(Received for publication, September 1, 1994; and in revised form, November 28, 1994)
From the
Thyroid hormone receptors (TRs) bind specific thyroid hormone response elements (TREs) as heterodimers with retinoid X receptors (RXRs) and act as transcriptional activators. As homodimers, TRs can bind a distinct set of sequences and function as ligand sensitive repressors. In our study, we compared the natural malic enzyme TRE (ME-TRE) as a model system for the TR/RXR heterodimer pathway to the chicken lysozyme silencer element F2-TRE which is strongly bound and regulated by TR/TR homodimers. Using electrophoretic mobility shift assays, transient transfections with a variety of natural and synthetic triiodothyronine and thyroxine derivatives as well as limited proteolytic analysis, we show that the natural homo- and heterodimeric pathways show similar ligand requirements. Furthermore, we observe that the ligand-induced conformational changes in the receptor proteins that either result in a loss of TR/TR homodimer binding and release of transcriptional repression or in transcriptional activation of TR/RXR heterodimers are indistinguishable. Therefore, we propose that in TR/TR homodimers and TR/RXR heterodimers very similar moieties of the receptors are involved in ligand binding and subsequent conformational changes that lead to loss of gene repression (TR/TR homodimer) and gain of gene activation (TR/RXR heterodimer).
Thyroid hormones regulate important biological processes such as
metamorphosis, development, growth, homeostasis and general metabolism (1) . The major forms of thyroid hormones comprise
3,5,3`,5`-tetraiodo-L-thyronine (L-T4) ()and
3,3`,5-triiodo-L-thyronine (L-T3), the latter is the most
active form. The biological effects of these hormones are mediated by
specific nuclear thyroid hormone receptors (TRs). Two genes encoding
two different receptor subtypes, TR
and TR
, have been
characterized (see for review, see (2, 3, 4, 5) ). The receptors are
members of the steroid hormone/retinoic acid receptor superfamily, a
large group of transcription
factors(6, 7, 8) . TRs have dual regulatory
roles and can function as transcriptional activators as well as
transcriptional repressors(5, 9) . Like other members
of the superfamily, TRs mediate T3 signals through specific DNA
sequences, the T3 response elements (TREs), usually found in the
promoter regions of responsive genes. The characterization of TREs
revealed that several configurations of two half-sites of the sequence
AGGTCA (or derivatives of this sequence) are possible, including a
palindrome with no spacer (10) , direct repeats with 4 base
pair (bp) spacer(11, 12, 13) , and inverted
palindromes/everted repeats spaced by 6 bp(14, 15) .
For effective DNA interaction, the TRs were found to require
association with a nuclear auxiliary factor, called
TRAP(16, 17, 18, 19, 20) ,
now identified as retinoid X receptors
(RXRs)(21, 22, 23, 24, 25, 26) .
The RXRs also play a central role in several other signal transduction
pathways since they heterodimerize with several other nuclear
receptors, including the retinoic acid receptors (RARs), the vitamin D
receptor (VDR), and the peroxisome proliferator activated receptors
(PPARs)(21, 22, 23, 24, 25, 26, 27, 28, 29) .
More recent evidence suggests that TRs can also form heterodimers
with other receptors(30, 31) . In addition TR/TR
homodimers (33, 34) ()and TR monomers (35, 36) have been proposed to confer T3 responsive
transcriptional regulation increasing the variety of possibilities for
T3 signal transduction mechanisms. However, so far it appears that most
TREs are activated by the TR/RXR heterodimeric pathway since the
heterodimers have the highest affinity for these TREs. TR/RXR
heterodimers were shown to form in solution in the absence of
ligand(21, 22, 25) , while the presence of
specific DNA binding sites like the
-myosin heavy chain TRE or the
malic enzyme TRE (ME-TRE) were shown to strongly enhance this
dimerization(37) . Interestingly, the chicken lysozyme TRE
(F2-TRE), an IP-6 type response element, initially characterized as a
silencer (14) can be bound by TR/TR homodimers and TR/RXR
heterodimers, the TR/TR homodimers forming the more stable complexes
with this TRE.
TR/TR homodimer binding to the F2-TRE,
however, is inhibited by T3(38)
allowing the
homodimers to function as T3 sensitive
repressors(5, 16) .
The major goal of this study was to compare these two major T3 signaling response pathways, the TR/RXR heterodimeric and the TR/TR homodimeric pathway, in terms of ligand mediated action. For this we analyzed the T3 induced release of repression by TR/TR homodimers using the F2-TRE, comparing it to TR/RXR heterodimers induced transcriptional activation on the ME-TRE, employing natural and synthetic thyroid hormone analogs. We show here that the TR/TR homodimeric and the TR/RXR heterodimeric pathways have very similar ligand requirements. Our data support the idea that the ligand-induced conformational changes in TR that lead to transcriptional activation by TR/RXR heterodimers and release of repression by TR/TR homodimers are identical or at least very similar. This implies that the orientation and spacing of the half-sites in the various natural TREs is a key determinant for either transcriptional activation or release of repression by TRs.
Figure 1:
Two natural TREs have distinct receptor
binding properties. A, sequences of wild type TREs used in
this analysis. The hexameric repeats involved in receptor binding and
function as demonstrated by previous studies are indicated by arrows.
The rat malic enzyme TRE (ME-TRE) sequence from 288 to 259 (45) and the chicken lysozyme silencer F2-TRE sequence from
-2354 to -2326 (14) are shown. B, binding
of homo- and heterodimeric complexes. P-Labeled ME-TRE and
F2-TRE were incubated with equal amounts of in vitro translated receptors. Binding of TR
homodimers in the
presence and absence of 10
M L-T3 and
TR
/RXR heterodimers were compared. Unprogrammed lysate (Lysate) served as a control.
Figure 2:
Analysis of TR/TR homodimer and TR/RXR
heterodimer response pathways in vivo. A, ligand
dependent transactivation of the ME TRE in CV-1 cells. The TR/RXR
heterodimeric complex was analyzed on a natural DR-4 element, the rat
malic enzyme (ME) TRE, in transient transfection experiments.
The ME-TRE was cloned upstream of the tk-promoter generating
the reporter construct pBLCAT2 ME TRE. 100 ng of pBLCAT2 ME TRE
together with 5 ng of expression vectors were cotransfected into CV-1
cells. The cells were treated with different concentrations of hormone (L-T3) and 24 h later tested for CAT activity. The mean of
four experiments is shown. The standard deviations are indicated. B, ligand dependent release of repression by TR/TR-homodimer.
The chicken lysozyme silencer F2-TRE, which has an inverted palindromic
structure with a spacing of 6 nucleotides, was cloned downstream the
TATA-box into the pBLCAT2 vector (pBLCAT2 TATA F2). This
construct has constitutive promoter activity which is repressed in the
presence of TR. T3 releases repression and results in the induction of
the reporter gene. 300 ng of pBLCAT2 TATA F2 were cotransfected into
CV-1 cells either with 50 ng TR expression vector or empty vector
as a control and 200 ng of
-gal expression vector. The cells were
treated with different concentrations of hormone (L-T3) and 24 h later
analyzed for reporter gene (CAT) activity. CAT values were
normalized to
-gal expression. The mean of four experiments is
shown. The standard deviations are indicated. The basal tk-promoter activity was repressed by TRs in the absence of
ligand by more than 80%.
When we analyzed the F2-TRE cloned upstream
of a tk-CAT gene, cotransfection of TR decreased the
basal CAT-activity. Addition of T3 not only reversed this effect, but
also up-regulated the CAT expression severalfold over the basal level,
consistent with previous observations (14) (data not shown). To
be able to separate the heterodimer response from the homodimer release
of repression response, we cloned the F2-TRE downstream of the TATA-box (Fig. 2B) such that the TRE-bound receptors would
inhibit the transcriptional machinery. Only the TR/TR homodimers could
interfere with the transcription initiation in a ligand responsive
manner. Indeed this experimental design allowed to separate the
predominantly TR/TR homodimeric effects from the TR/RXR heterodimeric
effects in transient transfection assays (Fig. 2B).
Repression of the F2-TRE was released in a ligand sensitive way,
reestablishing the basal expression without superactivation. Thus, the
TATA F2 construct is suitable for functional analysis of the release of
transcriptional repression important in the homodimer pathway.
Figure 3:
Dose response curves of natural and
synthetic T3 and T4 analogs. Repression release and transactivation
potencies of the different thyromimetics are shown. TR (A) and TR
(B) were analyzed. Transfections were
carried out essentially as in Fig. 2. CAT activity was measured
after 24 h of incubation in presence of different concentrations of the
T3 and T4 analogs. The values were normalized to
-gal expression.
High and medium activity compounds were compared to L-T3 (upper and middle panels) while low activity compounds (lower panels) were compared to
L-T4.
Figure 4:
Activities of T3 analogs. The EC values for the different ligands as determined by transcriptional
activation of the ME-TRE and release of repression on the F2-TRE are
listed. The structural formulas of the natural ligands (A) and
the synthetic T3 analogs (B) are shown. n.a., not
active.
We analyzed
[S]methionine-labeled TR
incubated with
different concentrations of chymotrypsin. As demonstrated in Fig. 5A, preincubation of labeled TR
with T3 gave
a different protease digestion pattern than unbound receptor. The
unliganded TR was more accessible to the protease; digestion with
chymotrypsin at a concentration of 10 µg/ml generated only two
fragments of low molecular weight, whereas the T3-induced conformation
protected the liganded TR from the protease activity. Chymotrypsin at a
concentration of 10 µg/ml digested the liganded TR in a distinct
pattern, yielding more and higher molecular weight fragments (Fig. 5A). In the presence of T3 a protected 30-kDa
peptide fragment appeared when a higher concentration of chymotrypsin
(10 µg/ml) was used. The protease resistant 30-kDa fragment was not
observed in the absence of hormone. The diverse proteolytic digestion
patterns and the T3-induced protection of a 30-kDa fragment reflect the
ligand induced conformational changes of the thyroid hormone receptor.
From immunocoprecipitation studies, it was previously shown that TR/RXR
heterodimers are formed in solution(37) . The protease
digestion pattern of the thyroid hormone receptor, when present as a
heterodimer with RXR in solution (Fig. 5B), showed a
slightly delayed digestion kinetic (a weak high molecular weight band
is still present at 10 µg/ml of chymotrypsin). Whereas the overall
proteolytic digestion pattern was identical to the unliganded thyroid
hormone receptor (Fig. 5B). Addition of T3 to the
TR/RXR heterodimer induced the same proteolytic digestion pattern as
observed for the T3 occupied TR. This delayed digestion kinetic might
be due to the interference of RXR with the digestion, but does not
suggest a major conformational alteration of TR since the protease
digestion pattern remained unchanged.
Figure 5:
Ligand-induced conformational changes
analyzed by limited proteolytic digestion. A, homodimer
analysis. S-Labeled TR
was analyzed by partial
chymotryptic digestion (2 µg/ml, 5 µg/ml, and 10 µg/ml).
Addition of 10
M L-T3 yielded a different
partial digestion pattern of TR
than in absence of ligand.
Liganded TR
was less accessible to the protease. Additionally, a
protected 30 kDa peptide fragment (triangle) was observed that
was not present in absence of ligand. The presence of 1 µM of F2-TRE double stranded oligo did not alter the ligand-induced
protection pattern. B, heterodimer analysis using limited
protease digestion.
S-Labeled TR
was preincubated
with equal amounts of RXR
and with either 1
buffer(-), F2-TRE (F2), TREp (TREp), or ME-TRE (ME) prior to limited chymotryptic digestion. The presence of
RXR
and/or the various TREs did not alter the digestion
pattern.
We were also interested on how the presence of different response elements could influence the conformation of the thyroid hormone receptor in the TR/TR homodimeric (F2-TRE) and TR/RXR heterodimeric (ME-TRE) pathways. Using the limited chymotrypsin digestion assay, the liganded TRs appeared to have the same structural configurations when interacting as a TR/TR homodimer with the F2-TRE (Fig. 5A) or as a TR/RXR heterodimer with the ME-TRE (Fig. 5B). Furthermore, TR/RXR heterodimer binding to the inverted palindrome F2-TRE or the palindromic TREp induced very similar structural changes in TR as judged from the limited proteolytic analyses. The unliganded TR yielded analogous digestion patterns in the presence of DNA as in its absence. These results suggest that the inverted palindromic F2-TRE, the DR-4 element ME-TRE and the palindromic TREp(9) , although different in their architecture, do not induce diverse structural states in TRs when analyzed in this system.
With the isolation and characterization of an increasing
number of TREs, different T3 signaling mediated by TRs have become
apparent(44, 53) In our study, we have
selected two mechanistically different TR response pathways to analyze
the molecular influence of the ligand. To facilitate this analysis we
have designed a new reporter construct that allows to follow the ligand
sensitive release of repression by TR/TR homodimers on the F2-TRE.
Interestingly, transcriptional activation of the TR/RXR heterodimer and
the release of transcriptional repression mediated by the TR/TR
homodimers appeared to be highly similar in terms of their ligand
requirements. Not only did the heterodimers and homodimers show very
similar T3 response curves, but they also reacted very similarly to T3
and T4 analogs.
Our analysis in addition suggests that the observed thyromimetic potencies of the T3 analogs reflect the binding affinities to the TRs. From studies of T3 and T4 analogs in quantitative structure-activity analysis (54, 55) it is known that the important 3`-residue interaction is strictly lipophilic and limited in size. The 4`-hydroxyl is suggested to donate a hydrogen bond to the ligand binding pocket of the TR. Consequently, when a hydrophilic group is introduced in 3`-position (compound 312810) or when the 4`-hydroxyl is blocked by a methyl group (compound 322010) the thyromimetic potencies should be eliminated, as observed here (Fig. 3). Interestingly, a lipophilic ring substitution of the Iodine at the 3` position (compound 322085) increased the biological activity (Fig. 5B). In contrast, introduction of a flexible and bulky phenyl group that increased the size of the 3` substituent (compounds 322450, 322094, and 312693) led to a considerable drop in biological activity. Changing the 3`-residue position to the 2`-position (compound 322010) led to a further reduction of the activity. Taken collectively, our experimental data confirm the known qualitative structure-binding affinity relationship for the interaction of L-T3 with TRs present in nuclear extracts (54, 55, 56) .
The subtle differences
observed between TR and TR
activation indicate that the
existing panel of T3 analogs does not yet allow sufficient molecular
discrimination to induce/repress TR subtypes selectively. This
situation may be comparable to the retinoid field several years ago,
when no satisfactory receptor selective ligands were available. The
recent discovery of selective retinoids for numerous functions (57, 58, 59, 60) could be followed
by a similar development in the thyromimetics field.
Recently, it
has been shown that L-T3 binding to TRs induces conformational changes
in the receptor, as measured by electrophoretic mobility analysis of a
receptor-DNA complex(33, 61, 62) , circular
dichroism (CD) spectroscopy(63) , or limited protease digestion
assays (64, 65) . Even though gel retardation analysis
showed enhanced migration of the monomeric and dimeric forms of the TRs
in the presence of ligand, these effects were rather small compared to
ligand induced disruption of TR/TR homodimers. While the gel
retardation technique is most frequently used to study ligand induced
effects, the CD spectroscopy method is a more effective analysis to
explore ligand induced secondary structure changes in TRs as
demonstrated for the chicken TR by Toney et al.(63) .
Interestingly, these investigators observed in addition to the ligand
effect also a measurable change in the CD spectrum when adding the
palindromic TREp, but no effect was detected in the presence of a DR-4.
These data could indicate that the TREp induces an additional
conformational change in the chicken TR. Other laboratories (64, 65) using partial tryptic digestions showed that
the T3 induced conformational changes generated trypsin-resistant
peptide fragments of the ligand binding domain. Using this same
method, we did not observe that DNA binding enhanced or decreased the
T3 induced protected chymotrypsin peptide fragments significantly.
Chymotrypsin produced a protected fragment (30 kDa) in the presence of
T3 that did not appear in absence of ligand, suggesting major
conformational differences between liganded and unliganded receptors.
Additionally, we also observed a T3 induced delayed chymotryptic
TR
degradation as previously demonstrated with trypsin, implying
that the conformational changes produced a more compact ligand binding
domain, less accessible, to the proteases. Importantly, our results
showed that the major conformational changes induced by L-T3 are at
least very similar in both heterodimers and homodimers.
In conclusion, the results presented here suggest that the ligand-induced changes in the TRs required for activation of the homodimeric and the heterodimeric response pathways are very similar. Thus the ligand binding pocket of TRs does not appear to be in very different configurations when TRs are complexed as heterodimers or homodimers on different structural elements. However, some structural changes, not detectable with the tools used here that might also contribute to differential TR function cannot be excluded. TRs are for instance also known to interact with the transcription factor AP-1 (66) . Recent results indicate (67) that this nuclear receptor/AP-1 interaction may require different ligand-induced conformational changes than transcriptional activation. Whether TR/RXR, TR/RAR(32) , TR/VDR (30) , and TR/PPAR (31) heterodimeric complexes have similar ligand responsiveness still needs to be analyzed.