Fig. 3D show that transfected TR-
2
bound to the biotinylated positive and negative TRE in a
ligand-independent manner. On the other hand, GS125 TR-
2 binding to
the positive and negative TRE was almost undetectable, which is
consistent with the results observed in gel-shift analysis.
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Because a putative site on the TSH- promoter for TR binding has not
been identified, the interaction of TR-
with the TSH-
promoter
was investigated using a modified chromatin immunoprecipitation (ChIP)
assay in transfected 293T cells (Fig. 3E). In this
procedure, DNA is cross-linked, isolated, and subjected to
immunoprecipitation with an antibody against a specific protein. By
using a TR-
antibody, association of TR-
with TSH-
promoter
was analyzed by PCR with specific primers for the TSH-
promoter. For
these experiments, the human TSH-
promoter pA3Luc plasmid was
cotransfected into 293T cells with either TR-
2 or GS125 mutant
TR-
2, and a PCR for the presence of the hTSH-
promoter was
performed. As demonstrated in the left panel, transfected
TR-
2 interacted equally well with the TSH-
promoter in the
absence or presence of T3. As a control experiment, TK 109 pA3Luc plasmid was transfected and immunoprecipitated with the same
procedure, and a PCR for the TK promoter was performed. As shown in the
right panel, no bands were detected from samples immunoprecipitated with TR-
antibody. In addition, a nonspecific antibody did not precipitate the hTSH-
promoter sequences with transfected wild type TR-
(NC, Fig. 3E). These
results clearly demonstrate that TR-
2 associated specifically with
the TSH-
promoter in cell culture. By contrast, transfected GS125
TR-
2 binding to the hTSH-
promoter in 293T cells was almost
undetectable. Thus, it is clear that the GS125 mutation disrupted the
ability of TR-
to bind to negative thyroid hormone-response elements.
A DBD Mutant of TR-2 Is Defective on Positive and Negative
TREs--
Ligand-dependent transcriptional activation by
the GS125 mutant TR was examined in transient transfection assays in
293T cells using three different pTREs as reporter constructs. As
demonstrated in Fig. 4A,
cotransfection of wild type TR-
2 increased DR+4 TK-Luc activity by
23-fold after treatment with T3. Interestingly,
cotransfection of RXR-
significantly reduced T3-mediated
activation on this reporter. In contrast, T3 stimulation of
this same reporter was not observed with GS125 TR-
2 regardless of
whether RXR-
was cotransfected. Similar results were obtained with
the other pTREs: LAP-TKLuc (Fig. 4B) and PAL-TKLuc (Fig.
4C) reporters. The GS125 DBD mutant, therefore, completely
abolished T3-dependent transactivation of
TR-
2 on pTREs.
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To determine whether negative regulation by T3 required TR
DNA binding, wild type or GS125 mutant TR-2 plasmids were
cotransfected into 293T cells with each of the three negatively
regulated gene promoters, TRH-Luc, TSH-
Luc, and TSH-
Luc, and a
control promoter, TK109 (Fig. 5). After
treatment with T3, wild type TR-
2 suppressed these
negatively regulated gene promoter activities to ~40% of basal
activities, whereas activity from the control reporter was not
significantly changed. Moreover, RXR-
cotransfection did not
significantly increase negative regulation by T3. However, the GS125 mutant TR-
2, which lacks DNA-binding activity,
paradoxically increased activity by 10-20%. Cotransfection of RXR-
did not alter these results. The reduction in transactivation and
transcriptional repression was not related to the quantity or stability
of the mutant TR-
2 protein in tissue culture, as the mutant receptor accumulated to a level similar to the wild type receptor as
demonstrated by Western blot analysis of nuclear extracts prepared from
transfected 293T cells (Fig. 5A). Thus, the GS125 DBD mutant
has no measurable repressive function on negatively regulated
genes.
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The GS125 DBD Mutant Is Functional on a Composite TR/GR-response
Element--
To demonstrate that the GS125 TR-2 is a functional
nuclear hormone receptor, we wished to determine whether it had
activity on a natural or hybrid thyroid hormone-response element.
Because the first two amino acids of the P box in GS125 TR-
2
resemble those found in the GR, we tested its function on a naturally
occurring glucocorticoid receptor element (GRE), mouse mammary tumor
virus promoter in 293T cells. After treatment with T3, the
GS125 mutant was unable to activate the mouse mammary tumor virus
promoter (data not shown). Next, the GS125 mutant TR-
2 was tested
for DNA binding on hybrid TR/GR-response elements. Hybrid TR/GREs contained mutations of the third and fourth nucleotides of the downstream half-site hexamer, as shown in Fig.
6B, whereas the upstream TRE
half-site and the direct repeat motif with four-nucleotide gap spacing
were preserved for binding of heterodimeric partner, RXR. As shown in
Fig. 6A, wild type TR-
2 and GS125 TR-
2 were able to
bind to all three hybrid TR/GRE elements as heterodimers with RXR-
.
For the most part, GS125 TR-
2 was able to bind to the hybrid TR/GRE
3 with much higher affinity than that of the wild type TR-
2, and
this element was chosen for further analysis.
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A composite TR/GR-response element 3 (AGGTCA cagg AGAACA) was inserted
as two copies into the TK-Luc reporter plasmid, and the transcriptional
activity of the GS125 TR-2 was tested on this reporter in 293T cells
with and without cotransfected RXR-
(Fig. 6C). The GS125
mutant in the presence of T3 was very active on this
element, whereas wild type TR-
2 showed an insignificant increase.
Moreover, the transcriptional activation of GS125 TR-
2 was
significantly increased after cotransfection with RXR-
, suggesting that binding of GS125 TR-
2 was enhanced in the presence of
cotransfected RXR-
. These data indicate that the GS125 DBD mutant
was expressed in the nucleus and acted as a functional TR on the
composite TR/GR-response element.
The GS125 DNA-binding Mutant Can Interact with Transcriptional
Cofactors--
To characterize the GS125 mutant function further,
interactions between GS125 TR-2 and transcriptional corepressors and
coactivators were examined in a mammalian two-hybrid assay.
Gal4DBD-NcoR and -SRC-1, which contain their respective nuclear hormone
receptor interaction domains, were tested for their ability to interact with wild type and GS125 mutant TR-
2 (Fig.
7). Nuclear corepressor protein (NCoR)
mediates ligand-independent repression of TRs by forming a complex with
histone deacetylase activities in the absence of ligand (48). Gal4-NCoR
interacts with VP16-TR-
1 and VP16-TR-
2 fusion proteins in the
absence of T3, resulting in a 5-6-fold activation of the
UAS-TK-Luc reporter. In the presence of T3, Gal4-NCoR
dissociated from VP16-TRs and as a result the activity of UAS-TK-Luc
was reduced to basal levels. Cotransfection of VP16-GS125TR-
2 led to
the same results as found with both isoform of wild type TR-
,
indicating that the mutant interacted normally with NCoR.
|
In the presence of T3, SRC-1 associates with TRs and
mediates ligand-dependent transcriptional activation by
TRs. In the presence of T3, a VP-16 fusion of the TR-
induced activity of UAS-TK-Luc by bound Gal4-SRC-1 by 6-8-fold. Again
the GS125 mutant TR-
2 displayed an equivalent interaction as
compared with wild type TR-
2. Finally, the heterodimerization
property of GS125 was examined with this assay. Coexpression of
Gal4-RXR-
and VP16-TR-
s resulted in a significant increase in
reporter gene activity and the addition of T3, enhancing
activities by 5-7-fold; equivalent results were observed with the
mutant. These data indicate that GS125 DBD mutation did not interfere
with corepressor, coactivator, or RXR interactions, indicating that all
of the important functional domains of this mutant TR, except DNA
binding, are intact.
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DISCUSSION |
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In the present study, we characterized the DNA binding domain
mutant GS125 TR-2, which has a two amino acid substitution within
the P box (GSG for wild type
EGG). GS125 TR-
2 exhibited functional ligand
binding and corepressor and coactivator binding properties similar to
wild type TRs, except for its inability to bind to positive and
negative TREs. The GS125 DBD mutant was defective in both
ligand-induced transactivation on pTRE reporter genes and
ligand-induced transrepression on the negatively regulated gene
promoters, crucial in regulating the H-P-T axis.
Negative feedback regulation of the H-P-T axis has been well studied
because of its physiological importance. A number of reports have
identified the element responsible for negative regulation and proposed
mechanisms and target cofactors based on the traditional "on-DNA"
hypothesis. The negative TRE within the human TSH- genes is composed
of single half-site motifs present near the TATA box and downstream of
the transcription start site, which interact with the TR monomer (21,
22). The human TRH promoter contains three nTREs, including separate
half-sites which are responsible for
T3-dependent negative regulation and binding of
TR. Site 4 in the TRH gene, which is capable of binding not only TR
monomers but also TR homodimers and TR-RXR heterodimers, may mediate
the enhancement of negative regulation by the RXR (13).
On the contrary, an "off-DNA" hypothesis for negative regulation
was proposed by Tagami et al. (49). They suggested that the
DNA-binding domain of TR- and/or direct DNA binding of TR was not
necessary in TR-mediated control of negatively regulated genes. A
question was raised, therefore, about whether negative TREs truly
existed. These investigators utilized a DBD mutant, however, which
would be predicted to disrupt the overall structure of the DBD, making
its effect on gene regulation difficult to interpret. In this study,
the GS125 DBD mutant provides a strong argument in support of the
on-DNA hypothesis even on the TSH-
promoter. Clearly, by
using this mutant, DNA binding by TR-
on negatively regulated gene
is essential for the T3-mediated transcriptional regulation.
Because nTREs do not contain high affinity TR DNA-binding sites when
tested by gel-shift analysis, it has been suggested that these regions
might function as a composite site involving the receptor and other
transcriptional cofactors. A newer model suggested that T3
induced recruitment of TR and histone deacetylase to the TSH- nTRE
resulting in negative regulation in GH3 cells (50). This
group suggested that TRs interacted directly with HDAC2 through the
DNA-binding domain on the negative TRE of the TSH-
promoter. We
employed an avidin-biotin complex DNA binding assay (ABCD assay) using
biotinylated oligonucleotides to test the effect of ligand on DNA
binding to the same nTRE. We succeeded in detecting direct DNA binding
of TR-
in GH3 nuclear extracts to the nTRE from human TSH-
gene using Western blot analysis and a TR-
-specific
antibody; T3 had no effect on the extent of TR-
binding.
DNA binding was abolished on a mutant human TSH-
fragment,
indicating that binding was specific to the TSH-
nTRE (Fig.
3A). These results are in accordance with other studies in
which DNA binding is required for TR-
-mediated negative regulation.
We found no evidence to support a T3-mediated recruitment
of TR to the TSH-
nTRE.
A number of studies have reported the effects of P box substitutions on
TR DNA binding or the responsiveness of TRs on thyroid hormone-response
elements (51-56). However, previously described DBD mutants would be
predicted to disrupt the overall -helical structure of the DBD or
other functional domain structures. With these DBD mutants, it is
difficult to conclude that loss of transcriptional activities is
specifically caused by the lack of DNA binding versus other
changes in the receptor. In this paper, the GS125 mutation was a P box
mutation substituting GSG for the wild type EGG sequence found in TR. This unique DBD mutant
TR-
, which was predicted to bind to a GRE half-site, was able to
bind to the hybrid TR/GR-response element and was fully functional on
this composite DNA element as a T3-responsive nuclear
receptor. T3-dependent transactivation of GS125
TR-
on the hybrid TR/GR-response element was dramatically increased
in the presence of RXR-
(Fig. 6). These data indicate that DNA
binding of GS125 TR-
was RXR-dependent in this
particular GR/TR composite response element. Finally, the GS125 mutant
preserved interactions with transcriptional cofactors as shown in the
mammalian two-hybrid assays. These results also support the conclusion
that the GS125 mutant TR is a fully functional nuclear hormone receptor.
TRs are encoded by two distinctive genes, c-erbA- and
-
(57, 58). Multiple isoforms of TRs are generated from two genes, and their expressions are regulated in specific temporal and spatial patterns (27). TR-
2 has tissue-specific expression in the anterior pituitary gland and the specific areas of the hypothalamus as well as
brain and inner ear (7, 46, 59). It has known that TR-
2 isoforms
play a key role in the feedback regulation of H-P-T axis (43, 44). It
is possible that GS125 DBD mutants TR-
might have various phenotypes
in tissue-specific pattern in the in vivo study. For
example, it was reported that DNA binding of the GR was necessary for
some but not all feedback effects in the hypothalamic-pituitary-adrenal
axis (60).
In summary, DNA binding of TR- is required for thyroid
hormone-mediated transcriptional regulation of both positively and negatively regulated genes. The GS125 DNA-binding mutant establishes the importance of DNA binding in negative regulation of gene
transcription mediated by thyroid hormone receptors.
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FOOTNOTES |
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* 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.
¶ To whom correspondence should be addressed: Section of Endocrinology, Dept. of Medicine, the University of Chicago, 5841 S. Maryland Ave., MC 1027, Chicago, IL 60637. Tel.: 773-702-6217; Fax: 773-834-0486.
Published, JBC Papers in Press, November 4, 2002, DOI 10.1074/jbc.M207264200
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ABBREVIATIONS |
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The abbreviations used are: H-P-T axis, hypothalamic-pituitary-thyroid axis; TRH, thyrotropin-releasing hormone; TSH, thyroid-stimulating hormone; T3, thyroid hormone; TR, thyroid hormone receptor; TRE, thyroid hormone-response elements; pTRE, positive thyroid hormone response element; nTRE, negatively regulated thyroid hormone response element; RXR, retinoid X receptor; DBD, DNA binding domain; LBD, ligand binding domain; NcoR, nuclear receptor corepressor; SRC-1, steroid receptor coactivator-1; GR, glucocorticoid receptor; ChIP, chromatin immunoprecipitation; UAS, upstream activating sequence; GRE, GR-response element.
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