From the
Isoform specificity likely plays a large role in the ability of
the thyroid hormone receptor (TR) to specifically regulate gene
expression in both the presence and absence of its cognate ligand,
triiodothyronine. To investigate further the mechanism of isoform
specificity of human TRs (TR
Thyroid hormone regulates target gene expression through cis-acting response elements or TREs,
Further complexity in
thyroid hormone signaling is added by the presence of multiple isoforms
of the TR(15) . The two major isoforms, TR
The cDNAs encoding human TR
Whole cell extracts were made from COS-1 cells
transfected as described above with 10 µg of expression plasmid per
100-mm plate. After 48 h, the cell cultures were harvested and
resuspended in 25 µl of binding buffer containing 1 mM of
phenylmethylsulfonyl fluoride and 1 mM of dithiothreitol.
Extracts were prepared by three freeze-thaw cycles.
Similar DNA binding specificity was observed on the PAL probe (Fig. 7B). However, the PAL element is more amenable to
heterodimerization than DR+4 (31) such that the preference
of the amino terminus of TR
We have used transient transfection in mammalian cells and
gel mobility shift assays to further explore the role of TR isoform
specificity on gene regulation. From our data, it is clear that
TR
A number of lines of evidence also support the
hypothesis that TR
1 and TR
1), we have examined
their functional effects on positive thyroid hormone response elements
(TREs) both in the presence and absence of ligand. TR
1 was greater
than 2-fold more potent than TR
1 on both TREs studied, in terms of
both ligand-independent repression and ligand-dependent stimulation. By
creating a number of chimeric and mutant receptors, we have established
that the increased functional potency of TR
1 is due to its unique
amino terminus. Deletion or substitution of the TR
1 amino terminus
leads to a loss of both its ligand-independent and -dependent functions
on positive TREs. Furthermore, the TR
1 amino terminus antagonizes
homodimer formation on the positive TREs studied. TR constructs, which
contain the TR
1 amino terminus, are unable to form homodimers and
form exclusively heterodimers with RXR
on direct repeat and
palindromic TREs. Deletion of the amino terminus from either TR isoform
leads to preferential homodimer formation, which suggests that the TR
amino terminus is important for relative heterodimerization capability.
From these data, we conclude that TR
1 isoform specificity on
positive TREs resides predominantly in its amino terminus through its
ability to favor heterodimerization with the retinoid X receptor or
other nuclear proteins.
(
)which bind the thyroid hormone receptor (TR) and
auxiliary proteins such as the retinoid X receptor
(RXR)(1, 2, 3) . Several investigators have
shown that TREs present in positively regulated elements are repeats of
a half-site consensus motif (AGGTCA) oriented as a direct repeat with a
4 base pair gap (DR+4), a palindrome (PAL), or an inverted
palindrome(4, 5, 6) . The TR interacts with
these response elements as a monomer, homodimer, or heterodimer with
RXR isoforms. Heterodimerization with RXR greatly enhances positive
induction of gene expression by thyroid hormone (7-10). Besides
its ligand-dependent properties, the TR functions as a
ligand-independent silencer on positive TREs(11, 12) and as ligand-independent activator on negative TREs (13, 14) in mammalian cells. These ligand-independent
properties appear to be dependent on TR
RXR heterodimer, as TR
receptor mutants that cannot form homodimers still possess potent
ligand-independent function(13) .
1 and TR
1,
are encoded by separate genes but are relatively homologous except for
distinct amino termini. Each of these receptors has alternatively
spliced variants TR
2 and TR
2, respectively, which may be
expressed in a tissue-specific fashion (16) to regulate gene
expression. Isoform specificity has been demonstrated on a unique
negative TRE present in the Rous sarcoma virus promoter where the
unique amino terminus of TR
1 allows for ligand-independent
activation(17) . The TR domain responsible for isoform-specific
effects on positive TREs has not been demonstrated, although TR
1
is more potent as a ligand-dependent activator on positive TREs (18,
19) and appears to be relatively defective in its ability to
homodimerize on positive TREs(20, 21) . Furthermore,
differential expression of TR isoforms has been observed during
development(22) , suggesting that TR isoform specificity plays a
role in differential gene expression. In the following report, we
demonstrate that isoform specificity on positive TREs is a function of
both ligand-independent and -dependent properties of the TR and that
these enhanced properties map to the unique amino terminus of TR
1.
In addition, the amino terminus of TR
1 inhibits homodimerization
on positive TREs favoring the formation TR
RXR heterodimers. These
distinct properties of the amino terminus of TR
1 allow for TR
isoform-specific regulation of target gene expression.
Plasmid Construction
The positive TREs
consist each of two copies of PAL (a gift of J. L. Jameson,
Northwestern University, Chicago) and DR+4 inserted upstream of
the TK109 promoter in the luciferase vector
pALUC(23, 24) , which contains a trimerized
SV40 poly(A) termination site that prevents transcriptional read
through.
1, TR
1, and chimeric
and mutated TRs were inserted into the expression vector pKCR-2, which
employs the SV40 early promoter(25) . Chimeric TRs were
constructed using SacI and PstI restriction sites
present in the TRs. A SacI site was introduced into TR
1
at amino acid 52 to allow for exchanges of the amino termini of the
different TR isoforms. Amino-terminal deletions were constructed using
the polymerase chain reaction to introduce a Kozak initiation sequence
at the beginning of the DNA binding domain of each isoform. The
integrity of all constructs was confirmed by restriction endonuclease
digestion and dideoxy sequencing.
Cell Culture and Transient
Transfection
The CV-1 cell line was 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 subsequently referred to
as culture medium. Transient transfections were performed in six well
plates on subconfluent cells. 1.6 µg of reporter construct with 80
ng of receptor expression vector and 80 ng of pKCR-2 vector alone
(except where noted) were introduced into each well using the calcium
phosphate technique without glycerol shock. 12-15 h after
transfection, the cells were washed with phosphate-buffered saline, and
the culture medium was replaced with culture medium with fetal bovine
serum treated with anion exchange resin and charcoal (type AGX-8,
analytical grade (Bio-Rad), and activated charcoal (Sigma)). 24 h after
transfection, 10 nM T was added. 42-48 h
after transfection, the cells were harvested and resuspended with 300
µl of extraction buffer (25 mM glycylglycine, 15 mM MgSO
, 4 mM EGTA, 1% Triton X-100, and 1
mM dithiothreitol) and assayed for luciferase
activity(26) . Transfection efficiency was monitored by a
thymidine kinase (TK) human growth hormone (GH) expression plasmid
(TKGH). GH levels were measured in 50 µl of media by
chemiluminescent assay (Nichols Institute, San Juan Capistrano, CA).
Luciferase expression was quantified as arbitrary light units/µl of
extraction buffer.
Gel Mobility Shift Assays
Double-stranded
DNA probes representing PAL (5`-TCAGGTCATGACCTGAC-3`) and
DR+4 (5`-TCAGGTCACTTCAGGTCAAC-3`) were radiolabeled
using the polymerase chain reaction and [P]dCTP
(400 µCi/m). Unincorporated
P was removed by G-25
Sephadex chromatography. The TR cDNAs were cloned into PGEM 3Z and the
RXR
cDNA was cloned into pBluescript (a gift of R. Evans, Salk
Institute). Proteins were made by in vitro translation in
rabbit reticulocyte lysate (TNT, Promega) using either T7 or T3
polymerase. To quantify protein production,
S
incorporation and direct visualization on SDS-polyacrylamide gel
electrophoresis (12%) was utilized. 3 µl of each protein was used
in each reaction with radiolabeled probe (75-100,000 cpm).
Binding reactions were performed in a 20-µl volume of binding
buffer (20% glycerol, 20 mM HEPES, pH 7.6, 50 mM KCl), 1 mM dithiothreitol, 1 µg of poly(dI-dC), 0.1
µg of salmon sperm DNA. Incubations were carried out at room
temperature for 30 min. Gel mobility shifts were resolved on 5%
non-denaturing polyacrylamide gels and visualized after
autoradiography.
Human TR
To evaluate the relative activity of the TR isoforms
on positive TREs, CV-1 cells were cotransfected with equal amounts of
TR isoform expression vectors and two separate positive TREs linked to
a luciferase reporter, DR+4 and PAL (see ``Materials and
Methods''). In the absence of ligand, TR1 Is More Active in Both
Ligand-independent and Ligand-dependent Function on Positive
TREs
1 was over 2-fold
more potent than TR
1 as a transcriptional silencer on the
DR+4 element (6.6- versus 2.6-fold) (Fig. 1A) and approximately 2-fold more potent in
ligand-independent activity on the PAL element (7.2- versus 3.7-fold). Similar isoform potency effects were seen in the
presence of 10 nM T
, where TR
1 was over
2-fold more potent in its ability to activate transcription on both PAL
and DR+4 (45- versus 20-fold and 9- versus 4.4-fold, respectively) (Fig. 1B).
Figure 1:
Ligand-independent
and -dependent effects of TR isoforms on positive TREs is isoform
specific. A, CV-1 cells were cotransfected with PAL and DR+4 TREs
either in the presence of TR isoform or PKCR-2 alone. The data are
presented as fold repression from basal expression (PKCR-2 alone) and
are the mean of three separate experiments. The errorbars indicate standard error of the mean (S.E.). B, CV-1 cells
were cotransfected with PAL and DR+4 and either TR isoform in the
presence or absence of 10 nM T. The data is
presented as fold stimulation (10 nM T
) over basal
(in the presence of TR isoform alone).
To control
for relative levels of TR isoform expression, we 1) transfected
increasing amounts of TR isoform in the presence of both TREs and
T and 2) isolated cellular extracts from transfected COS-1
cells (which are deficient in endogenous RXR(27) ) and assessed
relative amounts of protein produced in gel mobility shift assays. Fig. 2A demonstrates that the potency differences
between the isoforms, in the presence of T
, is not due to
increasing amounts of transfected TR
1 versus TR
1.
Ligand-independent repression was preserved at all doses of receptor
used. The receptor dose chosen for all future experiments (80 ng/well)
gave near maximal ligand-independent and -dependent effects on both
reporters studied.
Figure 2:
Isoform effects are independent of
transfection efficiency. A, CV-1 cells were cotransfected with
either PAL or DR+4 (1.6 µg/well) and increasing amounts of TR
isoform in the presence or absence of 10 nM T. The
data are presented as fold stimulation ± S.E. in the presence of
10 nM T
. The solidbars represent TR
1, and the closedbars represent TR
1. B, 5 µl of COS-1 cell whole cell
extracts of TR isoforms, chimeras and mutants, and PKCR-2 were
incubated with 3 µl of in vitro translated RXR
and
radiolabeled DR+4. The arrows identify the specific
bands. The indicated probe is shown below the autoradiograph. NS, nonspecific band.
Quantification of TR isoform expression was
performed in cellular extracts (Fig. 2B) from the
CV-1-related cell line COS-1 and supports the above data. A relative
increase in TR1 isoform-RXR heterodimer is formed with
reticulocyte lysate-produced RXR and whole cell extract-produced TR. As
TR
1 function is relatively preserved over a wide range of receptor
concentrations (Fig. 2A), receptor dose is not
responsible for the potency differences observed. The mobility of the
heterodimers produced are consistent with the different sizes of the
human TR isoforms (55 kDa for TR
1 and 46 kDa for TR
1) (28, 29) due primarily to their different sized amino
termini.
The Amino Terminus of TR
A number of
chimeric and mutated receptors were constructed to define the
functional domain of TR1 Is Responsible for the
Increased Function of TR
1 on Positive TREs
1, which was responsible for its increased
ligand-independent and -dependent function on DR+4 and PAL (Fig. 3). These chimeric receptors were used in an identical
cotransfection paradigm as described above. As Fig. 4A demonstrates, the addition of the TR
1 amino terminus to the
DNA and ligand binding domains of TR
1 (TR
,
) causes a reconstitution of the wild-type TR
1
effect on PAL, both in the presence and absence of ligand. Conversely,
swapping the TR
1 amino terminus with the amino terminus of
TR
1 to form TR
causes ligand-independent and
-dependent function to decrease to levels found with TR
1 on PAL.
Deletion of the TR
1 and TR
1 amino termini (TR
and TR
, respectively) leads to receptors that possess
significantly less ligand-independent and -dependent activity than
their wild-type originators on PAL.
Figure 3:
Structure of TR isoform chimeras and
mutants. A number of TR chimeras and mutants were created using
restriction enzyme digestion and polymerase chain reaction. The numbering indicates the amino acid number in each isoform. The
designation of each chimera with threesymbols indicates the origin of the amino terminus, DNA binding domain,
and ligand binding domain.
Figure 4:
Isoform specificity resides in the amino
terminus. A, CV-1 cells were cotransfected with PAL and the
various TR constructs in both ligand-independent and -dependent
paradigms as described in Fig. 1. The data are reported as % maximal
fold repression (where %100 equals the activity of TR1 (see Fig.
1)) for ligand-independent effect and % maximal fold stimulation for
ligand-dependent (10 nM T
) effect. The data are
the mean of three separate experiments and are displayed with the S.E. B, CV-1 cells were transfected as above with DR+4 as the
reporter. The data are displayed as above and are the mean of
three separate experiments.
Similar data are achieved in our
analysis on DR+4 (Fig. 4B). TR is
approximately 2-fold more active than TR
1 in ligand-independent
and -dependent function on DR+4 and has 75% of the effect of
TR
1 in both of these functions. In contrast, TR
is
more similar in both ligand-independent and -dependent activity to
TR
1 (it is in fact less active). However, TR
is
more potent in both ligand-independent and ligand-dependent properties
than TR
, suggesting that the DNA binding domain and the
hinge region may play some role in isoform specificity on this element
consistent with their well defined role in half-site recognition and
heterodimerization(30) . This is supported by the fact that
although deletion of the amino termini of both TR isoforms leads to a
significant loss of function, TR
is still more active
than TR
in all respects on DR+4.
The Amino Terminus of TR
To examine whether the amino
terminus of TR1 Inhibits Homodimer
Formation on DR+4 and PAL
1 could influence DNA binding, we performed gel
mobility shift assays with the wild-type, mutated, and chimeric
receptors. Analysis of in vitro translated receptor produced
was performed by [
S]methionine incorporation and
direct visualization (Fig. 5). The size differences and presence
of two translation products of the various receptors correspond to
previous work(28, 29) , and their known sizes (55 kDa
for TR
1 and 46 kDa for TR
1). The size of the chimeras are
dictated by the origin of their amino terminus. TR
is
smaller than all of the chimeric constructs and shows only one
translation product because of the deletion of its entire amino
terminus and the insertion of a single translational start site. In the in vitro translation system used, TR
1 amino terminus
containing receptors are produced at approximately 25% of the level of
TR
1 amino terminus containing receptors (Fig. 5, first and lastlanes). When the TR
1 translation
start site was converted to a Kozak consensus site, translation
efficiency was not improved (data not shown). In contrast, this same
Kozak site was used in generating TR
and
TR
where translation of both proteins is enhanced
relative to their respective full-length receptors. This suggests that
the quantity of TR
1 produced in vitro is intrinsic to the
amino terminus of the receptor itself. Therefore, initial examination
of the ability of TR
1 to homodimerize was performed using
increasing amounts of the in vitro translated product.
Figure 5:
In vitro translation of TR
isoforms, chimeras, and mutants. The different TR constructs were in vitro translated in reticulocyte lysate, and the
translation products were quantified by direct incorporation of
[S]methionine and then visualized on
SDS-polyacrylamide gel electrophoresis. The arrows demonstrate
the relative location of the TR proteins (dashedarrow, TR
1; solidarrow, TR
1.
Receptors containing either the TR
1 or TR
1 amino termini have
two translation products, while the truncated TR contains only one (see
text).
As
shown in Fig. 6, increasing amounts of TR1 leads to the
formation of a single migrating complex with increased mobility, which
represents monomer on both PAL and DR+4 (see Fig. 7for
comparison of distances of migrating complexes). The addition of RXR
leads to the formation of a strong heterodimer. Despite using up to
four times the amount of lysate used in other gel mobility shifts, no
homodimerization of TR
1 was seen.
Figure 6:
TR1 preferentially heterodimerizes on
PAL and DR+4. Gel mobility shift assay of increasing amounts of in vitro translated TR
1 as indicated and a constant
amount of RXR
with both radiolabeled DR+4 and PAL as probes. HD, heterodimer; M, monomer; NS, nonspecific
band.
Figure 7:
The
amino terminus of TR1 antagonizes homodimerization on DR+4
and PAL. A, gel mobility shift assay of radiolabeled DR+4
incubated with in vitro translated TR constructs and RXR
.
The concentration of T
used is 10 nM. Lane1 contains unprogrammed reticulocyte lysate. B,
gel mobility shift assay of radiolabeled PAL with the same in
vitro translated proteins as above. C, gel mobility shift
assay of radiolabeled PAL with in vitro translated
and RXR
with wild-type TR
used as a control. HD, heterodimer; D, homodimer; M, monomer; NS, nonspecific band.
When DR+4 was used as a
probe with the chimeric and mutated receptors, the TR1 amino
terminus inhibited homodimer formation (Fig. 7A) as
TR
only formed a heterodimeric complex with RXR while
TR
leads to the formation of homo- and heterodimeric
complexes. In contrast, the TR
amino terminus facilitates
homodimerization as TR
forms a strong homodimer, unlike
TR
1 (Fig. 6). Interestingly, the deletion of the amino
terminus of TR
1 (TR
) causes a loss of heterodimeric
capability on DR+4 and favors the formation of a strong homodimer.
A similar loss in heterodimeric capability was seen when the amino
terminus of TR
1 (TR
) was deleted (data not shown).
These data indicate that while both amino termini antagonize homodimer
formation, the TR
1 terminus is much more potent in this regard.
1 to allow for heterodimerization is
increased. Still, exchanging the amino termini of TR
1 and TR
1
(see TR
versus TR
) dramatically
changes the relative ratios of hetero- versus homodimer that
is not related to amount of receptor protein. TR
forms
both a weaker homodimer on PAL than DR+4 and, in contrast to its
activity on DR+4, a weaker homodimer than TR
1 on PAL.
TR
was able to form a strong homodimer and heterodimer
on PAL (Fig. 7C), consistent with the TR
1 amino
terminus antagonizing homodimer formation.
1 preferentially acts as a ligand-independent silencer and
ligand-dependent activator on the positive TREs studied. These studies
also further establish the important role of ligand-independent
repression on the relative level of target gene expression either in
the presence or absence of T
. It is also clear that the
distinct amino terminus of TR
1 allows for the isoform-specific
effects on both DR+4 and PAL. Inclusion of the TR
1 amino
terminus in TR chimeras allows for 2-fold increases in both
ligand-independent and -dependent functions on DR+4 and PAL. The
TR
1 amino terminus also inhibits homodimerization on DR+4 and
PAL, indicative of the fact that the relative changes in binding
specificity may underlie the functional effects seen (Fig. 8).
Figure 8:
Model relating DNA binding to functional
activity. The wild-type, chimeric, and mutant receptors are shown in
cartoon form as either homodimers (opencircle) or as
heterodimers with RXR (opensquare). The arrow indicates the favored conformation of the particular receptor
shown. The relative functional activity of each of the receptors in the
presence or absence of ligand is listed above.
The mechanism of ligand-independent silencing is not clear but is
presumed to result from the interaction of the TR with other proteins,
such as TFIIB(32, 33) . Recent data suggest that the
TRRXR heterodimeric complex is responsible for this
ligand-independent effect, as TR mutants that are defective in their
ability to homodimerize but still retain the ability to heterodimerize
either retain or are enhanced in their ability to act as
transcriptional silencers(13) . Also, the TR homodimer, which
only forms in the presence of a DNA binding site(34) , may
compete with the heterodimer, in the absence of T
, for TRE
binding. In this case, enhanced homodimerization would lead to less
silencing as these complexes may be transcriptionally
inactive(35) . One region of the TR specifically involved in
ligand-independent activity may lie in the hinge region (36) of
the TR in both isoforms as mutagenesis of a proline (amino acid 209 in
TR
1 and amino acid 160 in TR
1) causes a specific loss of
ligand-independent activity but preserves ligand-dependent function.
Thus, in addition to other previously characterized regions of the TR
important for heterodimerization(37, 38) , the TR
1
amino terminus by enhancing heterodimeric capability or inhibiting
homodimerization allows for increased ligand-independent function on
certain TREs. Whether this is due entirely to DNA binding or to the
exposure of a new activation domain in the amino terminus remains to be
determined.
RXR heterodimerization is essential for
ligand-dependent transcriptional activation. 1) T
binding
dissociates TR homodimers from positive TREs (39) (see Fig. 7A), allowing preferential binding of TR
RXR
heterodimers; 2) TR
RXR heterodimers have a stronger affinity for
TREs than TR homodimers(8) ; 3) cotransfection of RXR enhances
the positive transcriptional response to T
on a number of
different elements(10, 40) ; and 4) more recently yeast
expression systems demonstrate that TR
RXR heterodimerization is
necessary for a full ligand-dependent transcriptional
response(41) . The data presented here support this hypothesis
by demonstrating that the propensity for a receptor to heterodimerize
is influenced strongly by its amino terminus and determines its ability
to respond to ligand. Similar to their ligand-independent effects, TR
mutants that cannot homodimerize are still powerful ligand-dependent
transcriptional activators (13), which supports the notion that
propensity to heterodimerize determines ligand-dependent
transcriptional ability. Further work in this area will likely focus on
the mechanism by which the TR
RXR complex is able to cause
ligand-independent and -dependent effects. Specific co-activators are
likely to be involved, such as TFIIB (32, 33) and other
unidentified proteins, which may interact with the TR
RXR complex.
,
triiodothyronine.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.