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Originally published In Press as doi:10.1074/jbc.M306988200 on July 23, 2003

J. Biol. Chem., Vol. 278, Issue 41, 39383-39391, October 10, 2003
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Binding of the Thyroid Hormone Receptor to a Negative Element in the Basal Growth Hormone Promoter Is Associated with Histone Acetylation*

Aurora Sánchez-Pacheco and Ana Aranda {ddagger}

From the Instituto de Investigaciones Biomédicas "Alberto Sols," Consejo Superior de Investigaciones Científicas (CSIC) and Universidad Autónoma de Madrid, 28029 Madrid, Spain

Received for publication, July 1, 2003 , and in revised form, July 16, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nuclear thyroid hormone receptors (TRs) act as ligand-dependent activators, but paradoxically unliganded TRs can increase transcription of promoters containing negative response elements (nTRE), and hormone binding represses this activation. The rat growth hormone (GH) promoter contains a positive TRE and a nTRE. Ligand-dependent negative regulation mediated by the nTRE could play an important physiological role in restricting GH gene expression in non-pituitary cells that express TRs. With chromatin immunoprecipitation assays, we show here that the nTRE is responsible for binding of TR to the promoter in non-pituitary HeLa cells and that this element also governs transactivation by the unoccupied receptor and repression by triiodothyronine. Occupancy of the promoter by TR is concomitant with appearance of acetylated histone H3, and triiodothyronine causes release of the receptor as well as disappearance of the acetylated histone from the promoter. Although the nTRE overlaps the TATA box, the receptor does not exclude binding of TATA-binding protein, but could rather facilitate formation of the preinitiation complex. Furthermore, the proximal GH promoter is synergistically stimulated by unliganded TR and TATA-binding protein, whereas the ligand represses this cooperation. Constitutive receptor activity and synergism with TATA-binding protein require binding of corepressors. Furthermore, inhibitors of histone deacetylases enhance promoter activation by the unliganded receptor and reduce triiodothyronine-dependent repression, whereas expression of HDAC1 reverses promoter stimulation. This suggests that partitioning of histone acetylases and deacetylases between the receptors and basal transcription factors could be involved in regulation of the basal GH promoter by TRs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Thyroid hormones exert their actions in cells by binding to nuclear receptors (TRs),1 with act as ligand-dependent transcription factors (1). The mechanisms of ligand-dependent transcriptional activation by nuclear receptors are relatively well understood. Positively regulated genes contain thyroid hormone response elements (TREs), which preferentially bind heterodimers of TR with the retinoid X receptor (RXR). In these elements the receptors act as repressors in the absence of ligand and as ligand-dependent activators upon binding of thyroid hormone (T3). This is based on an exchange of corepressor complexes for coactivator complexes in response to the ligand (2, 3). Coactivator recruitment depends on a highly conserved motif in the C-terminal {alpha}-helix of the ligand-binding domain (LBD), referred to as AF2 (4). This helix extends away from the LBD in the unliganded receptor but upon ligand is tightly packed against the body of the LBD, creating together with residues located in helices 3, 5, and 6 a surface that facilitates coactivator interactions (5, 6). A signature LXXLL motif in the coactivator proteins mediates association with receptors LBDs (7, 8). Coactivators form large complexes, which act as chromatin remodelers through intrinsic histone acetylase activity (3). In addition, other complexes appear to act more directly on the transcriptional apparatus, suggesting that activation of gene expression by the receptors involves both chromatin modifications and direct recruitment of basal transcription factors to the regulated promoter (2). Transcriptional silencing by corepressor complexes also involves changes in histone acetylation. The corepressors SMRT (silencing mediator for retinoid and thyroid hormone receptors) (9) and NCoR (nuclear receptor corepressor) (10) assemble in complexes that include histone deacetylases (HDACs). The corepressors can interact indirectly with class I HDACs through the Sin3 protein (11–13), and directly with class II HDACs through a different domain (14, 15). Recruitment of deacetylases is believed to cause chromatin compaction and transcriptional repression.

In addition to stimulate transcription, TRs can also repress gene expression in a ligand-dependent manner. In some instances, this repressive effect is secondary to transcriptional antagonism with other transduction pathways or transcription factors (16, 17). However, in other cases, ligand-dependent repression could require binding to negative TREs (nTREs). A rather common finding is that on nTREs the unoccupied receptor increases transcription and the ligand reverses this stimulation (18–21). Although at the present time the properties of the negative hormone response elements are not totally understood, there is increasing evidence that corepressors and deacetylase activity could be also involved both in the stimulation caused by the unliganded receptor and in the ligand-dependent negative regulation (22, 23).

Repression of gene expression by nuclear receptors can also occur due to competition for DNA sites with other transactivators. Particularly, several examples of repression of gene activity by competitive binding at the TATA box have been described previously (24–26). In these cases, the overlap or close location of binding sites for the receptors and the TATA box suggests that repression results from competition between the TATA-binding protein (TBP) and the receptors. This could lead to the inhibition of formation of a functional preinitiation complex (PIC) by displacement of transcription factor IID (TFIID) from the TATA box. In contrast, TRs can directly interact with TBP (27), and in transient transfection assays ligand-dependent transactivation of simple promoters consisting of a hormone response element and a TATA box by other nuclear receptors can be enhanced in response to over-expression of TBP (28, 29).

Rat growth hormone (GH) gene transcription is strongly stimulated by T3 in pituitary cells. The GH promoter contains a positive TRE located at –167 to –190 bp upstream of the transcription start site, which appears to mediate the stimulation by T3 and retinoic acid (30, 31). In addition to the stimulation of GH promoter constructs containing the positive hormone response elements, T3 inhibits the activity of constructs containing only the proximal GH promoter sequences (19, 32). The unliganded TR causes a strong activation of the core GH promoter, which is reversed after ligand addition, and this inhibitory effect of T3 appears to be mediated by a nTRE adjacent to the TATA box (33). This nTRE could play a role in repressing GH promoter activity in non-pituitary cells. In the present study we show by chromatin immunoprecipitation (ChIP) assays that the nTRE is responsible for binding of TR to the GH promoter in non-pituitary cells, whereas the distal element is dispensable. Unliganded TR binds to the promoter and causes transactivation, and T3 releases the receptor from the promoter and represses activation. In addition, occupancy by TR causes an increase of binding of acetylated histone H3, and the release of receptor from the GH promoter upon ligand binding is concomitant with a reduction on the levels of acetylated histone bound. These results demonstrate the important role of histone acetylation on regulation of the core GH promoter by TR. Despite the location of the nTRE overlapping the TATA box, TR does not exclude binding of TBP. Furthermore, TBP strongly potentiates ligand-independent activation of the GH promoter by TR in transient transfection assays. The synergistic effect of TBP with the receptors does not require the AF2 domain. In contrast, mutants with reduced ability to bind corepressors do not display constitutive activity and do not cooperate with TBP, suggesting the involvement of corepressors on ligand-independent activation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—The constructs –39GH-CAT, –145GH-CAT, and –530GH-CAT containing 5'-flanking sequence of the rat GH promoter cloned into the polylinker region of the pUC8 vector in which an AP-1 site has been deleted, and TATA-CAT containing a consensus TATA box in the same vector have been described previously (19). –530mutGH-CAT was generated with the QuikChangeTM site-directed mutagenesis kit (Stratagene) with the oligonucleotide 5'-GGAAAACCGGTTGGGTATAAAACGGGTATGCAAGGG-3'. This mutation removes the proximal nTRE located between nucleotides –34 and –18. Expression vectors for TR{alpha} and TR{beta} contain the cDNA sequences of {alpha} chick TR and the {beta} isoform of human TR{beta}, respectively. Chimeras between v-erbA and TR{alpha}, as well expression vectors for the mutants chick TR{alpha} E401Q, E401K, and K232I were described previously (4, 32). In the AHT receptor 3 residues in helix 1 of the LBD of TR{beta} have been mutated (10). In the TR{alpha} mutant C51G, a conserved cysteine residue was substituted by a glycine residue by site-directed mutagenesis. This mutant receptor does not bind DNA and was a gift from A. Pascual. Expression vectors for TBP and the NCoR (12, 28) were also used.

Cell Culture, Transient Transfections, and CAT Assays—HeLa cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and transfected with calcium phosphate with 5 µg of the reporter plasmid as described previously (19). The reporter plasmid was cotransfected with 0.5 µg of the TBP vector and/or 1.5 µg of TR or the TR mutants. The amounts of other expression vectors used are shown in the legends to the corresponding figures. The total amount of DNA from each transfection was kept equal by the addition of the corresponding empty expression vectors. Treatments with 100 nM T3 were administered in Dulbecco's modified Eagle's medium containing 10% AG1 x 8 resin-charcoal stripped newborn calf serum. After 48 h, CAT activity was determined and quantified. Each experiment was performed with triplicate cultures and was repeated at least twice with similar relative differences in regulated expression. The data are expressed relative to the CAT values obtained in control untreated cells and represent mean ± S.D. values.

Mobility Shift Assays—Gel retardation assays were carried out with highly purified preparations of TR/RXR obtained from vaccinia infection of HeLa cells, and with recombinant TBP, TFIIA, and TFIIB expressed in bacteria and purified as described previously (34). As probes we used an oligonucleotide corresponding to –39/+12 bp of the rat GH promoter (19) and the oligonucleotide 5'-GTGACGACTTATAAAACCCCAGGG-3' containing the consensus TATA box of the Hsp70 gene. Mobility assay conditions were optimized for simultaneous binding of the receptors and TBP. For this purpose the poly(dI-dC) was replaced by poly(dG-dC), and 10 mM MgCl2 was used in the binding buffer. For the binding assays the purified proteins were incubated on ice for 15 min in a buffer (10 mM Hepes, pH 8.4, 90 mM KCl, 1 mM dithiothreitol, 5 µg/ml bovine serum albumin, 10% glycerol) containing 500 ng poly(dG·dC) and then for 20–30 min at room temperature with ~30,000 cpm double-stranded oligonucleotide end-labeled with [{gamma}-32P]ATP using T4-polynucleotide kinase. DNA·protein complexes were resolved on 5% polyacrylamide gels in 0.5x Tris borate EDTA buffer. The gels were then dried and autoradiographed.

Protein-Protein Interactions—Wild type and mutants TR{alpha} cloned in pSG5 were used for in vitro transcription and translation following the manufacturer's recommendations of the TNT7 quick-coupled transcription/translation system (Promega). Reactions were performed in the presence of 40 µCi of L-[35S]methionine (Amersham Biosciences). TBP and TBP deletions mutants fused to GST (35) were expressed in the bacterial strain BL21 (DE3). They were grown at 37 °C in LB until the absorbance reached 0.6. Then the induction was performed at 30 °C for 2h with 0.4 mM isopropyl-1-thio-{beta}-D-galactopyranoside. GST and GST fusion proteins were purified by standard techniques following the recommendations of Pharmacia. The expression of correctly sized proteins was monitored by SDS-PAGE. GST pull-down assays were performed with 5 µl of the in vitro translated L-[35S]methionine-labeled receptors as described previously (36). These proteins were incubated with 1 µg of the GST fusion protein, or with the same amount of GST as a control, immobilized in glutathione-Sepharose beads. The reaction with the beads was performed for 1 h at 4 °C in a binding buffer containing 25 mM Hepes KOH, pH 7.9, 1% glycerol, 5 mM Mg2Cl, 1 mM dithiothreitol, 0.05% Triton X-100, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride. Free proteins were washed from the beads with a buffer containing increasing concentrations (100, 200, and 500 mM) of KCl, and the bound proteins were analyzed by SDS-PAGE and autoradiography.

Chromatin Immunoprecipitation—HeLa cells were cotransfected with the reporter plasmids –530GH-CAT, –530mutGH-CAT, or –145GH-CAT, and expression vectors for TR{alpha} (1.5 µg) and/or TPB (0.5 µg) as indicated in the corresponding figures. ChIP assays were performed essentially as described previously (17). Chromatin was immunoprecipitated with antibodies against TR{alpha} (FL-408, Santa Cruz), TBP (SC-273, Santa Cruz), or acetylated histone H3 (C#06–599, Upstate Biotechnology) (1 µg/sample). An equivalent amount of anti-GFP antibody (SC-8334) was used as a negative control. The precipitated DNA was quantified by real-time PCR using SYBR Green. A 230-bp fragment of the rat GH gene was amplified using the primer pair 5'-GTTTTCCCAGTCACGAC-3' and 5'-GCTTCCTTAGCTCCTGAAAATCTCG-3'. PCR signals were always corrected for the PCR signal obtained with the total DNA IP input. Expression of the receptor or TBP did not alter results obtained with the negative control GFP antibody. The ratio obtained with the corresponding antibody in untreated cells was arbitrarily set as 1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Binding to the nTRE Is Required for Regulation of GH Promoter Activity by TR—In non-pituitary HeLa cells, which express low TR levels, treatment with T3 did not affect the activity of a reporter plasmid containing the fragment –530 to +8of the GH promoter. However, and in agreement with our previous observations (19), when this plasmid was transfected along with an expression vector for TR{alpha}, a ligand-independent stimulation of transcription, which was reversed by T3, was observed (Fig. 1A). To analyze the contribution of the positive TRE located at –167/–190 and the nTRE that overlaps the TATA box to this regulation, a plasmid in which the proximal element was mutated was also used. Interestingly, TR stimulated only weakly activity of the mutated promoter, suggesting that the proximal element is mostly responsible for the observed regulation. This was confirmed with the construct –145GH-CAT, which contains only the nTRE, which was induced by the unliganded receptor and repressed by T3 to a similar extent as the plasmid containing both DNA elements. To analyze whether binding to DNA was required for activation of the minimal GH promoter, the influence of a TR{alpha} mutant in a cysteine of the first zinc finger of the DNA binding domain was also analyzed. As illustrated in Fig. 1B, whereas the unliganded wild type receptor significantly increased basal promoter activity, the mutant receptor C51G that does not bind DNA was unable to increase CAT activity. As a consequence, a ligand-dependent repression was not observed either. These results indicate that binding of the receptor to the nTRE is necessary for ligand-independent stimulation, as well as for negative regulation by T3.



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  		FIG. 1.
Role of the nTRE on regulation of the GH promoter by TR. A, HeLa cells were transfected with 5 µg of the plasmid –530GH-CAT (–530) or with a construct in which the nTRE located between nucleotides –34 and –18 has been mutated (–530 mut). The reporter plasmids were cotransfected with 1.5 µg of an expression vector for TR{alpha} or with the same amount of an empty expression vector. In B, the cells were cotransfected with the –145GH-CAT plasmid and with wild type TR{alpha} (wt) or with the C51G mutant. Following transfection the cells were treated with medium alone or with 100 nM T3 (black bars) and after 48 h were harvested for CAT assay. CAT activities, shown as the mean values ± S.D., are expressed relative to the levels obtained in untreated cells transfected with the corresponding reporter construct alone.

 

In Vivo Binding of TR to the GH Promoter—Chromatin immunoprecipitation assays with an anti-TR{alpha} antibody were performed in HeLa cells transfected with different GH promoter plasmids. As shown in Fig. 2, upon expression of TR{alpha}, a significant binding to the construct that contains both TREs was found. Promoter occupancy by the receptor paralleled transcriptional activity and, accordingly, TR{alpha} binding to the promoter was reversed by T3. In addition, no binding of the receptor to the promoter fragment in which the nTRE has been mutated was observed. This is also in agreement with the transactivation data shown in Fig. 1A and suggests again that the nTRE is responsible for receptor binding and regulation of GH promoter activity in non-pituitary cells. This was confirmed by the finding that TR{alpha} bound strongly to the minimal GH promoter that contains only the nTRE. Again, promoter occupancy was observed only with the unliganded receptor, and T3 inhibited binding.



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  		FIG. 2.
In vivo binding of TR to the GH promoter. ChIP assays were performed in HeLa cells cotransfected with the GH reporter plasmid indicated and an expression vector for TR{alpha} (1.5 µg) or the same amount of a noncoding vector. Cell lysates from untreated cells and cells treated with 100 nM T3 for 24 h were immunoprecipitated with anti-TR{alpha} and anti-GFP antibodies. The amount of TR recruited to the promoter in cells expressing only endogenous receptor levels was essentially undetectable, being enriched less than 1.5-fold with respect to the levels obtained with the negative control antibody. The data are the means of three experiments performed in duplicate. Values were corrected with the inputs and are expressed as fold activities as compared with values obtained in untreated cells transfected with empty vector only.

 

Unliganded TR{alpha} Causes Recruitment of Acetylated Histone H3 to the Minimal GH Promoter—It has been proposed that ligand-independent transcriptional stimulation by TR is associated with increased histone acetylation (22) and that ligand-induced recruitment of a HDAC is involved in negative regulation by T3 (23). To analyze the effect of histone acetylation on TR-mediated regulation of the minimal GH promoter, the cells were incubated in the presence of inhibitors of HDACs or transfected with an expression vector for HDAC1. Incubation with the HDAC inhibitor trichostatin A (TSA) at a 25 nM concentration did not alter basal activity but, as shown in Fig. 3A, increased significantly the promoter response to unliganded TR{alpha}. Incubation with a 200 nM concentration of TSA resulted in basal promoter stimulation and also in a significantly reduced response to T3 (Fig. 3A). Although this difference is better observed at this high concentration of TSA, T3-dependent repression was also partially inhibited with 25 nM TSA. Similar results were obtained with butyrate, another deacetylase inhibitor (data not shown). In addition, expression of HDAC1 decreased strongly stimulation by the unoccupied receptor, and consequently also reduced T3-dependent repression (Fig. 3B). This effect appears to be specific, because expression of the deacetylase did not affect basal promoter activity.



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  		FIG. 3.
Influence of acetylation on activation of the core GH promoter by TR. A, the GH promoter plasmid –39GH-CAT was cotransfected with TR{alpha}, and the cells were treated with T3 in the presence of increasing concentrations of TSA. B, the cells were transfected with TR{alpha} in the presence and absence of an expression vector for HDAC1 (1.5 µg) as indicated. CAT activity was determined in cells treated during 48 h with T3.

 

These results strongly suggest that histone acetylation is involved in stimulation of transcription by the unliganded receptor and inhibition by T3. To directly analyze in vivo binding of acetylated histones to the minimal GH promoter, ChIP assays were performed with an antibody recognizing acetylated histone H3. As shown in Fig. 4A, expression of TR{alpha} caused a significant increase in the amount of acetylated histone H3 bound to the promoter, which was strongly inhibited in the presence of T3. This inhibition was rapid, and acetylated H3 was already reduced after 30 min of incubation with T3, and was maximal after 4 h. Binding of acetylated histone H3 paralleled promoter occupancy by the receptor. Thus, as shown in Fig. 4B, TR{alpha} was released from the promoter with the same kinetics after incubation with T3.



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  		FIG. 4.
Recruitment of acetylated histone H3 in response to TR. HeLa cells were transfected with the –145GH promoter plasmid in the presence and absence of 1.5 µgofTR{alpha} and treated with T3 for the time periods indicated. Aliquots of the cell lysates were used for ChIP assays with antibodies against acetylated histone H3 (A) and TR{alpha} (B). An anti-GFP antibody was used as a negative control. Results are expressed relative to the levels obtained in the corresponding untreated cells. Values are mean of duplicates, and similar results were obtained in an additional experiment.

 

Binding of TR and TBP to the Proximal GH Promoter— Because the nTRE overlaps the TATA box, we were interested in investigating whether the receptors affect binding of TBP to the GH promoter. For this purpose, binding of recombinant TBP in the presence and absence of purified receptors to a probe spanning nucleotides –39/+1 of the GH gene was analyzed by gel retardation assays. The nTRE is located between nucleotides –34 and –18, and we have shown that this element binds preferentially TR/RXR heterodimers (19). As illustrated in Fig. 5A, both the receptor heterodimer and TBP bound independently to this promoter causing the appearance of retarded complexes. In addition, when TR/RXR and TBP were mixed, the formation of a new complex with a slower mobility was observed (lane 5). Binding was slightly decreased in the presence of T3 (lane 6). The presence of TBP and the receptors in this complex was analyzed by competition with specific binding sites and by incubation with antibodies against both components (data not shown). The super-retarded complex, whose abundance increased as the concentration of the heterodimer in the assay augmented (lanes 8-10), was not formed when a consensus TATA box corresponding to the human Hsp70 gene was used (lanes 12 and 13). The affinity of the protein-DNA complexes was assessed by off-rate experiments challenging the performed complexes with an excess of the unlabeled GH promoter fragment. As shown in Fig. 5B, binding of the receptor heterodimer was rather stable, with a significant binding being still observed after 30 min of incubation with the oligonucleotide. In addition, the slow mobility complex formed in the presence of TBP was inhibited with the same kinetics as TR/RXR alone, showing that the interaction with TBP does not alter the apparent affinity of binding of these factors to the promoter. Together, these results suggest that binding of receptor heterodimers to the nTRE does not exclude binding of TBP to the overlapping TATA box. To demonstrate whether TR and TBP can bind also simultaneously in vivo to the proximal GH promoter, ChIP assays were performed in HeLa cells transfected with vectors for both proteins alone or in combination. As shown in Fig. 5C, expression of TR{alpha} or TBP caused an increase in promoter occupancy by these proteins after immunoprecipitation with the corresponding antibodies. Furthermore, these factors did not appear to compete for in vivo binding to the promoter, as association of TR and TBP was not decreased when both factors were expressed together. Therefore, the receptors and TBP can form a ternary complex on the GH promoter, which may be facilitated by the existence of direct protein to protein interaction between these factors (27).



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  		FIG. 5.
Binding of TBP and TR to the TATA-associated nTRE of the rat GH promoter. A, in lanes 1–6, a labeled oligonucleotide spanning fragment –39/+12 of the GH promoter was incubated with 50 ng of the purified TR/RXR heterodimer, 5 ng of recombinant TBP, or the combination of both. When indicated, the binding reactions were carried out in the presence of 1 µM T3. In lanes 8–10, this oligonucleotide (TATAGH) was incubated with TBP in the presence of increasing amounts (25, 50, and 100 ng) of the receptor heterodimer. In lanes 12 and 13 the consensus TATA box of the Hsp70 gene (cTATA) was incubated with 5 ng of TBP alone or in the presence of 100 ng of TR/RXR. Lanes 1, 7, and 11 show the mobility of the unretarded oligonucleotides. The mobility of TR/RXR, TBP, and the super-retarded ternary complex is indicated by arrows. B, the labeled GH promoter fragment was incubated with 100 ng of TR/RXR (lanes 2-6), 5 ng of TBP (lanes 7–11), or the combination of both (lanes 12–16), and the protein·DNA complexes were allowed to form. A large excess of unlabelled oligonucleotide was added at time 0, and aliquots were loaded in the polyacrylamide gel at the indicated time points. C, ChIP assays were performed with HeLa cells transfected with the –145CAT plasmid and expression vectors for TR{alpha} (1.5 µg), TBP (0.5 µg), or the same amount of empty vectors. After 24 h, aliquots of the cell extracts were immunoprecipitated with antibodies against the receptor ({alpha}-TR{alpha}), the basal factor ({alpha}-TBP), or GFP (used as a negative control). Data are expressed relative to the values obtained in cells transfected with the empty vector alone and are the mean obtained in two separate experiments performed in duplicate. D, gel retardation assays with the GH oligonucleotide was performed with TR/RXR (100 ng) and/or TBP (5 ng) in the presence or absence of recombinant TFIIA (50 ng) and TFIIB (50 ng).

 

During the formation of the PIC, several other basal factors interact with TBP and stabilize binding of TFIID. We then tested by gel retardation assays, whether accessibility of TFIIB and TFIIA to the GH promoter was altered by the presence of the receptor heterodimer. As shown in Fig. 5D, TBP formed the expected complex with TFIIA (lane 4) or with the combination of TFIIA plus TFIIB, which presented an increasingly reduced mobility (lane 5). Incubation with TR/RXR significantly increased the abundance of the retarded complexes formed in the presence of TFIIA and TFIIB (lanes 7 and 8). These results suggest that binding of the receptors to the nTRE adjacent to the TATA box could facilitate the assembly of the PIC on the GH promoter.

TBP Synergizes with the Unoccupied TR to Stimulate the Proximal GH Promoter—The functional interaction between the receptor and TBP was examined in HeLa cells transfected with a reporter CAT gene containing sequences –39/+12 of the GH promoter. As shown in Fig. 6, expression of TBP enhanced promoter activity, and this effect was not influenced by T3 in the absence of transfected TR. In addition, the level of promoter activity achieved in cells expressing both TR{alpha} and TBP was greater than that produced by each factor alone. Incubation with T3 significantly reversed the stimulation to levels similar to those obtained with TBP alone. These results show that the unliganded receptor and TBP synergistically activate the basal GH promoter, which contains binding sites for both factors. In contrast, a construct containing the consensus TATA box, which is not stimulated by TR (19), did not cooperate with TBP (not illustrated). In addition, the functional cooperation of TBP and TR was specific, because expression of other components of the basal transcriptional machinery such as TFIIB or TFIIA did not increase the stimulatory effect of TR{alpha} on the promoter (data not shown).



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  		FIG. 6.
Synergistic activation of the proximal GH promoter by unliganded TR and TBP. HeLa cells were transfected with 5 µgofthe reporter plasmid containing 39 bp 5' from the start of transcription of the GH promoter (–39GH-CAT). The plasmid was cotransfected with 1.5 µg of an expression vector for TR{alpha} alone or in combination with 0.5 µg of an expression vector for TBP.

 

The AF2 Domain Is Dispensable for Synergism of TR and TBP, but Is Required for the Reversal of Stimulation by Thyroid Hormone—To establish whether mutations that diminish or abolish T3-dependent transactivation also affected the ability of the receptor to cooperate with TBP, different chimeras of the v-erbA oncogene, which is the viral counterpart of TR{alpha}, the c-erbA protooncogene, were used (Fig. 7). TBP cooperated strongly with construct C1, an otherwise wild type TR{alpha} containing the C terminus of v-erbA, in the absence of ligand. However, in contrast with the results obtained with the wild type receptor, no significant reversal of stimulation upon incubation with T3 was found in cells expressing this mutant. Conversely, the V1 chimera, in which the C terminus of TR{alpha} containing the AF2 domain has been introduced in the v-erbA background, was able not only to display constitutive activity and synergism with TBP, but also gained the ability to mediate T3-dependent repression of promoter activity. Other TR{alpha}/v-erbA chimeras shown in Fig. 3 were able to cooperate with TBP with potency similar to that shown by the native receptor. However, T3 only repressed activity in cells transfected with constructs that possess the TR{alpha} C-terminal region.



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  		FIG. 7.
Synergism of TR{alpha}/v-erbA chimeras with TBP for activation of the proximal GH promoter. Schemes of the chimeras between TR{alpha} and v-erbA are depicted in the left part. V-erbA-derived sequences are shown as dark boxes, with the dots representing the residues that are mutated in the viral protein. The black box indicates the viral gag region, and the open triangle the C-terminal deletion present in v-erbA. CAT activity was determined in HeLa cells transfected with the reporter construct –39GH-CAT and expression vectors for TR{alpha}/v-erbA chimeras and/or TBP.

 

The above results suggested that helix 12 of the LBD, which contains the core AF2 domain, is dispensable for ligand-independent stimulation and cooperation with TBP, although it appears to be required for ligand-dependent repression. To further analyze the role of the AF2 domain, the influence of mutation of a conserved glutamic acid residue in helix 12, which is required for binding of coactivators and ligand-dependent transcriptional activation, was also examined. In addition, the effect of this mutation was compared with that caused by mutation of a conserved lysine residue in helix 3. Both residues form a "charge clamp" that positions the LXXLL motif of the coactivators into the hydrophobic pocket formed by the surfaces of receptor helices 3, 5, and 6 (3). As shown in Fig. 8A, mutants E401K and E401Q, were able to synergize with TBP to stimulate the core GH promoter with a potency similar to that shown by the native receptor. However, the stimulation was not inhibited by T3 in cells expressing the mutant receptors. The lack of ability to suppress does not correlate with a reduced ability to bind ligand. E401Q binds T3 with a normal affinity (Kd of 0.1 nM), and although the E401K mutant shows a decreased affinity with a Kd of 3.3 nM (4), T3 was used in the experiments at 100 nM, a high enough concentration to occupy the receptor. In contrast with the results obtained with the helix 12 mutants, K232I mutant in helix 3 was unable to cooperate with TBP to stimulate the promoter. Furthermore, promoter activity was consistently lower in cells expressing the combination of TBP and the mutant receptor than in cells expressing TBP alone.



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  		FIG. 8.
The TR{alpha} AF2 domain is not required for cooperation with TBP. A, activation of the –39GH-CAT construct by transfection of 1.5 µg of wild type (wt)TR{alpha} or the same amount of the AF2 defective mutants E401K and E401Q in helix 12, and K232I in helix 3 of the ligand-binding domain. The receptors were transfected alone or in combination with TBP as indicated. Following transfection, the cells were treated with T3 and after 48 h were harvested for CAT assay. B, interaction of TR{alpha} with TBP. GST-TBP and the GST-TBP fragments 1–176 and 95–339 were immobilized in glutathione agarose beds. GST alone was used as a control. TR{alpha} wild type or the TR{alpha} mutants E401K, C1 (lacking helix 12) or K232I, labeled with [35S]methionine by in vitro translation were incubated with 1 µg of the GST fusion proteins. The upper panel shows the input (20%) for each labeled receptor. After incubation the beads were washed, and the eluted proteins analyzed by SDS-PAGE and visualized by autoradiography.

 

One possible explanation for the lack of stimulatory effect of the K232I receptor could be the inability of this mutant to interact with TBP. However, a normal interaction of the helix 3 mutant with TBP could be observed. As shown in Fig. 8B, not only TR{alpha} and the helix 12 mutants E401K and C1, but also the mutant K232I efficiently bound TBP in in vitro GST pull-down assays. This interaction mapped to the C-terminal domain of TBP, as the different receptors failed to interact specifically with the N-terminal portion of TBP (amino acids 1–167), whereas they associated with the C terminus of this factor (amino acids 95–339).

A Mutation of TR that Reduces Corepressor Binding Affects Stimulation of the Basal GH Promoter and Synergism with TBP—It has been recently shown that mutation of the conserved lysine residue in helix 3 also impairs interaction of corepressors with the receptors (37–39). Therefore, we also examined the influence of other mutant receptor defective in interaction with corepressors on GH promoter activity. This receptor, the AHT mutant, has a 3 amino acids substitution in the so-called CoR box located in helix 1 of the LBD (10). The AHT mutation has been introduced in the background of the TR{beta} gene and abolishes binding of corepressors. As shown in Fig. 9A, wild type TR{beta} also caused a ligand-independent activation and cooperated with TBP to stimulate the promoter. The synergism between TR{beta} and TBP was also reversed in the presence of T3. In contrast, the unliganded AHT mutant was unable to stimulate the promoter and did not cooperate with TBP.



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  		FIG. 9.
A, a TR{beta} mutated in the corepressor box is devoid of ligand-independent activation and does not cooperate with TBP. The GH promoter plasmid was cotransfected with 1.5 µg of an expression vector for TR{beta} or with the same amount of the AHT mutant. The receptors were transfected alone or in combination with the expression vector for TBP. CAT activity was determined in cells incubated with and without T3. B, influence of corepressors on the synergism between TR and TBP. CAT activity was determined in cells cotransfected with the reporter plasmid and expression vectors for TR{alpha} and/or TBP. These plasmids were co-transfected alone or in combination with 1.5 µg of expression vectors for the corepressor NcoR.

 

In view of the finding that the mutant receptors K232I and AHT that are defective for corepressor binding are unable to stimulate the promoter, the influence of expression of the corepressors on promoter activity was also analyzed. As shown in Fig. 9B, the corepressor NCoR did not increase TBP-mediated stimulation, and caused a modest increase in ligand-independent activation by TR. However, NcoR was unable to further induce the synergism between TR and TBP, and in fact the levels of CAT activity were lower in cells expressing the combination of the three factors than in cells transfected with TR plus TBP. Therefore, although corepressor binding appears to be involved in ligand-independent activation of the GH promoter, the endogenous cellular levels of corepressors appear to be sufficient to mediate the observed effect on the GH promoter. In keeping with the observation that mutations in the AF2 helix did not impair stimulation by the unliganded receptor, expression of the coactivator steroid receptor coactivator-1 did not increase T3-independent activation of the GH promoter (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
T3 significantly increases transcription of the endogenous GH gene in pituitary cells. This induction is mediated by a well defined positive TRE located in the 5'-flanking region, between nucleotides –169 and –190 (30). However, the rat GH promoter also contains a negative TRE overlapping the TATA box (33). In this element the unoccupied receptor causes transactivation and the ligand reverts stimulation (19). We have previously shown (19) that GH promoter activity is repressed by liganded TR, even in the presence of the positive TRE, in cells that do not express the pituitary-specific transcription factor GHF-1/Pit-1. Interestingly, the ligand-independent inhibition is transformed into a synergistic activation in non-pituitary cells expressing both the receptors and GHF-1/Pit-1 (19, 36). The GH gene is specifically transcribed in cells of the anterior pituitary, which express both the receptors and GHF-1/Pit-1, and the nTRE could contribute to restrain GH gene expression in non-pituitary cells expressing TRs. To further examine the role of the nTRE on GH promoter activity in this work we have used HeLa cells, which express low receptor levels and do not express the pituitary factor. Our results show that, upon expression of TR, the receptor binds in vivo to the GH promoter, and that deletion of the distal TRE does not affect TR recruitment to the promoter, whereas mutation of the proximal element abolishes binding. This demonstrates that the positive TRE is non-functional in non-pituitary cells and that the nTRE governs both TR binding to the GH promoter and transactivation by the unliganded receptor. We have also observed that the ligand causes release of the receptor from the GH promoter, in parallel with the decrease in transcriptional activity.

Both DNA-binding dependent and independent mechanisms have been proposed to explain negative regulation of gene expression by T3. Although recent data indicate that binding of TR to DNA is required for regulation of the thyroid-stimulating hormone {alpha} (TSH{alpha}), TSH{beta}, and thyrotropin releasing hormone promoters (37), it has also been proposed that binding of TR to the TSH{alpha} promoter is not required for regulation that would be mediated by TR interaction with promoter bound proteins, as opposed to TR binding directly to DNA sequences (22). Particularly, a cyclic AMP response element (CRE) appears to play a critical role in regulation of this promoter by TR. We have also demonstrated that TR can inhibit CRE-mediated transactivation. Thus, T3 inhibits GHF-1/Pit-1 expression by interference with the activity of CRE motifs in the promoter (38). TR does not bind directly to these motifs, but the receptor is recruited to the promoter upon ligand binding (17). In contrast, our present results demonstrate that TR binds in vivo to the GH promoter in the absence of ligand and that T3 cancels this binding. Furthermore, mutation of the nTRE abolishes binding, and mutation of the receptor DNA binding domain renders a TR unable to regulate the GH promoter. Therefore, binding to the nTRE appears to be crucial for the action of TR on this promoter. The different results obtained with the different promoters indicate the existence of alternative mechanisms by which genes are negatively regulated by T3.

Histone acetylation appears to play a critical role both in stimulation by the unoccupied receptor and in ligand-dependent reversal of activation. Thus, an increase in histone acetylation caused by inhibitors of HDACs significantly enhances promoter activation by the unliganded receptor. It should be noted that this occurs with low concentrations of inhibitors that induce small and transient changes in histone acetylation (39) and have little if any effect on basal promoter activity. In addition, expression of HDAC1 reverses promoter stimulation by the receptor, reinforcing the idea of the importance of histone modification in this activation. Also consistent with a role for HDACs in negative regulation, HDAC inhibitors attenuate T3-dependent repression of the TSH{alpha} (22) and TSH{beta} promoters (23), and we have observed that the same occurs with the GH promoter. Accordingly, measurement of in vivo occupancy of the GH promoter by acetylated histone H3, demonstrates that binding of the unliganded receptor causes a concomitant increase in the appearance of the acetylated histone. Our ChIP assays also show that treatment with T3 causes a rapid depletion of acetylated histone H3 from the GH promoter. This decrease coincides with the release of TR, and it is most likely involved in ligand-dependent transcriptional repression. This is contrary to the expectation based on positive response elements in which HDACs are dissociated from the receptors upon ligand binding. However, it has been shown that the nTRE of the TSH{beta} promoter constitutively binds HDAC1, and that upon addition of T3, this element recruits other deacetylase, HDAC2, which in in vitro assays interacts directly with the receptor DNA binding domain (23).

It has been hypothesized that ligand-independent activation of the TSH{alpha} promoter is the consequence of withdrawal of HDACs from other target sites, resulting in a net increase of histone acetylation and in transcriptional stimulation (22). However, we observe stimulation by unliganded TR of minimal promoter sequences in which no known binding sites for other transcription factors are present. The GH promoter sequences used in our study essentially consist of binding sites for the receptors and the basal transcriptional machinery. This suggests that partitioning of HDACs between the receptors and basal transcription factors could be involved in GH promoter activation. The finding that basal transcription factors can be acetylated (40) supports the notion that acetylation of these factors could play a role in activation by unliganded TRs. We have previously shown that the inhibitors of histone deacetylation also potentiate ligand-dependent activity of the endogenous GH gene and of reporter plasmids containing the positive TRE in pituitary cells (39). Thus, taken together, our results demonstrate that histone acetylation appears to play a key role in both ligand-dependent and -independent stimulation of the GH promoter by TRs.

The nTRE of the GH gene overlaps the TATA box. In this study we show that despite the proximity of the binding sites for the TR/RXR heterodimer and TBP, these factors appear to bind simultaneously forming a stable ternary complex on the promoter. The existence of the receptor-binding site appears to be a prerequisite for formation of the complex containing the receptor heterodimer and TBP, because it is not observed in a consensus TATA box. That TR does not exclude TBP from binding to the promoter was also suggested by ChIP assays. Although in these assays the fraction of transfected templates occupied by either protein is undetermined, binding of TR or TBP to the promoter was not decreased when both proteins were expressed together. That these factors could bind at the same time on the promoter might be explained by the particular structure of TBP and its DNA binding manner. The overall shape of TBP is saddle-like, and the concave surface of the saddle binds to the minor groove of DNA over the region of the TATA box (41). Binding induces a drastic bend of the DNA, which could allow binding of receptors to the major groove on the nTRE. Direct protein to protein interactions between the receptors and TBP (27), which we have confirmed in our pull-down assays, most likely contribute to stabilization of the complex on DNA. However, simultaneous interaction of TBP with nuclear receptors on promoters containing binding sites for both does not appear to be a general property. Thus, the glucocorticoid receptor binds to an element that overlaps a noncanonical TATA box in the human osteocalcin gene, and the receptor and TBP bind to their cognate overlapping elements in a mutually exclusive manner resulting in glucocorticoid-dependent repression of transcription (24, 25).

Previous results (42) have shown that a TR{beta}-RXR heterodimer interacts with a TFIIB·TBP·TATA complex. Our results additionally show that the receptor heterodimer, at least in vitro, can interact with a complex containing also TFIIA on the core GH promoter. Interactions of unliganded receptors with TBP and TFIIB had been interpreted to result in disruption of transcriptional initiation of promoters containing positive TREs (27, 43, 44). However, more recent data have suggested that liganded TR activates transcription by recruiting TBP and the remaining basal transcription factors of the preinitiation complex rather than by activating the complex already formed on the promoter (45). Our data suggest that binding of the unliganded heterodimer to the nTRE could also result in recruitment of the PIC complex to the GH promoter that contains the negative element. This is based not only on in vitro binding studies, but also on functional studies in which we observe a strong synergism between unoccupied TR and TBP. These results show that TBP is limiting for TR-activated transcription of this minimal promoter. Potentiation of activation by unliganded receptors has not been previously reported, but in other studies it has been shown that ligand-dependent activation of simple promoters consisting of an estrogen response element and a TATA box can be enhanced in response to over-expression of TBP (29). Although this potentiation appears to be independent of spacing between the estrogen response element and the TATA box, the influence of overlapping elements for the estrogen receptor and TBP has not been examined.

We have previously reported (19) that ligand-independent activation of the core GH promoter by TR does not require the receptor AF2 helix. Our present results demonstrate that helix 12 is also dispensable for synergism of the unoccupied receptor with TBP. Because the AF2 motif is required for coactivator recruitment, these results suggest that they do not play an important role in constitutive activation by TR. In contrast, the AF2 domain appears to be important in mediating ligand-dependent repression. This is based in the finding that T3 reversed the synergistic actions of TR and TBP on the promoter, but this effect was abolished in TRs in which helix 12 of the LBD is deleted, or mutated in a conserved glutamic acid residue critical for binding of coactivators. These results were confirmed with the use of TR{alpha}/v-erbA chimeras lacking the AF2 helix, which were able to increase basal promoter activity and to cooperate with TBP, but were unable to mediate ligand-dependent reversion of this stimulation.

In contrast with the results obtained with helix 12 AF2 mutants, mutation of a conserved residue in helix 3 of the LBD renders a receptor that was unable both to mediate a constitutive T3-independent activation of the core GH promoter (19) and to cooperate with TBP to enhance promoter activation. This receptor interacts normally with TBP, showing that the direct interaction between TR and TBP is not sufficient for the synergism between both factors. The lysine residue in helix 3 of TR was originally demonstrated to be critical for coactivator recruitment (46, 47). However, more recent observations have suggested an additional role for this residue in binding of corepressors. Interestingly, corepressors possess a motif similar to the signature LXXLL motif in coactivator proteins, which is sufficient for corepressor binding and ligand-induced release. This allows binding of coactivators and corepressors to partially overlapping binding sites in the receptor. Accordingly, amino acids of helixes 3, 5, and 6, among them the conserved Lys-232 residue, that directly participate in coactivator binding are also involved in corepressor association (48–50). That corepressors could participate in T3-independent activation of the GH promoter is further suggested by the finding that a TR{beta} mutant in the CoR box was also unable to constitutively activate the promoter and to synergize with TBP. Although this mutation also appears to affect overall structure of the receptor LBD (51), the mutated residues in the CoR box are essential for interaction of receptors with the corepressors (9, 10). It has been described that overexpression of NCoR or SMRT paradoxically enhances rather than suppresses basal activation of the TSH{alpha} and thyrotropin releasing hormone (TRH) promoters by unliganded TR (20). However, we find that expression of corepressors has little effect on the stimulation of the basal GH promoter by unliganded receptor and that, furthermore, it does not enhance the synergism with TBP. A different promoter context and/or cell-specific effects could explain these differences.

In summary, our results show an important role of the nTRE on the regulation of GH promoter activity by TR. Binding of unliganded TR to this element causes a paradoxical stimulation that is associated with recruitment of acetylated histones to the promoter. T3 represses this activity by releasing the receptor from the promoter and inhibiting histone acetylation. Furthermore, this hormone represses cooperation of the receptor with TBP. Therefore, the existence of the nTRE could play a role in preventing GH gene expression in non-pituitary cells expressing TRs under physiological conditions in which the thyroid hormones are present.


   FOOTNOTES
 
* This work has been supported by Grant BMC2001-2275 from the Dirección General de Enseñanza Superior e Investigación. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Instituto de Investigaciones Biomédicas, CSIC, Arturo Duperier 4, 28029 Madrid, Spain. Tel.: 34-91-5854453; Fax: 34-91-5854401; E-mail: aaranda{at}iib.uam.es.

1 The abbreviations used are: TR, thyroid hormone receptor; GH, growth hormone; nTRE, negative thyroid hormone response element; ChIP, chromatin immunoprecipitation; TBP, TATA-binding protein; HDAC, histone deacetylase; RXR, retinoid X receptor; LBD, ligand-binding domain; SMRT, silencing mediator for retinoid and thyroid hormone receptors; NCoR, nuclear receptor corepressor; PIC, preinitiation complex; GST, glutathion S-transferase; T3, triiodothyronine; CAT, chloramphenicol acetyltransferase; GFP, green fluorescent protein; TSA, trichostatin A; TSH, thyroid-stimulating hormone; CRE, cyclic AMP response element; TF, transcription factor. Back


   ACKNOWLEDGMENTS
 
We thank D. Barettino, R. Evans, A. Pascual, and M.Meisterernst for plasmids used in this study



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