From the Division of Endocrinology and Metabolism, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0678
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ABSTRACT |
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Thyroid hormone receptors are ligand-modulated transcription factors that can repress or activate transcription depending upon the absence or presence of thyroid hormone and the nature of the hormone response element to which the receptors are bound. The ability of thyroid hormone receptors to repress transcription in the absence of ligand is thought to be due to associations with nuclear hormone receptor corepressors. Ligand binding by the thyroid hormone receptor is believed to dissociate these corepressors and recruit coactivators to promote transcription from target promoters. We hypothesize that variations in response element architecture may influence both the association and dissociation of corepressors from DNA-bound thyroid hormone receptors. Using a chimeric corepressor, we find that ligand alone does not fully relieve corepressor-mediated repression, particularly in the presence of thyroid hormone receptor and its heterodimerization partner, the retinoid X receptor. Interestingly, the steroid receptor coactivator 1 together with ligand is able to mediate full release of corepression, but this relief is dependent upon the architecture of the response element to which the nuclear receptor dimer-corepressor complex is bound. These studies suggest that other cellular factors in addition to ligand may be required for the release of corepressors from thyroid hormone receptor dimers.
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INTRODUCTION |
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Thyroid hormone receptors (TRs)1 are ligand-modulated transcription factors in the erbA superfamily that are found constitutively in the nucleus, bound to specific DNA sequences known as thyroid hormone response elements (TREs). TRs can bind TREs as monomers, homodimers, or heterodimers with the retinoid X receptor (RXR) and positively or negatively regulate gene expression depending on the nature of the TRE and the presence of thyroid hormone (T3). TREs are generally composed of two or more TR binding half-sites arranged as direct repeats spaced by four nucleotides, palindromes without a nucleotide spacer, or inverted palindromes spaced by six nucleotides (1). The optimal TR binding half-site is the octamer TAAGGTCA, an extended version of the common core response element hexamer AGGTCA (2). Using Saccharomyces cerevisiae as a system devoid of endogenous RXR, we have shown that TREs composed of optimal octameric half-sites can be activated by TR alone. In contrast, TREs composed of sub-optimal hexameric half-sites require the expression of RXR with TR for substantial gene activation (3).
In mammalian cells, unliganded TRs can function as transcriptional repressors when bound to positive TREs (4). Upon T3 binding, however, TRs direct the activation of gene expression from target promoters. Recent studies have characterized numerous TR-associated proteins that play important roles in converting unliganded TR transcriptional repression into liganded TR activation. In the absence of bound ligand, TRs associate with the nuclear receptor corepressor (N-CoR) or the silencing mediator of retinoid and thyroid hormone receptors (SMRT), which mediates the repression of basal levels of gene expression from target promoters (5, 6). TR binding of T3 is thought to induce a conformational change in the receptor, which results in dissociation of the corepressor complex and allows recruitment of coactivators such as the steroid receptor coactivator 1 (SRC-1) (7) and the cAMP response element-binding protein-binding protein (8). The association of coactivators with the liganded TR tethers activation domains into the vicinity of target promoters, resulting in transcriptional activation. Gene activation in response to thyroid hormone is, therefore, both a release of repression and a recruitment of activation functions. The molecular mechanisms by which ligand binding modulates these opposed functions are only beginning to be understood.
All mammalian cell lines examined to date contain endogenous corepressors and coactivators, which complicates studies designed to address the roles of these cofactors in TR-dependent gene regulation. To delineate the relative contributions of release of repression and recruitment of coactivation functions in T3-dependent gene activation, we have reconstituted TR-dependent gene expression in the lower eukaryote S. cerevisiae. Since S. cerevisiae lacks endogenous nuclear hormone receptors, nuclear receptor corepressors, and nuclear receptor coactivators, it provides a null background in which the functions of these molecules can be studied independently or in combination.
It has been shown that unliganded TR is a transcriptional activator
in S. cerevisiae (3, 9-11), a finding likely due to the
absence of endogenous nuclear receptor corepressors in yeast. Ligand-dependent gene activation (above this basal
activation) from single copy TREs requires the presence of coactivators
such as SRC-1 (3). Interestingly, the orientation of the half-sites composing single TREs and the 5
dinucleotides flanking the hexameric half-sites modulate the ability of SRC-1 to induce gene expression in
response to ligand (3). Given this influence of TRE architecture on
coactivation, we hypothesized that similar TRE variations also may
modulate the ability of corepressors to repress transcription from
target promoters. To test this hypothesis we have examined the
influence of a nuclear receptor corepressor on TR function in a panel
of yeast reporter strains bearing different TREs.
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EXPERIMENTAL PROCEDURES |
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Yeast Reporter Strains and Expression Vectors--
All yeast
strains were derived from the parental strain SEY 6210 (MAT;
ura3-52; leu2-3, -112; his3-
200; trp1-
901; lys2-801; suc2-
9).
-Galactosidase reporter constructs containing
octameric (optimal) or hexameric (suboptimal) direct repeats or
palindromes (Table I) were inserted into
the yeast chromosome at the leu2 locus by homologous
recombination (3). Strains bearing single insertions were identified by
Southern blot analysis (data not shown). TR
1 (12) and RXR
(13)
were subcloned into pRS-GalH on opposite sides of a bi-directional
galactose-inducible promoter (3). SRC-1 (7) was subcloned into the
plasmid p413-TEF, and T-CoR (see below) was subcloned into the plasmid
p424-GPD (14). Yeast transformations were performed using a high
efficiency lithium acetate protocol (15), and single transformants were isolated on agar plates containing appropriate selective synthetic medium.
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Construction of the Fusion Protein T-CoR--
The chimeric
corepressor protein T-CoR was created containing the repression domain
of the yeast repressor protein Tup1 fused to the TR interaction domains
of N-CoR. The SV40 nuclear localization sequence, hemagglutinin epitope
tag, and the coding sequence of amino acids 1-200 of Tup1 were
amplified by the polymerase chain reaction using YCp91-Tup1 (16) as the
template. The 5 oligonucleotide primer encoded a 5
EcoRI
restriction site, start codon, and 18 nucleotides of the SV40 nuclear
localization sequence; the 3
oligonucleotide primer contained an
ApaI site and the reverse complement of Tup1 codons
195-200. The amplified fragment was digested with EcoRI and
ApaI, purified by agarose gel electrophoresis, and then
ligated to the gel-purified ApaI-ClaI fragment of
pBKS-N-CoR (5), which contains N-CoR codons 1819-2453 with two copies of FLAG epitope sequences attached. This ~2.6 kilobase pair fragment, designated T-CoR, was purified by agarose gel electrophoresis and then
ligated into the multiple cloning site of the expression vector
p424-GPD using the EcoRI and ClaI sites.
Hormone Inductions and Reporter Gene Assays--
Yeast
strains were grown in selective synthetic media containing a
nonrepressive carbon source (3% glycerol, 3% ethanol) to limit the
levels of nuclear receptor expression before hormone inductions. Yeast
cultures were diluted to an absorbance at 650 nm
(A650) of 0.2, nuclear receptor expression was
induced by the addition of 3% galactose, and the cultures were grown
overnight at 30 °C in the presence or absence of 1 µM
3,5,3-triiodothyroacetic acid (triac), a T3 analog. The next day the
A650 of each culture was determined. 1.5 ml of
each culture was pelleted by microcentrifugation, the supernatants were
removed, and the pellets were frozen at
20 °C. The
-galactosidase activity of each pellet was measured as described
previously (3).
-Galactosidase activities were normalized to the
level of activity present in each yeast reporter strain containing an
empty nuclear receptor expression vector and the indicated p413-TEF and
p424-GPD constructs. Data represent the results of at least five
independent experiments and are expressed as the mean ± S.E.
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RESULTS |
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N-CoR and SMRT Do Not Function in S. cerevisiae-- Transformation of yeast with N-CoR or SMRT expression vectors failed to repress reporter gene activation by TR or RXR + TR (data not shown). Although N-CoR expression was detected by immunoprecipitation (data not shown), it is difficult to know whether the lack of functional repression was due to an insufficient level of N-CoR expression or incompatibilities with potential downstream targets in yeast (or lack of such targets in yeast). To overcome this problem, the chimeric protein T-CoR was constructed and studied as a corepressor of TR-mediated gene activation in yeast.
T-CoR Represses TR and RXR + TR Activity on the Optimal Direct
Repeat TRE--
In the absence of ligand, TR-TR homodimers and RXR-TR
heterodimers function as constitutive activators in yeast, presumably due to the lack of nuclear corepressors (3, 9-11). A yeast strain
bearing an optimal direct repeat TRE (8DR4) -galactosidase reporter
construct was activated 8.0-fold by the expression of TR alone in the
absence of triac (Fig. 1A).
T-CoR repressed this activity to just a 2.7-fold stimulation, which
represents a 76% repression. This repression was dependent on the
presence of TR
1, as T-CoR expression had essentially no effect on
the levels of reporter gene activity in reporter strains bearing an
empty nuclear receptor expression plasmid (data not shown) or in the
context of RXR alone (Fig. 1B). In addition, native Tup1 did
not repress TR
-induced
-galactosidase activity (data not
shown).
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T-CoR Represses RXR+TR Activity on the Sub-optimal Direct Repeat
TRE 6DR4--
T-CoR function also was studied in the context of the
sub-optimal direct repeat 6DR4, which requires the coexpression of RXR with TR for substantial gene activation (3). Thus, although expression
of TR or RXR alone had little or no effect on reporter gene activity
(Fig. 2, A and B),
coexpression of TR and RXR led to a 5.7-fold induction of
-galactosidase in the absence of triac (Fig. 2C). This
5.7-fold RXR + TR induction was repressed to 2.3-fold (73% repression)
upon expression of T-CoR. The addition of triac, however, was unable to
relieve this repression, as reporter gene induction remained
essentially unchanged at 2.4-fold (Fig. 2C). Thus T-CoR
repressed gene activation mediated by an obligate RXR-TR heterodimer
from the sub-optimal TRE 6DR4. However, in contrast to the situation
with TR-TR homodimers on 8DR4, this T-CoR-mediated repression of RXR-TR
heterodimers on 6DR4 was unaltered by the addition of ligand. This
suggests that other factors in addition to ligand may be required to
relieve corepressor-mediated repression, particularly in the context of
the RXR-TR heterodimer.
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T-CoR Represses TR and RXR + TR Activity on the Optimal Palindrome-- To assess the importance of half-site orientation on corepressor function, we expressed various combinations of T-CoR, TR, and RXR in a yeast strain containing a reporter construct driven by an optimal palindromic TRE (8Pal0) or an optimal inverted palindromic TRE (8IP6). T-CoR had no effect on reporter gene activity from 8IP6 in the presence of TR alone, RXR alone, or TR with RXR (data not shown). However, on the palindrome 8Pal0, T-CoR mediated a 73% repression of TR-TR homodimer activity and a 74% repression of RXR + TR activity in the absence of triac (Fig. 3, A and C). Thus, in contrast to the results with the optimal direct repeat 8DR4, T-CoR was equally effective in mediating repression in the presence of TR alone and TR with RXR. The addition of triac modestly relieved T-CoR-mediated repression from 8Pal0 in the context of TR alone (from 73 to 49% repression; p = 0.01), but did not significantly do so in the presence of TR with RXR (p = 0.2). Taken together, these data would suggest that corepressor-mediated repression may be influenced by TRE half-site orientation, and the release of corepressors from TR may require other events in addition to ligand binding, particularly in the context of RXR-TR heterodimers.
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SRC-1 Does Not Augment the Release of T-CoR-mediated Repression of
RXR+TR Activity on Direct Repeat TREs--
Baniahmad et al.
(17) demonstrated that disruption of the AF-2 activation domain in the
extreme C terminus of TR1 did not destroy the ability of the
receptor to bind ligand but did abolish the ability of ligand to
relieve the silencing function of TR
1. These authors suggested the
potential importance of ligand-dependent trans-activating
factors, which interact with the AF-2 domain, in the release of
receptor-associated corepressors. In yeast, since no endogenous nuclear
receptor coactivators exist to associate with the liganded nuclear
receptor complex, it might be predicted that ligand alone would fail to
relieve corepressor activity fully. We hypothesized that the presence
of a nuclear receptor coactivator in addition to T-CoR might be
sufficient to restore complete ligand-dependent relief of
T-CoR-mediated corepression in the yeast reporter strains. To test this
hypothesis, we coexpressed the mammalian coactivator SRC-1 with T-CoR
and examined its ability to augment the release of T-CoR-mediated
corepression in response to the addition of ligand.
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SRC-1 Augments Relief of T-CoR-mediated Corepression from the
Optimal Palindromic TRE 8Pal0--
In contrast to the results seen
with the DRs, on 8Pal0, SRC-1 is able to substantially enhance the
triac-mediated release of T-CoR repression. In the case of the
expression of RXR plus TR without SRC-1, T-CoR repressed
-galactosidase induction from 4.8-fold to 1.9-fold, and triac did
not significantly relieve this repression (Fig. 3C). In the
presence of SRC-1, T-CoR was still a strong repressor of RXR+TR
activity, as expected, reducing reporter gene induction in the absence
of triac from 4.7- to 2.2-fold (Fig.
5C; Figs. 5, A and
B, will be discussed subsequently). Now, however, the
addition of triac allowed the 2.2-fold induction to rise to 5.9-fold,
which approaches the level seen with TR, RXR, SRC-1, and triac in the
absence of T-CoR (7.9-fold). Thus, triac plus SRC-1 was more effective
than triac alone at relieving T-CoR-mediated repression of RXR + TR
activity on 8Pal0 (p = 0.017).
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DISCUSSION |
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Thyroid hormone receptors are found constitutively bound to DNA at response element sequences. In general, unliganded TRs function as transcriptional repressors of nearby target promoters. Thyroid hormone binding serves to convert the TR from a repressor to an activator of transcription.
The interaction of unliganded TRs with corepressor proteins seems a likely explanation for the repression exhibited by unliganded TRs bound to positive TREs. Thyroid hormone binding by TR is thought to induce a conformational change in the receptor, which in some way promotes corepressor dissociation. This ligand-induced destabilization of the TR-corepressor interaction has been demonstrated in vitro (5, 6, 18) and is assumed to occur in vivo. However, the identification of TR mutants that retain the ability to bind ligand but fail to release silencing functions and fail to activate gene expression suggested that ligand alone may not be sufficient to release corepressor activities from TR (17).
Little is known regarding the functional importance of TRE architecture in regulating unliganded TR-mediated repression. Given the influence of TRE architecture on the function of coactivators (3, 19), we hypothesized that variations in the sequence and orientation of TRE half-sites also would modulate corepressor function. S. cerevisiae provided an excellent null background in which to examine the regulation of corepressor release from TR-TR homodimers and RXR-TR heterodimers, since, in contrast to mammalian cells, endogenous nuclear receptors, nuclear receptor corepressors, and nuclear receptor coactivators are absent in this organism.
Our initial efforts were directed at studying wild type SMRT or N-CoR in yeast. However, neither SMRT nor N-CoR functioned as a corepressor in our system. Whether this was due to poor expression of these molecules or incompatible differences between mammalian repression domains and yeast basal machinery targets is not clear. To circumvent this problem, we have constructed a fusion protein referred to as T-CoR in which the repression domain of the yeast repressor Tup1 is fused in-frame to the nuclear receptor interaction domains of N-CoR. In yeast, T-CoR functioned as a corepressor of constitutive TR-TR and RXR-TR activity, and this repression could be modulated to varying degrees by the addition of ligand. How relevant is T-CoR to the study of mammalian corepressor function? Recent studies have shown that repression by N-CoR and SMRT involves an interaction with Sin3A and a histone deacetylase (20, 21). Repression by Tup1 does not appear to utilize the yeast homologs of Sin3A and histone deacetylase (22). Therefore, T-CoR is not likely to repress transcription by the identical molecular mechanisms used by N-CoR and SMRT in mammalian cells. However, evidence suggests that interactions with Sin3A and histone deacetylase do not fully account for N-CoR- and SMRT-mediated repression (23). Therefore, it remains possible that the mechanism of repression by these proteins is at least partially related to the mechanism of repression by Tup1. More importantly, the mode of repression by N-CoR is unlikely to alter the mechanisms by which it is recruited and released from TR, as that information appears to be encoded in distinct interaction domains contained within its C terminus (24, 25). Since N-CoR and SMRT do not function in our yeast system, T-CoR is a reasonable tool for dissecting the recruitment and release of corepressors from nuclear receptors bound to the response elements of target genes.
We found that the ability of T-CoR to repress TR-mediated gene activation depended upon the composition of the nuclear receptor dimer (TR-TR or RXR-TR) and the sequence and orientation of the half-sites to which the nuclear receptor dimer was bound. Although T-CoR repressed unliganded TR-TR homodimers on optimal direct repeat and optimal palindromic elements equally well, the ability of ligand alone to relieve this repression was substantially better from the optimal direct repeat than from the optimal palindromic TRE. The functional interaction of mammalian corepressors with nuclear hormone receptors has been shown to require the involvement of two nuclear receptor ligand binding domains (26). Conformational differences imposed upon a TR-TR homodimer by binding a direct repeat TRE versus a palindromic TRE may alter the positioning of the ligand binding domains in such a way as to account for the unequivalent release of T-CoR-mediated repression upon addition of ligand. Importantly, however, on 8Pal0, SRC-1 enhanced the ability of triac to release corepressor activity, achieving an effect similar to that of triac alone on 8DR4.
On the other hand, unliganded RXR-TR heterodimers were repressed by T-CoR, but ligand alone failed to substantially relieve T-CoR-mediated repression of heterodimers bound to sub-optimal direct repeat and optimal palindromic TREs. Moreover, SRC-1 did not augment the ligand-dependent relief of T-CoR-mediated repression from RXR-TR heterodimers bound to direct repeat TREs. This contrasts with the potent ability of SRC-1 to augment the relief of T-CoR-mediated corepression on 8Pal0. This would suggest that the conformational differences in the TR-dimer dictated by its binding to different TREs influences the ability of SRC-1 to augment the release of the corepressor in response to ligand. Additionally, this suggests that other mammalian nuclear factors, perhaps other coactivators, are required for the release of corepressors from RXR-TR heterodimers bound to direct repeat TREs.
It is possible that differences in corepressor release from TR homodimers and RXR-TR heterodimers may have important biological functions. Sequestration of the promiscuous heterodimerization partner RXR either by RXR-RXR homodimer activation in response to 9-cis-retinoic acid or through heterodimerization with other nuclear receptor family members may increase the relative importance of TR-TR homodimers in mediating T3 responses from high affinity TREs. This interplay might be particularly important during development, given the essential roles of both thyroid hormone and retinoic acid in this process. In addition, N-CoR interactions with TR have been suggested to play an important role in mediating ligand-independent activation from negative regulatory elements in the TRH promoter (27). Therefore, regulating the release of N-CoR from TR may be particularly relevant in overall T3 homeostasis.
Our data demonstrate that the requirements for the release of corepressors from TR-TR homodimers and RXR-TR heterodimers are different and may be modulated by the architecture of the TRE to which the TR dimer is bound. We propose that the variations in half-site sequence, number, and orientation within naturally occurring TREs serve to modulate the composition of the bound nuclear receptor dimers, the recruitment and release of associated corepressor proteins, and the recruitment and function of coactivator proteins. These multiple layers of integrated regulation may be important in determining the complexity of responses to a seemingly simple thyroid hormone signal.
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ACKNOWLEDGEMENTS |
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We would like to thank Keith Koch, Dennis Thiele, and Diane Robins for helpful discussions and careful reading of this manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK44155.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: University of Michigan
Medical Center, 5560 MSRB II, 1150 W. Medical Center Dr., Ann Arbor, MI
48109-0678. Tel.: 313-763-3056; Fax: 313-936-6684; E-mail:
rkoenig{at}umich.edu.
1
The abbreviations used are: TR, thyroid hormone
receptor; TRE, thyroid hormone response element; RXR, retinoid X
receptor; T3, thyroid hormone (3,5,3-triiodothyronine); triac,
3,5,3
-triiodothyroacetic acid; N-CoR, nuclear receptor corepressor;
T-CoR, Tup1 N-CoR chimeric protein; SMRT, silencing mediator of
retinoid and thyroid hormone receptors; SRC-1, steroid receptor
coactivator 1; DR, direct repeat; Pal, palindrome; IP, inverted
palindrome.
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REFERENCES |
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