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INTRODUCTION |
Thyroid hormone receptors
(TRs)1 are ligand-activated
transcription factors that mediate the biological effects of thyroid hormone (T3). TRs, along with the receptors for steroid
hormones, retinoids, and vitamin D, belong to the nuclear hormone
receptor superfamily (1). Like other nuclear receptors, TRs exhibit a
modular structure with separable functional domains. A
ligand-independent transcription activation function (AF-1) is located
in the highly variable N-terminal A/B domain (2). The highly conserved
DNA binding domain containing two zinc finger motifs is centrally located and characterizes the members of the superfamily (1). The
moderately conserved C-terminal ligand binding domain (LBD) possesses a
strong receptor dimerization interface and a
ligand-dependent activation function, AF-2 (3-5).
TRs exert their effects on gene expression through direct interaction
with specific DNA sequences known as thyroid hormone response elements
(TREs), usually found in the 5'-flanking regions of
T3-responsive genes. Unlike steroid hormone receptors, TRs interact with TREs even in the absence of ligand. TREs usually are
composed of two or more receptor-binding hexameric half-sites related
to the sequence AGGTCA arranged as direct repeats (6) or, less
commonly, everted repeats (7).
Although TRs are capable of binding to TREs without auxiliary factors,
they preferentially bind to many TREs as heterodimers with retinoid X
receptors (RXRs) (8, 9). Based on this observation, it is thought that
TRs activate gene expression primarily as heterodimers with RXR
(reviewed in Refs. 1, 10, and 11). However, the physiological role of
RXR in TR action remains to be elucidated, largely because RXR-null
mammalian cells do not exist. It has been shown that TRs and RXRs
prefer to bind to different DNA sequences T(A/G)AGGTCA and GGGGTCA (12,
13), respectively. Therefore, we postulate that certain TREs are
primarily responsive to TR, whereas others require RXR-TR heterodimers
for gene activation. To test this hypothesis, Saccharomyces
cerevisiae was used as a model system. S. cerevisiae is
devoid of endogenous nuclear receptors and thus can be manipulated to
express TRs and/or RXRs, which allows for the analysis of receptor
requirements dictated by the TRE for TR-mediated gene expression.
Transcriptional regulation by TRs or other nuclear receptors is
accomplished by the concerted action of an array of coregulatory proteins including coactivators and corepressors (14). In mammalian cells, the binding of unliganded TRs to "positive" TREs results in
repression of transcription, which is mediated by interaction of the TR
with a corepressor complex (15, 16). The binding of ligand triggers
conformational changes in the TR LBD that result in release of the
corepressor complex and recruitment of coactivators (17, 18), thereby
leading to gene activation. Little is known about the potential
influence of TRE sequence on coactivator function. Yeast lack
endogenous proteins homologous to mammalian corepressors and
coactivators and thus provide a null background that should be
advantageous for gaining insight into this issue.
In the present report, 10 naturally occurring mammalian TREs were
studied in a yeast model system to test the importance of RXR for the
transcriptional activity of TR. The data demonstrate that thyroid
hormone response element sequence plays an important role in
determining the nuclear hormone receptor and coactivator requirements
for TR action.
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EXPERIMENTAL PROCEDURES |
Yeast Reporter Strains, Expression Plasmids, and Site-directed
Mutagenesis--
Yeast strains bearing chromosomally integrated
reporter genes were constructed as described previously (19). In brief,
double-stranded oligonucleotide response elements were inserted into
the BglII site upstream of a basal cytochrome c
promoter (cyc1) linked to a
-galactosidase reporter gene
in the yeast integrating plasmid p83:305. The constructs were confirmed
by sequencing and were integrated into the chromosome of S. cerevisiae strain SEY6210 (MAT
;
ura3-52; leu2-3,-112; his3-200; trp1-901; lys2-801; suc2-9) by
homologous recombination at the leu2 locus. Strains
bearing single reporter construct insertions were identified by
Southern analysis and were selected for further study. The
pRSGalH-TR
1, pRSGalH-TR
1-RXR
, and p413-TEF-TIF2 expression
plasmids were described previously (19). pRSGalH-TR
1(E457A)
and pRSGalH-TR
1(L454A) were generated using the Stratagene
QuikChange mutagenesis kit and confirmed by sequencing.
Induction of Reporter Gene Expression and
-Galactosidase
Activity Assays--
Yeast strains were transformed with
pRSGalH-TR
1, pRSGalH-TR
1-RXR
, p413-TEF-TIF2, or the empty
expression vectors. Resultant transformants were grown overnight in an
appropriate selection medium supplemented with 3% glycerol and 3%
ethanol, a nonrepressive carbon source that does not induce nuclear
receptor expression. Cultures were diluted to an absorbance of 0.2 at
650 nm and grown overnight at 30 °C in the presence of 3%
D-galactose (to induce nuclear receptor expression) with or
without 1 µM 3,5,3'-triiodothyroacetic acid (triac), a
T3 analog. Following overnight growth, the
A650 of each culture was measured as an
indicator of cell density. One ml of each culture was pelleted by
microcentrifugation, and the pellets were frozen at
20 °C for
-galactosidase activity assay (20). In brief, frozen cells were
thawed, resuspended in 0.7 ml of reaction mixture containing 25 mM Tris, pH 7.5, 125 mM NaCl, 2 mM
MgCl2, and 12 mM
-mercaptoethanol, and
solubilized with 2 drops of both chloroform and 0.1% SDS (21). After
vortexing, 40 µl of cell suspension were incubated with 160 µl of
reaction mixture containing 0.3 mM
4-methylumbelliferyl-
-D-galactoside (Sigma) at 37 °C.
After 1 h, 1.9 ml of stop solution containing 133 mM
glycine, 83 mM Na2CO3 (pH 10.7) was
added to terminate the reaction. Fluorescence was measured with 50 nM 4-methylumbelliferone (Sigma) used as a standard to read
500 (excitation wavelength 365 nm, emission wavelength 460 nm).
Production of TR
1, RXR
, and TIF2 in E. coli--
Full-length rat TR
1, full-length mouse RXR
, and the
human TIF2 nuclear receptor interaction domain (amino acids 624-869) (22) were expressed in Escherichia coli as glutathione
S-transferase (GST) fusion proteins using the vector pGEX-KG
(23). E. coli strain BL21 carrying the fusion protein
expression vectors were grown at 30 °C. The cultures were induced
with 0.1 mM
isopropyl-
-D-thiogalactopyranoside for 2.5 h at
30 °C. To increase the solubility of the receptor fusion proteins, a
plasmid expressing E. coli thioredoxin (24) was coexpressed
with GST-TR
1 or GST-RXR
. After harvesting, the cells were
resuspended in a buffer containing 150 mM NaCl, 10 mM sodium phosphate, pH 7.5, 2 mM EDTA, 5 mM dithiothreitol, and 1 tablet of Protease Inhibitor
Mixture (Roche Molecular Biochemicals) per 25 ml, and passed through a
French press twice at 1200 lb/in2. The bacterial lysates
were centrifuged at 10,000 × g for 15 min, and the
supernatants were incubated with 1 ml of glutathione-agarose beads at
4 °C for 1 h. After washing three times with cold phosphate buffer and once with thrombin cleaving buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl, 2.5 mM CaCl2, 2 mM dithiothreitol, the
agarose beads were resuspended in 1 ml of thrombin cleaving buffer. The
TR
1, RXR
, and TIF2 receptor interaction domains were cleaved from
GST on the agarose beads by addition of 3.6 units of thrombin (Novagen,
Madison, WI), incubated for 30 min at room temperature, and purified by centrifugation.
Electrophoretic Mobility Shift Assays--
To perform EMSAs,
double-stranded synthetic oligonucleotides were radiolabeled with
[32P]dCTP by fill-in reactions using the Klenow fragment
of DNA polymerase. The oligonucleotide probes used are listed in Table
I. 1.5 × 104 cpm of radiolabeled oligonucleotides
were incubated with 0.25 µg of purified TR
1, RXR
, or both
together. Where indicated, incubations also included 0.2 µg of the
purified TIF2 nuclear receptor interaction domain. Binding reactions
were carried out at room temperature for 40 min in a total volume of
17.5 µl in binding buffer containing 20 mM HEPES, pH 7.8, 50 mM KCl, 1 mM dithiothreitol, 0.1% Nonidet
P-40, 20% glycerol, and 0.9 µg of poly(dI·dC). After incubation,
the mixtures were analyzed on 6% nondenaturing acrylamide gels using
0.25× Tris borate/EDTA buffer. The gels were fixed, dried, and
analyzed by autoradiography and/or PhosphorImager analysis.
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RESULTS |
Influence of the TRE Sequence on the Role of RXR in TR-mediated
Gene Expression--
Ten naturally occurring direct repeat TREs (Table
I) were examined in yeast. The TREs were
inserted upstream of a basal cyc1 promoter linked to a
-galactosidase reporter (19) and then integrated into the chromosome
of S. cerevisiae.
-Galactosidase activity assays were
performed with yeast reporter strains, in which TR
1, RXR
, or both
together were expressed. Data are expressed as
-galactosidase
activity relative to identical yeast strains with an empty nuclear
receptor expression vector (fold induction). As shown in Fig.
1, TR mediated reporter gene induction
from each of these TREs in the absence of thyroid hormone. This
ligand-independent reporter gene stimulation reflects the function of
the AF-1 domain (2), as it is uninfluenced by mutations in AF-2 (see
Fig. 9) or by the expression of AF-2-dependent coactivators
(data not shown). The focus of these studies is not on the absolute
magnitude of reporter gene induction but on the relative reporter gene
induction seen in the presence of RXR + TR versus that seen
in the presence of TR alone. TREs exhibit a continuous spectrum of RXR
dependence, which is reflected in the ratio of
-galactosidase
activity induced by RXR + TR to TR alone (Fig.
2). The higher the ratio, the more the
RXR dependence. At one end of this spectrum, RXR is virtually required
for reporter gene activation, as illustrated by the rat
-myosin
heavy chain (r
MHC) TRE. Coexpression of RXR
and TR
1 resulted
in a 4.2-fold increase (from 1.7- to 7.3-fold induction) in reporter
gene activation as compared with expression of TR
1 alone (Fig. 1,
lane 1, and Fig. 2). At the other end of the spectrum, reporter gene activation from the rat uncoupling protein 1 (rUCP) TRE
was almost entirely independent of RXR. TR
1 alone activated reporter
gene expression 5.3-fold, and the coexpression of RXR only resulted in
a further increase of 1.2-fold (Fig. 1, lane 10, and Fig.
2).
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Table I
Gene abbreviations and T3 response element sequences
The top strands of the TRE oligonucleotides are shown without the
5'-GATC overhangs. The receptor binding half-sites are underlined and
the dinucleotides TA or TG upstream of the 5' half-sites are in
boldface.
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Fig. 1.
Different RXR requirements for
ligand-independent TR-mediated reporter gene activity in yeast.
-Galactosidase activities of yeast strains bearing chromosomally
integrated single copies of the lacZ gene driven by a basal
cyc1 promoter and the naturally occurring direct repeat
response elements (TREs) were measured while TR 1, RXR , or both
together were expressed. All data are represented as -galactosidase
activity (fold induction) relative to identical yeast
strains with an empty nuclear receptor expression vector. TREs are
arranged from being almost fully dependent on RXR for TR induction of
reporter gene activity (r MHC) toward being
essentially independent of RXR (rUCP). The expression of RXR
alone did not induce -galactosidase activity from any of these TREs
(data not shown). Results are the mean ± S.E. of four independent
experiments.
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Fig. 2.
RXR dependence of TR-mediated reporter gene
expression. The ratio of -galactosidase activity induced by RXR
plus TR to TR alone was calculated from the data of Fig. 1 to reflect
the RXR dependence of TR-mediated reporter gene expression. The
sequences of the TRE oligonucleotides are shown without the 5'-GATC
overhangs. The dinucleotide TG or TA upstream of the 5' half-site is in
boldface, and the receptor binding half-sites are
underlined.
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Comparison of the TRE sequences with in vivo reporter gene
activities (Fig. 2) indicates that independence of RXR for reporter gene activation correlates relatively well with the presence of the
dinucleotide TG or TA 5' to the upstream half-site. For example, as
shown in Fig. 2, rUCP and hPL possess a TG upstream of the 5' core
hexamer and display minimal RXR dependence for TR-mediated reporter
gene expression. In contrast, RXR dependence is maximal for r
MHC,
rGH, and rP450, none of which contains the dinucleotide T(A/G)
upstream of the 5' core hexamer. Since the polarity of RXR-TR
heterodimers is such that RXR occupies the 5' half-site (25, 26), it is
reasonable that the TRE 5' half-site sequence is the one that
influences RXR activity.
To assess further the importance of the 5' T(A/G) upstream of the core
hexamers, we generated a mutant r
MHC reporter construct in which the
first G of the 5' dinucleotide GG was replaced by T, creating
r
MHC(TG) (Table I). As predicted and shown in Fig. 3, expression of TR
1 alone led to a
5-fold induction of
-galactosidase activity from r
MHC(TG), and
addition of RXR resulted in a further increase of only 1.4-fold (Fig.
3A). In contrast, expression of TR
1 alone led to only a
1.7-fold induction from wild type r
MHC, but this increased 4.3-fold
with the addition of RXR (Fig. 3B). These data demonstrate
that a single substitution of T for G two nucleotides 5' to the
upstream core hexamer is capable of shifting the nature of the r
MHC
TRE from more RXR dependence to less RXR dependence.

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Fig. 3.
The effect of thymine 2 bp upstream of the 5'
core hexamer on TR-mediated reporter gene expression. A,
-galactosidase activity of the r MHC(TG) reporter construct was
measured while TR 1, RXR , or both together were expressed in the
absence of ligand. The expression of RXR alone did not induce
-galactosidase activity (data not shown). The sequence of the
oligonucleotide is shown at the top of the panel. The
dinucleotide TG upstream of the 5' half-site is in boldface,
and the receptor binding half-sites are underlined. Results
are the mean ± S.E. of four independent experiments.
B, -galactosidase assays were carried out as per
A but with the wild type r MHC reporter construct. The
expression of RXR alone did not induce -galactosidase activity
(data not shown). The sequence of the oligonucleotide is shown at the
top of the panel. The receptor binding half-sites are
underlined. Results are the mean ± S.E. of four
independent experiments.
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In summary, the results confirm that RXR is not always required for
TR-mediated gene activation. The dinucleotide T(A/G) 5' to the upstream
TRE half-site is important in determining the RXR requirement for
TR-mediated gene expression. However, RXR dependence is not all-or-none
but, rather, varies continuously. This suggests that, in addition to
the 5' T(A/G), the sequence and spacing of the TRE half-sites may
modify the RXR dependence of TR-mediated gene expression from certain TREs.
Differential Binding of TR Alone or RXR-TR Heterodimers to T3
Response Elements--
To determine whether the different requirements
of RXR for TR-mediated gene expression correlate with the different
binding abilities of RXR-TR heterodimers versus TR alone to
these TREs, we performed EMSAs using purified recombinant TR
1 and
RXR
. Fig. 4A shows that
TR
1 itself is able to bind to almost all these natural response
elements, although the level of binding varies considerably. The
strongest binding of TR
1 is seen with hDI, rSerCa2, hPL, and rUCP
(lanes 6, and 8-10). These TREs generally exhibit a relatively high
-galactosidase activity induced by TR
1
alone or less RXR dependence for TR-mediated gene activation (Figs. 1
and 2). However, the binding of RXR-TR heterodimers to rSerCa2, hPL,
and rUCP was essentially undetectable (Fig. 4B, lanes
8-10). In contrast, TREs such as r
MHC, rP450, and rME that display more dependence on RXR for gene activation support the formation of RXR-TR heterodimers (Fig. 4B, lanes 1, 3, and 4) but bind TR
1 alone weakly relative to
rSerCa2, hPL, and rUCP (Fig. 4A, lanes 1, 3, and
4 versus 8-10). hDI binds well both to RXR-TR
heterodimers and to TR alone and thus is intermediary in RXR dependence
for gene activation. The correlation between RXR-TR DNA binding and RXR
dependence of yeast reporter gene activity was evaluated by plotting
the ratio of (RXR + TR heterodimer DNA complexes)/(TR DNA complexes)
determined by EMSA versus the reporter gene induction data
(Fig. 4C). This plot shows that TREs capable of forming
stronger RXR-TR heterodimers are more heavily dependent on RXR for gene
activation.

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Fig. 4.
A, binding of TR 1 to natural TREs
analyzed by EMSA. EMSAs were performed with purified E. coli-derived full-length TR 1 and 32P-labeled
double-stranded probes corresponding to the TREs described in Table I.
Protein-DNA complexes were separated by nondenaturing polyacrylamide
gel electrophoresis. This experiment was repeated once with identical
results. B, binding of RXR -TR 1 to natural TREs
analyzed by EMSA. EMSAs were performed with purified E. coli-derived full-length TR 1, RXR , and
32P-labeled double-stranded probes corresponding to natural
TREs described in Table I. Protein-DNA complexes were separated by
nondenaturing polyacrylamide gel electrophoresis. Arrows
indicate the mobility of DNA complexes with RXR-TR heterodimers, TR
alone, and RXR alone. Complexes were identified by comparing the
results to parallel EMSAs with TR alone or RXR alone (data not shown).
This experiment was repeated once with identical results. C,
plot of RXR dependence of yeast reporter gene activity and TR-DNA
binding by EMSA. EMSA quantitations of RXR-TR-DNA complexes and TR-DNA
complexes were carried out by PhosphorImager analysis. The data of the
ratio of -galactosidase activity induced by RXR plus TR to TR alone
in the absence of triac were derived from Fig. 2. D, binding
of RXR and TR 1 to the r MHC and r MHC(TG) TREs analyzed by
EMSA. EMSAs were performed with purified E. coli-derived
full-length TR 1, RXR , or both together and the
32P-labeled double-stranded probes r MHC and r MHC(TG).
Protein-DNA complexes were separated by nondenaturing polyacrylamide
gel electrophoresis. Arrows indicate the mobility of RXR-TR
heterodimers, RXR-DNA complexes, and TR-DNA complexes. This experiment
was repeated twice with identical results.
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Since we observed that the mutant TRE r
MHC(TG) exhibits increased
reporter gene induction by TR alone and less RXR dependence, we were
interested in determining if the binding of TR to this mutant TRE also
is altered. EMSA showed that, as expected, the binding of TR
1 to
r
MHC(TG) was greatly enhanced when compared with the binding to wild
type r
MHC (Fig. 4D). This indicates that a single base
change in a TRE can regulate the importance of RXR for both TR binding
and gene activation.
Taken together, these data suggest that the different binding
affinities of TR alone versus RXR-TR heterodimers account in large part for the different requirements of RXR for TR-mediated gene
expression. The importance of the 5' dinucleotide T(A/G) in determining
the RXR requirement for TR-mediated gene expression appears to be
through enhancing TR binding, at least for certain TREs.
Effect of Coactivators on TR-mediated Reporter Gene
Expression--
Nuclear hormone receptors activate transcription in a
ligand-dependent manner through an interaction of their
AF-2 domains with coactivator proteins. The p160 coactivator family has
previously been shown to induce hormone-dependent
transcriptional activation on the artificial T3 response
element 8DR4 (TAAGGTCATCTAAGGTCA) in a yeast expression system (19). We
asked whether the related p160 coactivator TIF2 (18) could potentiate
hormone-dependent gene activation from these natural TREs.
To do so, the previously studied yeast strains were transformed with an
expression vector for TIF2 (or empty vector), and
-galactosidase
activity was measured following incubation with or without triac, a
T3 analog.
In the absence of coactivator,
-galactosidase activity driven by all
of these natural TREs was not increased by triac. Instead, triac caused
a small (20%) but reproducible suppression of
-galactosidase activity (data not shown). As shown in Fig.
5, ligand induction of reporter gene
expression from almost all these TREs could be restored by coexpression
of TIF2 and TR
1. However, the level of ligand-induced reporter gene
expression varied considerably among the TREs. For example,
-galactosidase induction from rUCP was stimulated 6.1-fold by triac
(Fig. 5, lane 10, solid bar), whereas induction from r
MHC
was stimulated only 1.6-fold (lane 1, solid bar).
Interestingly, coexpression of RXR
with TR
1 and TIF2 reduced the
ligand induction of reporter gene expression on nearly all TREs. For
example, the triac induction from rUCP fell from 6.1- to 2.9-fold when
RXR was coexpressed (lane 10). Thus, in general, TIF2
mediated ligand-dependent reporter gene expression is
stronger when TR alone is present than TR and RXR.

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Fig. 5.
Restoration of ligand-dependent
TR-mediated reporter gene expression by the coactivator TIF2.
-Galactosidase activities of the reporter constructs were measured
while TR 1 ± RXR were expressed in the absence or presence of
triac. The data are represented as the ratio of reporter activity with
triac to without triac. Results are the mean ± S.E. of 4-6
independent experiments. Triac inductions were significantly reduced by
the expression of RXR for lanes 3-11 (p 0.01 except lane 5 p = 0.03, t
test). A reporter construct lacking a TRE lacked triac induction
(0.9-fold; data not shown).
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In addition, a strong triac-dependent increase (3.4-fold)
in reporter gene activation was observed from r
MHC(TG) when TIF2 and
TR
1 were coexpressed (Fig. 5, lane 11, solid bar). In the context of wild type r
MHC, such TIF2-mediated
ligand-dependent activation was only 1.6-fold (Fig. 5,
lane 1). This difference is consistent with the enhanced
ability of r
MHC(TG) to bind TR in the absence of RXR and
substantiates the preference of TIF2 as a coactivator for TR alone
rather than RXR-TR.
Strong triac induction was seen only with the hDI, rUCP, and MHC(TG)
TREs, all of which bind TR well in the absence of RXR. However, the
rSerCa2 and hPL TREs also bind TR well, even though they support only
minimal triac induction. To investigate this further, EMSAs were
performed with these TREs and recombinant TR ± TIF2 (Fig.
6). Two distinct TR-TIF2-TRE complexes
were identified, the slower of which correlated with triac induction in
the yeast reporter gene assay. This slower complex was abolished by
mutation of the 5' TRE half-site (lane 8), suggesting that
it represents a TR homodimer-TIF2 complex.

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Fig. 6.
Formation of TR-TIF2 complexes on TREs
analyzed by EMSA. EMSAs were performed with TR 1, T3, and
32P-labeled double-stranded probes corresponding to the
TREs described in Table I, ± the TIF2 nuclear receptor interaction
domain (TIF2 amino acids 624-869). This gel was run longer than usual
to resolve the two TR-TIF2-DNA complexes, resulting in the free DNA
running off the bottom of the gel. Thus, all free probe lanes
demonstrated no radioactivity; this is shown only for SerCa2,
lane 1. The TR-DNA complexes are at the bottom of the gel,
as illustrated by lane 10, which contains TR without TIF2
(similar results were obtained with all TREs, data not shown). TIF2
alone failed to bind to these TREs (data not shown). The TR-TIF2-DNA
complexes are marked by arrows. This experiment was repeated
twice with identical results.
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The decrease in triac induction of reporter gene expression when RXR
was coexpressed with TR also was investigated by EMSA. The TR-TIF2
complex with the rUCP TRE was greatly inhibited by the addition of RXR
(Fig. 7, lane 2 versus 4), consistent with the loss of triac
induction.

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Fig. 7.
Inhibition of TR-TIF2-DNA complex formation
by RXR. EMSAs were performed with TR 1, T3, and
32P-labeled rUCP TRE, ± the TIF2 nuclear receptor
interaction domain (TIF2 amino acids 624-869) and RXR as indicated.
The TR-TIF2-DNA complex is marked by the arrow. This
experiment was repeated twice with identical results.
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Mutational and x-ray crystallographic studies have demonstrated that
the AF-2 domain of nuclear hormone receptors plays a critical role in
mediating ligand-dependent transcriptional activation (5,
27). To assess whether the restoration of ligand-dependent reporter gene expression by TIF2 in these studies also is mediated through the AF-2 domain, the point mutations E457A or L454A were introduced into the TR
1 AF-2 domain. Previous studies have shown that either E457A or L454A impairs AF-2 function in mammalian cells but
does not affect the ability of the receptor to bind to DNA or to
thyroid hormone (28). As shown in Fig. 8,
these mutations impaired the ability of TIF2 to support triac induction of
-galactosidase activity. Although the effect of the E457A mutation was modest, L454A decreased triac induction by ~80%. This
is not likely due to diminished expression, as L454A has wild type
triac-independent AF-1 activity. Thus, as expected, TIF2 function is
AF-2 dependent in yeast as it is in mammalian cells.

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Fig. 8.
Effect of TR 1 AF-2
mutations on TIF2-mediated ligand stimulation of reporter gene
expression. -Galactosidase activities of reporter constructs
were measured while TR 1 or TR 1 point mutants (E457A or L454A)
were expressed with TIF2 in the absence or presence of triac. Results
are the mean ± S.E. of four independent experiments.
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In the absence of coactivators such as TIF2, triac modestly represses
TR-mediated reporter gene activation. Surprisingly, as shown in Fig.
9, the TR
1 AF-2 mutations relieve this
triac repression seen in the absence of TIF2. As expected, however, the
TR
1 AF-2 mutations have no effect on ligand-independent gene activation, as this is mediated through the N-terminal AF-1 domain.

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Fig. 9.
Effect of TR 1 AF-2
mutations on reporter gene expression in yeast. -Galactosidase
activities of reporter constructs were measured while TR 1 or TR 1
point mutants (E457A or L454A) were expressed in the absence or
presence of triac. Results are the mean ± S.E. of four
independent experiments.
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DISCUSSION |
It is widely held that, in mammalian cells, gene activation by
thyroid hormone receptors is mediated via RXR-TR heterodimers (reviewed
in Refs. 1, 10, and 11). This model has developed from two
observations. 1) RXRs were identified as proteins that heterodimerize
with and increase the DNA binding of TRs (8, 9, 29). 2)
Cotransfection of mammalian cells with RXR can increase the
T3 induction of reporter genes (29). However, the importance of variations in TRE sequences was not recognized when the
"RXR hypothesis" was developed. In fact, the above studies utilized
either the rat
-myosin heavy chain TRE (29) or inverted repeat
sequences that function as promiscuous nuclear receptor response
elements in transfections but that are not known to function as natural
TREs in vivo (9, 30, 31). Naturally occurring TREs generally
are direct repeats (or occasionally everted repeats), and as shown in
this paper, the
MHC direct repeat is more dependent on RXR for
TR-DNA binding than are most other direct repeat TREs. Thus,
inadvertently, initial investigations were performed using response
element sequences that favored the role of RXR-TR heterodimers.
There is only one published report that systematically assessed the
effect of cotransfected RXR on T3 inductions from several TREs in mammalian cells (32). In that publication, COS, ES, and JEG-3
cells were transfected with 7 different TRE-chloramphenicol acetyltransferase reporter plasmids along with TR ± RXR. The 7 TREs are difficult to compare because they differ not only in sequence
but also in the number of receptor-binding sites (2, 3, or 4) and the
orientations of the binding sites. Nevertheless, certain trends were
apparent. In JEG-3 cells, RXR failed to enhance gene expression from
any of the TREs. In COS and ES cells, reporter gene inductions
increased anywhere from nil to 3-fold, with most TREs showing small
increases of 25-50%. Furthermore, stronger effects did not strictly
correlate with lower endogenous RXR levels. For example, the TRE that
was most affected by cotransfected RXR in COS cells (rGH TRE) showed a
3-fold increase in gene induction. However, gene expression from this
TRE was not affected by exogenous RXR in ES cells, even though ES cells
express less endogenous RXR than COS cells. Despite this inconsistency,
the lack of effect of RXR could be interpreted to indicate that
endogenous RXR is sufficient for T3 responsiveness. This would be
consistent with the fact that JEG-3 cells have the most endogenous RXR
of the 3 cell lines and showed no effects with exogenous RXR. However, transfection of JEG-3 cells with an RXR response element-reporter construct does not result in 9-cis-retinoic acid (the RXR
ligand) induction of the reporter gene unless exogenous RXR is
cotransfected (33). It is difficult to understand how cells can have
enough endogenous RXR to support maximal gene inductions from TREs, yet be unable to support even minimal gene inductions from RXR response elements.
In any case, the above experiments address the function of exogenous
RXR on TR function but do not really address whether RXR is
fundamentally needed. This is because all mammalian cells express RXR
endogenously. There are three RXR genes, denoted
,
, and
.
Mice and cell lines that are null for 5 of these 6 RXR alleles have
been created, but it has not been possible to create animals or
mammalian cell lines that are truly null for RXR protein. Thus, it has
not been possible to determine how the absence of RXR would influence
TR-dependent gene expression in mammalian cells.
It is for this reason that we used S. cerevisiae as a model
system for our experiments. Since yeast lack nuclear receptors (and
coactivators), they represent a null system into which TR, RXR, and
coactivators can be added or not, thus allowing a true test of the
consequences of RXR deficiency. Although it is appropriate to interpret
results in S. cerevisiae with caution, the track record of
yeast as a model system for nuclear receptor action has been
impressive. Several laboratories have shown that TRs and other nuclear
hormone receptors can function as ligand-independent or
-dependent transcription factors in S. cerevisiae (19, 34-37). The essential role of SWI/SNF proteins in
nuclear receptor action was first demonstrated in an S. cerevisiae model system (38) and was subsequently confirmed in
mammalian cells (39). Similarly, the role of Ada proteins in
glucocorticoid receptor action was first demonstrated in S. cerevisiae (40). In both of these cases, the yeast model system
was uniquely valuable because yeast cells existed that were null for
the SWI/SNF or Ada proteins when equivalent mammalian systems did not
exist. We have chosen to utilize a yeast model system to study the role
of RXR in TR function for analogous reasons.
As many as 8% of genes expressed in liver may be
T3-responsive (41), but only a small number of TREs have
been well characterized. We selected 10 naturally occurring direct
repeat response elements for study. These TREs vary considerably in
sequence, but all have two directly repeated half-sites. Thus, by
comparing the sequences of the TREs with their activities in yeast, we
may decipher the sequence features for RXR-independent gene expression
from direct repeat response elements without the complexities caused by
variations in half-site orientation and number.
In mammalian cells, the unliganded TR generally functions as a
transcriptional repressor, which is mediated through interaction with a
corepressor complex. In yeast, the corepressors that interact with TR
are absent, thus allowing TR to display ligand-independent transcriptional activity (2, 19, 37). This study demonstrates that
TR-mediated reporter gene stimulation from natural TREs is differentially enhanced by RXR in the absence of ligand. These TREs
exhibit a continuous spectrum of RXR dependence. Comparison of the TRE
sequences with in vivo gene expression patterns suggests that independence of RXR generally is associated with the presence of
the 5' dinucleotide TG or TA upstream of the 5' half-site. In addition,
the importance of the 5' T(A/G) was confirmed by creating a mutant
reporter construct r
MHC(TG) that is identical to the wild type
r
MHC except the dinucleotide TG replaces the wild type GG upstream
of the 5' half-site. In contrast to wild type r
MHC, r
MHC(TG) is
largely RXR-independent.
If the 5' T(A/G) were the sole determinant of RXR independence, TREs
would fall into two discrete groups, complete independence of RXR or
complete dependence. This, however, is not what was observed. Rather,
the importance of RXR varies in a continuous fashion from near total
dependence to near total independence. This indicates that there must
be modifying influences beyond the presence or absence of the 5'
T(A/G). Of the 10 response elements in Fig. 2, the 5 at the extremes
all follow the T(A/G) rule (the three TREs with the greatest RXR
dependence all lack T(A/G), and the two TREs with the greatest RXR
independence contain T(A/G)). The TREs in the middle may or may not
contain the 5' T(A/G), suggesting that minor differences in TRE
sequence can modify the influence of the T(A/G), thus moving the TRE
from one of the extremes toward the middle of the spectrum of RXR
dependence. A comparison of the 10 TRE sequences does not reveal any
clear rules as to what these modifying influences may be, and it is
likely that many sequence differences each may contribute a little.
In vitro DNA binding assays revealed that TR itself is able
to bind to almost all these natural response elements. Functional independence of RXR generally correlates with the strong binding of TR
alone to the TREs. Importantly, we also show that the conversion of the
r
MHC TRE from more RXR dependence to less RXR dependence (r
MHC(TG)) is due to the increase in TR binding to DNA. The hDI TRE
is unusual in that it binds well to TR alone and to RXR-TR heterodimers, which explains why its RXR dependence is intermediate. The relationship between receptor-DNA binding and reporter gene activation for the hDI TRE is similar to that of the other TREs (Fig.
4C). However, for all TREs, a perfect quantitative
relationship between receptor-DNA binding and reporter gene activation
does not exist, suggesting that DNA binding may not be sufficient to lead to transcriptional activation of the target genes.
In mammalian cells, TR functions as a ligand-activated transcription
factor. TR modulation of gene expression involves the coordination of
an array of coregulatory proteins including coactivators and
corepressors. In the absence of ligand, TR interacts with a corepressor
complex and inhibits positively regulated gene transcription. The
binding of ligand triggers a conformational change in the TR that
results in replacement of the corepressor complex by a coactivator
complex, thereby leading to activation of transcription. In the yeast
model system, addition of thyroid hormone is unable to elevate reporter
gene expression above the level achieved by the ligand-independent
activity of TR (AF-1 activity), due to the lack of
AF-2-dependent nuclear receptor coactivators. As expected, however, coexpression of the coactivator TIF2 in yeast can restore ligand-dependent gene activation from nearly all these TREs.
Although three of the TREs supported triac inductions of 4-fold or
more, most showed inductions of 2-fold or less (Fig. 5). This low level
of hormone induction is not surprising, as thyroid hormone has very
modest effects on endogenous gene expression in mammalian cells. For
example, a cDNA microarray analysis identified 14 hepatic genes
induced by T3 in vivo; of these, only one was induced more than 4-fold and 8 were induced less than 3-fold (42). Furthermore, in mammalian cells T3 induction is a
consequence of both AF-1 and AF-2 activities. However, as noted above,
yeast lack corepressors, resulting in constitutive AF-1 activity. Thus, in yeast, hormone induction reflects only AF-2 activity.
Surprisingly, we observed that the effect of TIF2 on
ligand-dependent activity is more potent in the presence of
TR alone than RXR plus TR, regardless of the nature of the TRE. The
observation that TIF2-mediated ligand-dependent activity is
markedly increased in the context of r
MHC(TG) relative to wild type
r
MHC is consistent with this, because the ability of r
MHC(TG) to
bind TR alone is much stronger than that of wild type r
MHC. Gel
shift studies with the rUCP TRE demonstrate that RXR inhibits formation
of the TR-TIF2-DNA complex, consistent with the yeast reporter gene data.
Overall, the data suggest that TRE sequence variations influence the
roles of RXR and specific coactivators in TR-mediated gene expression.
The availability of RXR and/or specific coactivators thus could
differentially regulate the T3 induction of subsets of hormonally
responsive genes within a cell. It will be important to develop
mammalian model systems to test these principles.