From the
The role of retinoid X receptors (RXRs) on negative thyroid
hormone response elements (nTREs) is not well understood. In this
report, we demonstrate that an orientation-specific monomeric thyroid
hormone receptor (T
Thyroid hormone mediates its effect through cis-acting response
elements. Several investigators
(1, 2, 3, 4) have shown that these elements are repeats of a half-site
consensus motif ((A/G)GG(T/A)CA). The orientation and spacing of these
half-sites determine the specificity of nuclear receptor binding and
the outcome of receptor-DNA
interactions
(4, 5, 6) . Umesono et al.(4) have proposed that a direct repeat of this half-site
containing a 4-bp
Unlike the role of
RXR to increase heterodimer binding and enhance thyroid hormone
stimulation from a pTRE, its effect on the negative TRE (nTRE) is not
well understood. A major nTRE in the human, mouse, and rat TSH-
Recently, Naar et al.(5) and Hallenbeck et al. (23) have suggested that negative regulation of the mouse
TSH-
Transient
transfection studies were performed in JEG-3 cells without and with
cotransfected human c-erbA-
DR+5 configuration at site 1, however, resulted in a different
pattern of expression (DR+5, Fig. 5). Ligand-independent
activation by T
To determine whether the orientation
of site 1 is important for T
To investigate further
the effect of RXR on thyroid hormone receptor function, experiments
were performed with medium containing serum treated with both anion
exchange resin and activated charcoal to remove both endogenous thyroid
hormones and retinoids. We first compared the effect of RXR-
R) DNA-binding site mediates thyroid
hormone inhibition in the thyrotropin
subunit gene (TSH-
)
from human and murine species. Unlike positive TREs, addition of the
ligand 9-cis retinoic acid (9-cis RA) to cells transfected with a
T
R
1 expression vector significantly reduces thyroid
hormone inhibition of the TSH-
gene, indicating that endogenous
retinoid receptors antagonize T
R function. Cotransfection
of an RXR-
but not a retinoic acid receptor-
expression
vector further antagonizes thyroid hormone inhibition, but only in the
presence of 9-cis RA. Antagonism by RXR requires both an intact DNA-
and ligand-binding domain. Removal of monomeric T
R binding
to the TSH-
nTRE also requires both RXR domains. A model is
proposed whereby monomeric T
R is removed from a nTRE by RXR
occupied by its ligand 9-cis RA. This is the first report of 9-cis
RA-dependent antagonism of thyroid hormone inhibition via negative
TREs.
(
)
gap (DR+4) functioned as
a positive thyroid hormone response element (pTRE), while DR+3 and
DR+5 arrangements functioned as vitamin D and retinoic acid
response elements, respectively. Nuclear hormone receptors interact at
these sites as either homodimers or heterodimers in association with
nuclear auxiliary proteins. Auxiliary proteins have been described for
T
R
(7, 8, 9) . Characterization of
these proteins reveal that some (RXRs) are related to retinoic acid
receptors (RARs) and bind the ligand 9-cis
RA
(10, 11, 12, 13, 14) . On
positive TREs (pTREs), heterodimer formation between T
R and
RXR increased T
-stimulated
transcription
(11, 12, 15, 16, 17) .
This effect is thought to be mediated by enhanced binding affinity of
RXR
T
R heterodimers to pTREs.
subunit genes has been
described
(18, 19, 20, 21) . The human
nTRE consists of two consensus half-sites situated 24 bp apart and an
additional degenerate half-site (3/6) positioned 4 bp downstream from
the first half-site in the first exon
(20) . These sites are
identical in the mouse and rat TSH-
genes, but their individual
roles in thyroid hormone inhibition have not been fully characterized.
The more 5`-T
R half-site (site 1) of this nTRE is of higher
affinity than the 3`-half-site (site 2), but both sites appear to be
functionally important for thyroid hormone
inhibition
(20, 21) . Moreover, at site 1, T
R
structurally interacts with the proto-oncogene c-jun during
thyrotropin-releasing hormone stimulation of this gene
(22) .
subunit gene was mediated by formation of unique
T
R homodimeric or heterodimeric complexes on a somewhat
different nTRE. Their proposed nTRE was a DR+2 element located at
-18 to -5 bp in the mouse TSH-
gene. The more
5`-half-site of this nTRE is relatively degenerate, however, and is not
conserved in either the human or rat TSH-
subunit genes. We
provide evidence that the human and mouse TSH-
nTREs bind
monomeric T
R, and RXR, in a ligand-dependent manner,
antagonizes T
-mediated inhibition.
Probe Design
Double-stranded
P-labeled probes were synthesized using polymerase chain
reaction and [
-
P]dCTP (400 Ci/mM)
and incorporated 4-6 [
-
P]dCTP
residues. Unincorporated
P was removed by Sephadex G-25
chromatography, and the probes were purified on 5% non-denaturing gels
as described
(22) . Fig. 1illustrates the DNA probes used
in this study.
Figure 1:
Wild type and mutant nTREs
from the human and mouse TSH- genes utilized in this study.
Nucleotide position relative to the start of transcription is indicated
above the DNA sequences. T
R half-sites in the WT
nTREs are boxed, and nucleotide mutations are
underlined. Mut, mutant.
Gel Shift Assay
Human c-erbA--1 in
pGEM3 and RXR-
in pBS (gift of R. Evans, Salk Institute, San
Diego) were synthesized in an in vitro transcription/translation reaction (TnT kit, Promega).
Unprogrammed translation reactions were used as a control. Quality and
quantity of translated proteins were assessed by
[
S]methionine labeling and SDS-polyacrylamide
gel electrophoresis analysis. Binding reactions were performed in a
15-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. 30,000-50,000 cpm of
probe was added to each reaction with 3 µl of programmed or
unprogrammed reticulocyte lysate for 30 min at room temperature.
``Supershifts'' were performed using 2 µl of either
polyclonal anti-TR
-1 (Affinity Bioreagents, Meshanic St., NJ) or
anti-c-jun (Santa Cruz Biotechnology, Santa Cruz, CA) with an
additional 30 min of incubation at 4 °C. Electrophoresis was
performed on a 5% non-denaturing gel containing 5% glycerol as
previously described
(22) .
Plasmid Construction and DNA
Transfections
Mutations noted in Fig. 1were
introduced into either a -125/+37-bp human TSH- or a
-124/+4-bp mouse TSH-
promoter fragment utilizing
polymerase chain reaction and mutagenic oligodeoxynucleotides. All
constructs were confirmed by restriction enzyme analysis and DNA
sequencing. Human or mouse TSH-
promoter fragments were cloned
into a luciferase vector at the HindIII site as previously
described
(20) . Human T
R
1 and RXR-
cDNAs
were inserted as EcoRI fragments into the pKCR2 vector as
described
(22) . RXR-
deletion mutant cDNAs were constructed
by deleting either an NcoI or StuI fragment from the
full-length cDNA and inserting these cDNAs into pKCR2. The human
RAR-
cDNA in the pCDM8 expression vector was a gift of Dr.
Benjamin Neel (Beth Israel Hospital, Boston). Transient transfection of
choriocarcinoma cell line JEG-3 was performed using methods previously
described
(20, 22) . Cell culture medium contained 10%
fetal calf serum (Life Technologies, Inc.) treated with either anion
exchange resin (50 mg/ml AGX-8, Bio-Rad) for two separate periods of at
least 5 h each or anion exchange resin as before with the addition of
activated charcoal (100 mg/ml, Sigma) with the second anion exchange
resin treatment. The resin and charcoal were then removed, and the
serum was filter-sterilized prior to its addition to the cell culture
medium.
The Human TSH-
The focus of this study was site 1 in the nTRE of the
TSH- nTRE Binds T
R
Monomer, and RXR-
Removes Monomeric T
R
Binding
gene from different species. The WT nTRE and mutations within
and around site 1 in the nTRE were tested for their ability to bind
T
R and RXR in gel mobility studies. The WT nTRE
(Fig. 2) bound in vitro translated T
R (human
c-erbA-
-1) as a monomer (M); the migration position of a
monomeric T
R complex was established by comparison to the
nTRE containing a DR+4 mutation (Fig. 3), which formed both
monomeric and dimeric (D) T
R complexes. Using the
3/7 mutation, which eliminated T
R binding at site 1,
we have previously shown
(20, 22) that monomeric
T
R bound predominantly to this half-site in the WT nTRE. As
expected, when site 1 was reversed (M1R), a monomeric
T
R binding pattern identical to the WT nTRE was observed
(data not shown). The nonspecific complex (NS) found with
unprogrammed lysate migrated slightly above the heterodimer band and
was not ``supershifted'' by the anti-TR
-1 antiserum
(Fig. 2). Interestingly, the DR+5 DNA fragment
(Fig. 4), which contains an additional consensus T
R
binding half-site 5 bp downstream from site 1 (Fig. 1), bound
both one (M) and two monomers (2M) of T
R.
10 nM T
did not alter monomeric T
R
binding on the WT nTRE (Fig. 2) but reduced dimeric
T
R binding on the DR+4 element (Fig. 3), as has
been previously reported
(24, 25) . Note that unlike the
DR+4 probe, 10 nM T
did not dissociate the 2
M complex on the DR+5 probe. Polyclonal anti-TR
-1
but not anti-c-jun antiserum supershifted the monomeric, two
monomeric, and dimeric T
R complexes, confirming their
identity. In fact, polyclonal anti-TR
-1 antibody supershifted two
distinct complexes with the DR+4 probe (Fig. 3,
arrows), representing a dimeric and monomeric complex but only
a single complex with the WT nTRE (Fig. 2, arrow) or the
DR+5 probe (Fig. 4, arrow).
Figure 2:
Gel mobility shift assay of the wild type
nTRE in the human TSH- gene. Radiolabeled DNA probe containing
-18 to +37 bp of the human TSH-
gene was utilized in a
gel mobility shift assay with in vitro translated proteins (3
µl) as noted by the +. 10 nM T
,
c-erbA-
, or c-jun antiserum was used in some binding
reactions. UP, unprogrammed; M, monomer; NS,
nonspecific band; arrow, supershifted T
R
complex.
Figure 3:
Gel
mobility shift assay of the DR+4 mutant nTRE. Radiolabeled DNA
probe containing -18 to +37 bp of the human TSH- gene
and the DR+4 mutation (Fig. 1) was utilized in a gel mobility
shift assay with in vitro translated proteins (3 µl) as
noted by the +. 10 nM T
, c-erbA-
, or
c-jun antiserum was used in some binding reactions. M,
monomer; D, dimer; HD, heterodimer; arrows,
supershifted T
R complexes.
Figure 4:
Gel mobility shift assay of the DR+5
mutant TRE. Radiolabeled DNA probe containing -18 to +37 bp
of the human TSH- gene and the DR+5 mutation (Fig. 1) was
utilized in a gel mobility shift assay with in vitro translated proteins (3 µl) as noted by the +. 10
nM T
, c-erbA-
, or c-jun antiserum was used in
some binding reactions. M, monomer; 2M, two monomers;
NS, nonspecific band; and arrow, supershifted
T
R complex.
In vitro translated human RXR- did not form a specific complex with
the WT nTRE (Fig. 2) but, when added with T
R,
eliminated monomeric T
R binding. These data suggest that
heterodimerization between T
R and RXR in solution prevents
T
R binding to the WT nTRE. Identical results were obtained
with the M1R DNA probes (data not shown). In contrast, the DR+4
element (Fig. 3) formed a strong heterodimeric complex when both
T
R and RXR were added to the binding reaction. The
DR+5 element (Fig. 4) behaved like the WT nTRE except that
RXR inhibited formation of both monomeric complexes (M and
2M). 10 nM T
did not alter heterodimer
complexes on the DR+4 probe. Since the ligand for RXR is 9-cis RA
(gift of A. Levin, Hoffman-LaRoche, Nutley, NJ), we also tested whether
this ligand
(13, 14) would alter the pattern of
T
R or T
R
RXR heterodimeric binding on
these elements. 1 µM 9-cis RA did not alter RXR antagonism
of T
R monomeric binding on the WT nTRE or DR+5 probes
or T
R
RXR heterodimeric binding on the DR+4
element (data not shown).
The Orientation and Surrounding Nucleotides of Site 1
in the Human TSH-
JEG-3 cells were chosen for these studies
because they are functionally deficient in T nTRE Are Important for Thyroid Hormone
Inhibition
R and have been
used to investigate negative T
regulation of the common
glycoprotein
subunit and TSH-
subunit
genes
(22, 26) . Luciferase reporter constructs
containing -125 to +37 bp of either the WT or mutant human
TSH-
nTRE were utilized in these studies. As a positive control, a
luciferase reporter construct containing two copies of a T
palindromic element (TRETK, Ref. 27, gift of J. L. Jameson,
Northwestern University Medical School, Chicago) upstream of a minimal
thymidine kinase promoter was utilized. Transfection efficiency was
monitored using a cotransfected human growth hormone reporter plasmid
(pTKGH); luciferase activity was expressed relative to the basal
activity of the human TSH-
WT construct, except for the pTRETKLUC
activity, which was expressed relative to its own basal activity.
Relative luciferase activity (relative light units measured over 20
s/human growth hormone measured in culture medium in ng/ml) of TRETK,
WT human TSH-
, and ``promoterless'' luciferase
constructs was 36, 427, 2115, and 25, respectively.
-1 (pKCR2-c-erbA-
,
T
R). Luciferase activity from the WT -125/+37-bp
human TSH-
reporter plasmid (Fig. 5) was increased by
cotransfected T
R; the addition of 10 nM T
reduced luciferase activity by 70%. 10 nM T
,
in the presence of cotransfected vector, did not inhibit luciferase
expression from this construct, confirming the functional absence of
T
R in this cell line. We have previously shown that
mutation of site 1 (Fig. 1,
3/7) abolished
T
-mediated inhibition of the -125/+37-bp human
TSH-
reporter plasmid in this cell line in the presence of
cotransfected T
R
(22) . As expected, the TRETK
construct was stimulated 16-fold by T
in the presence of
cotransfected T
R (Fig. 5). Note that T
R
cotransfection resulted in ligand-independent repression, not
stimulation, of this construct containing a pTRE.
Figure 5:
Functional studies of the WT and mutant
nTREs in a transiently transfected human placental cell line (JEG-3).
The WT and mutant TREs in the context of -125 to +37 bp of
human TSH--luciferase construct were tested in JEG-3 cells. The
construct is named for the mutation in the nTRE and corresponds to the
nomenclature in Fig. 1. A positive control, the pTRETK plasmid (2
Pal), which contains two palindromic pTREs upstream of a
minimal thymidine kinase promoter, was also utilized. Cotransfected
plasmid(s) are indicated to the left of each graph.
All luciferase activity was corrected for transfection efficiency by
measuring secreted human growth hormone in the medium and expressed as
% activity of the WT -125 to +37 construct in the absence of
cotransfected T
R, RXR, and T
, except for TRETK,
which was expressed relative to its own basal activity. Data are mean
± S.E. of three to six independent
determinations.
To determine
whether a DR+4 configuration at site 1 would alter
T-mediated inhibition, this mutation was constructed in the
context of the human TSH-
nTRE (Fig. 1). Unlike the WT
construct, cotransfection of T
R with this construct
resulted in a 2-fold reduction in basal activity; T
stimulated expression from this construct 5-fold (DR+4,
Fig. 5
). These data, together with those from the WT construct,
indicated that unliganded monomeric T
R stimulated, while
dimeric or heterodimeric T
R inhibited, transcription from
the WT and mutant TSH-
TREs, respectively. Moreover, the WT nTRE
was converted to a pTRE by adding a second half-site 4 bp downstream
from site 1, indicating that the composition of the TRE and not its
location determined the direction of thyroid hormone action.
R was impaired relative to the WT construct,
but T
-mediated inhibition was preserved. Since the
DR+5 probe bound T
R as either one or two monomers (see
Fig. 4
), these data indicated that the ability of T
R
to form homo- or heterodimers (DR+4 arrangement) and not just a
second nearby half-site (DR+4 or DR+5 arrangement) was
essential for thyroid hormone stimulation. The DR+5 arrangement,
unlike the DR+4 element, would bind both RARs and RXRs present in
the JEG-3 cell line, and this could explain the difference in
ligand-independent activation.
-mediated inhibition, we
constructed the M1R mutant (Fig. 1) in the -125/+37-bp
human TSH-
reporter plasmid. Interestingly, this mutation
abolished T
-mediated inhibition, even though the only
difference between this construct and the WT construct is the
orientation of the T
R-binding site 1 (M1R,
Fig. 1
and Fig. 5). Since this mutant only bound monomeric
T
R in gel mobility shift experiments, it suggested that the
presentation of certain domains of the T
R to the
transcription complex at site 1 was crucial for T
-mediated
inhibition.
The Mouse TSH-
Unlike the rat and human
TSH- nTRE Bound T
R Monomer
and T
R/RXR Heterodimers
subunit genes, Naar et al.(5) have proposed
a somewhat different nTRE in the mouse. Their proposed mouse TSH-
nTRE includes site 1 present in the rat and human genes but also
includes a second degenerate half-site more 5` to site 1, which is not
found in either the rat or human genes (Fig. 1). When tested in
the gel mobility shift assay, the WT mouse TSH-
DNA fragment
(identical to that in Ref. 5) formed a specific complex with
T
R that migrated at the position of a T
R
homodimer (Fig. 6, lane1). Using the WT mouse
probe, unprogrammed lysate (data not shown) and the RXR only (lane7) did not form this band, confirming it was due to
T
R binding. Two lines of evidence suggested, however, that
this was not a true homodimer but represented binding of two monomers
to this element. First, 10 nM T
dissociated
T
R homodimers on the mouse DR+4 element and resulted
in strong monomeric binding (compare Fig. 6, lanes5 and 6) but did not dissociate the WT mouse nTRE or an
idealized mouse DR+2 element (Fig. 6, lanes1 and 2 and lanes3 and 4,
respectively). A lack of T
R-T
R interaction on
the WT and DR+2 elements can be inferred because T
dissociated T
R homodimers on the DR+4 but not
the WT or DR+2 elements. Furthermore, T
altered
homodimeric but not monomeric T
R binding on the DR+4
element, indicating monomeric T
R-DNA interactions were not
disrupted by T
. Second, we were unable to cross-link
T
R on the mouse nTRE or an idealized DR+2 element
using 1,6-bismaleimidohexane and previously described methods (data not
shown and Ref. 20). These data are consistent with that of Naar et
al.(5) who demonstrated no significant cross-linking of
T
R on the WT mouse TSH-
nTRE.
Figure 6:
Gel mobility shift assay of the WT and
mutant mouse TSH- nTREs. Radiolabeled DNA probes, containing
-20 to +4 bp of either the WT or mutant (DR+2 and
DR+4) mouse TSH-
gene (Fig. 1), were utilized in gel mobility
shift assay with in vitro translated proteins as noted by the
+. The position of the two monomer (2M), homodimer
(D), or heterodimer (HD) complex is indicated.
NS, nonspecific band.
Mutation of the 5`-Degenerative Half-site in the
Mouse TSH-
Luciferase reporter constructs containing
-125 to +37 bp of either the human TSH- nTRE Does Not Alter Thyroid Hormone
Inhibition
, mouse
TSH-
, or a mutant mouse TSH-
gene nTRE were utilized in these
studies. As shown in Fig. 7, the WT human construct was inhibited
90% by 10 nM T
in the presence of cotransfected
T
R in these experiments. The WT mouse construct was
inhibited 77% by 10 nM T
in the presence of
cotransfected T
R (Fig. 7). The more 5`-degenerative
half-site in the mouse nTRE, however, was not important in mediating
thyroid hormone inhibition, since the mutant mouse nTRE (shown in
Fig. 1
), which resembles the human nTRE, was inhibited to a
greater extent (93%) than the WT mouse construct by 10 nM
T
(Fig. 7). These data indicate that the core
monomeric site in the mouse TSH-
gene nTRE and not the DR+2
arrangement dictates negative regulation.
Figure 7:
Functional comparison of the WT human,
mouse, and mutant mouse (MT) TSH- promoters in JEG-3
cells. The WT human TSH-
nTRE in the context of the human promoter
(-125 to +37 bp) or the WT and mutant mouse TSH-
nTREs
in the context of -124 to +4-bp promoter were tested in
JEG-3 cells. The construct named corresponds to the nomenclature in
Fig. 1. Data are expressed as a percent of a relative luciferase
activity ratio (+T
/-T
). Data are
mean ± S.E. of three to six independent
determinations.
RXR Does Not Antagonize Thyroid Hormone Inhibition in
the Absence of 9-cis RA
Having defined the importance of
the core monomeric TR-binding site (site 1) of the mouse
and human TSH-
gene nTREs, we next explored the role of RXR in
thyroid hormone inhibition. Both Hallenbeck et al.(23) and Carr and Wong
(28) have reported that
cotransfection of RXR-
and T
R expression vectors
abolished thyroid hormone inhibition of either the mouse or rat
TSH-
gene nTRE. It is unclear, however, how the cell culture
medium was treated in these studies to remove endogenous thyroid
hormones and retinoids present in the fetal calf serum. We initially
performed experiments with the WT human promoter in medium containing
fetal calf serum that was treated with an anion exchange resin to
remove endogenous thyroid hormones. When both T
R and RXR
expression vectors were cotransfected in JEG-3 cells, no significant
thyroid hormone inhibition of the WT human construct was observed (2%
inhibition by 10 nM T
).
or
RAR-
and their ligand 9-cis RA on T
R activation of the
WT human construct (Fig. 8). Ligand-independent activation by
T
R was significantly reduced by RXR cotransfection but only
in the presence of 9-cis RA. In contrast, RAR cotransfection actually
enhanced ligand-independent activation by T
R in the absence
of 9-cis RA and had no significant effect in the presence of 9-cis RA.
These data indicate that ligand-bound RXR functionally removes the
ligand-independent activation function of T
R.
Figure 8:
Effect of 9-cis RA on ligand-independent
activation by TR of the WT human TSH-
promoter after
cotransfection of RXR and RAR expression vectors. The effect of 9-cis
RA on basal expression of the WT human TSH-
nTRE in the context of
the human promoter (-125 to +37 bp) was tested in JEG-3
cells after cotransfection of either T
R,
T
R/RXR, or T
R/RAR expression plasmids. Data are
expressed as a percent of relative luciferase activity in the absence
of 9-cis RA after cotransfection of T
R alone. Data are mean
± S.E. of six independent
determinations.
We next
compared the ability of RXR- or RAR-
to antagonize thyroid
hormone inhibition of the WT human construct in the absence or presence
of 9-cis RA (Fig. 9). As expected, transfection of either an
RXR-
or RAR-
expression vector alone resulted in no T
inhibition in either the absence of presence of 9-cis RA.
Transfection of T
R alone resulted in 75% T
inhibition, which was reduced to 50% T
inhibition
after the addition of 9-cis RA. JEG-3 cells express the RXR-
isoform almost exclusively
(29) ; these data indicate that
endogenous RXR-
in JEG-3 cells can antagonize thyroid hormone
inhibition when occupied by its ligand, 9-cis RA. This effect was
specific for RXR since cotransfection of RXR and T
R
completely abolished T
inhibition in the presence of 9-cis
RA, while cotransfection of RAR and T
R yielded results
similar to T
R transfection alone.
Figure 9:
Effect of 9-cis RA on
T-mediated inhibition of the WT human TSH-
promoter
after cotransfection of RXR and RAR expression vectors in JEG-3 cells.
The effect of 9-cis RA on T
-mediated inhibition of the WT
human TSH-
nTRE in the context of the human promoter (-125
to +37 bp) was tested in JEG-3 cells after cotransfection of
either T
R, RXR, T
R/RXR, RAR, or
T
R/RAR expression plasmids. Data are expressed as a percent
of a relative luciferase activity ratio
(+T
/-T
). Data are mean ± S.E.
of three to six independent determinations.
These data are in
contrast to those obtained with the TRETK construct. After
cotransfection of RXR and TR, T
stimulation of
this construct was similar in the absence or presence of 9-cis RA (9.3
± 0.4- versus 9.5 ± 7-fold, respectively). Rosen
et al.(30) demonstrated that cotransfection of RXR
enhanced T
stimulation on a palindromic element, and this
enhancement was dependent on 9-cis RA. While our data do not confirm
9-cis RA enhancement of T
stimulation in JEG-3 cells, they
do demonstrate an antagonistic role for the same ligand in T
inhibition in these cells.
RXR Antagonism of Thyroid Hormone Inhibition Requires
an Intact DNA- and Ligand-binding Domain
To determine the
mechanism of RXR antagonism of TR action, two deletions of
the RXR-
isoform were constructed and translated in a coupled
in vitro transcription/translation reaction. As shown in
Fig. 10
, these mutants were synthesized to a similar degree in
the lysate as determined by [
S]methionine
labeling. The DBD mutant lacks amino acids 29-197, and the LBD
mutant lacks amino acids 403-462 and are correspondingly smaller
in molecular size than WT RXR-
of approximately 52 kDa. Both
mutants contain the region important in nuclear localization in this
superfamily or receptors
(31) , and these mutants localize to the
nucleus based on deletion studies with the RXR-
isoform
(32) .
Figure 10:
RXR mutants used in this study. An
autoradiogram of S-labeled full-length human RXR-
and
its deletion mutants is shown. The location of the deletion is
illustrated. RXR-LBD (StuI fragment removed) deletes part of
the ninth heptad in the C-terminal region, and RXR-DBD (NcoI
fragment removed) deletes part of the N terminus and the entire
DNA-binding domain.
We next determined whether these isoforms would
antagonize thyroid hormone inhibition by TR of the WT human
TSH-
promoter. JEG-3 cells were cotransfected with T
R
and RXR-
expression vectors as noted in Fig. 11. As
expected, cotransfection of the full-length RXR-
expression vector
abolished T
inhibition in the presence but not in the
absence of 9-cis RA. In contrast, both mutant RXRs were defective in
this property. Cotransfection of the LBD mutant resulted in no
significant additional antagonism of thyroid hormone inhibition by
9-cis RA compared with cotransfection of T
R alone. This
result was expected since the LBD mutant lacks the ninth heptad repeat
in the C terminus and does not bind as a heterodimer with
T
R
(33, 34) . Cotransfection of the DBD
mutant resulted in some additional antagonism of T
inhibition in the presence of 9-cis RA as compared with
T
R alone but clearly less than the full-length RXR.
Figure 11:
Effect of 9-cis RA on
T-mediated inhibition of the WT human TSH-
promoter
after cotransfection of RXR mutant expression vectors in JEG-3 cells.
The effect of 9-cis RA on T
-mediated inhibition of the WT
human TSH-
nTRE in the context of the human promoter (-125
to +37 bp) was tested in JEG-3 cells after cotransfection of
either T
R, T
R/RXR, T
R/RXR-DBD, or
T
R/RXR-LBD expression plasmids. Data are expressed as a
percent of a relative luciferase activity ratio
(+T
/-T
). Data are mean ± S.E.
of three to six independent determinations.
Given that both an intact DNA and ligand binding domain of RXR were
necessary for ligand-dependent antagonism of thyroid hormone
inhibition, we wanted to determine how these RXR mutants affected
heterodimer binding to both positive and negative TREs. Both RXR
mutants were defective in heterodimer formation with TR on
a DR+4 probe (Fig. 12) but did not affect homodimer binding
to this element. In contrast, the WT RXR and DBD mutant (to a lesser
extent) but not the LBD mutant removed monomeric T
R binding
to the WT TSH-
nTRE (Fig. 12). Addition of 9-cis RA did not
alter the effect of RXR-
and its mutants on either the DR+4
or TSH-
probes (data not shown). Because reticulocyte lysate
should contain 9-cis RA, it is unclear whether 9-cis RA affects
heterodimer formation between RXR and T
R on DNA or in
solution.
Figure 12:
Gel mobility shift assay of RXR mutants
on a DR+4 and WT human TSH- nTRE. Radiolabeled DNA probes,
containing either the DR+4 mutant or WT human TSH-
gene (Fig.
1), were utilized in gel mobility shift assay with in vitro translated proteins (3 µl) as noted by the +. The
position of the a monomer (M), homodimer (D), or
heterodimer (HD) complex is indicated. NS,
nonspecific band.
A Model of RXR Antagonism of Thyroid Hormone
Inhibition
Fig. 13
is a model of RXR action on
positive and negative TREs. RXR enhances TR binding to
pTREs via a DNA-binding element containing appropriately spaced
half-sites (A) in gel shift studies. Both T
and
9-cis RA may activate transcription by altering the conformation of the
heterodimer and allow it to interact with either an adaptor protein or
the basal transcriptional machinery (B). In contrast, we
suggest that the TSH-
nTRE is a monomeric T
R
DNA-binding element in human and murine species, binding one or several
molecules of T
R. RXR removes T
R binding to this
nTRE and antagonizes both ligand-independent activation by
T
R as well as T
-mediated inhibition (C and D). The importance of the monomer in this process is
further strengthened by the orientation dependence of the half-site in
the nTRE for inhibition and the ability of a second appropriately
spaced half-site to convert a nTRE to a pTRE. RXR antagonism of
negative TREs is strictly dependent on an intact DNA- and
ligand-binding domain of RXR and the ligand 9-cis RA. In conclusion,
this is the first demonstration of 9-cis RA-dependent antagonism of
thyroid hormone inhibition via negative TREs.
Figure 13:
Model of RXR action on positive and
negative TREs. TR and its ligand T
are shown as
ovals, and RXR and its ligand 9-cis RA are shown as
rectangles.
R, thyroid hormone receptor; WT, wild type; TSH-
,
thyrotropin
subunit; DR, direct repeat; RAR, retinoic acid
receptors.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.