©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ligand Modulates the Interaction of Thyroid Hormone Receptor with the Basal Transcription Machinery (*)

Guo-Xia Tong , Michael R. Tanen , Milan K. Bagchi (§)

From the (1) Population Council and Rockefeller University, New York, New York 10021

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We investigated the molecular mechanisms underlying the transcriptional silencing and the hormone-induced activation of target genes by thyroid hormone receptor (TR-). We developed a cell-free transcription system containing HeLa cell nuclear extracts in which unliganded human TR- represses basal transcription from a promoter bearing thyroid hormone response elements. Binding of hormonal ligand to the receptor reverses this transcriptional silencing. Specific binding of TR- to the thyroid hormone response element at the target promoter is crucial for silencing. Studies employing TR- mutants indicate that the silencing activity is located within the C-terminal rather than the N-terminal domain of the receptor. Our studies reveal further that unliganded TR- inhibits the assembly of a functional transcription preinitiation complex (PIC) at the target promoter. We postulate that interaction with TR- impairs the function(s) of one or more assembling transcriptional complexes during the multistep assembly of a PIC. Consistent with this hypothesis, we observe that, in the absence of thyroid hormone, TR- or a heterodimer of TR- and retinoid-X-receptor undergoes direct protein-protein interactions with the transcription factor IIB-TATA binding protein complex, an early intermediate during PIC assembly. Binding of hormone to TR- dramatically reduces the interaction between the receptor and the transcription factor IIB-TATA binding protein complex. We propose that the role of ligand is to facilitate the assembly of functional PICs at the target promoter by reducing nonproductive interactions between TR- and the initiation factors.


INTRODUCTION

The nuclear receptors for thyroid hormone belong to the superfamily of ligand-inducible transcription factors that also include receptors for steroid hormones, retinoids, and vitamin D(1, 2, 3) . The specificity of target gene recognition by two major types of thyroid hormone receptors (TRs),() TR- and TR-, is determined by the interaction of the receptors with short, enhancer-like sequences termed thyroid hormone response elements (TREs) located near the target gene promoter (4, 5) . TREs are composed of repeats of a consensus hexanucleotide motif, AGGTCA, arranged in a variety of ways: palindromic, direct repeat, or inverted repeat (6, 7, 8, 9, 10) . TR can bind to a TRE in vitro as a monomer, as a TR-TR homodimer, or as a heterodimer of TR with other nuclear factors termed TR-auxiliary proteins (11, 12, 13, 14) . The recently discovered nuclear receptors of 9- cis-retinoic acid, the retinoid X receptor (RXR) proteins, behave as TR-auxiliary proteins (15, 16, 17, 18, 19, 20, 21, 22) . TR binds to TRE in a ligand-independent manner and functions either as a transcriptional activator or a repressor of a TRE-linked promoter depending on the hormonal status of the receptor. In the absence of hormone, TR functions as a silencer of basal level of transcription from the target promoter (23, 24, 25, 26) . Ligand binding to the receptor releases transcriptional silencing and leads to the activation of target gene expression (23, 27, 28) .

The biological relevance of transcriptional silencing of cellular target genes by unliganded TR was suggested by recent analyses of the transcriptional properties of the avian erythroblastosis virus-encoded oncoprotein v- erbA, which is a mutated form of TR that has lost the ability to bind hormonal ligand (23, 29) . It is believed that v- erbA induces neoplastic transformation by arresting normal erythroid differentiation (30, 31) . In cell culture experiments, v- erbA acts as a constitutive silencer of TRE-linked genes and is a dominant negative repressor of the functions of wild-type TRs (23, 29, 32) . It has been reported that v- erbA may also interfere with the natural functions of other closely related members of the nuclear receptor superfamily such as the receptors of all- trans-retinoic acid, which are known regulators of cell development and differentiation (33) .

Transcriptional silencing may also contribute to the physiological perturbations associated with the naturally occurring mutations in the gene encoding TR- in human patients suffering from a genetic disorder termed generalized resistance to thyroid hormone (GRTH) (for review, see Refetoff et al.(34) ). All the GRTH mutations characterized so far result in either failure to bind T or reduced binding affinity of the hormone. The heterozygous kindreds harbor one mutant and three (one TR- and two TR-) normal alleles. It has been suggested that the product of the mutant allele inhibits normal TR function in a dominant negative manner. In vitro studies indicate that most of these mutants heterodimerize with RXR and bind to target DNA sites efficiently. Baniahmad et al.(32) using transient transfection assays demonstrated that two different GRTH mutants displaying drastically reduced or no T binding activity, respectively, functioned as constitutive repressors of target genes with strong silencing activity. These results favor the hypothesis that the dominant negative GRTH mutant is not simply an intrinsic transcriptionally nonfunctional receptor. It rather is a receptor that has lost the ability to transactivate but fully retains an active and constitutive silencing function. This viewpoint is consistent with the observation that a homozygous GRTH patient with two mutant (non-hormone-binding but active repressor) TR- alleles displayed much more severe clinical symptoms than a homozygous patient who has a complete deletion of the TR- gene (35, 36) .

The molecular basis of transcriptional silencing by TR and the hormonal modulation of this activity remains unclear. Previous studies reported that steroid hormone receptors stimulate initiation of transcription by facilitating the assembly of a preinitiation complex (PIC) at the target promoter (37, 38) . A prediction that follows directly from these studies is that the mechanism of gene repression by TR, a related nuclear receptor, may involve inhibition or destabilization of the initiation complex assembly at the TATA box. The formation of a functional initiation complex is a multistep process that proceeds through sequential assembly of RNA polymerase II and several other general transcription factors at the TATA promoter (39, 40, 41, 42) . The TATA binding protein TFIID is the first factor to enter into the complex (43) . The TFIIDTATA complex is then recognized by TFIIB producing the DBTATA complex (43) . The presence of TFIIB is critical for the subsequent recruitment of RNA polymerase II. The association of RNA polymerase II with the DB complex is mediated by TFIIF (44) . The resulting complex, DBpolF, is then recognized by TFIIE and TFIIH (45) . This creates DBpolFEH that, in the presence of ribonucleoside triphosphates, directs a basal level of RNA synthesis (46, 47) . It is conceivable that each of these steps in the complex assembly process can be a potential target of regulation by a transactivator or a repressor. Consistent with this scenario, previous studies indicated that (i) unliganded TR efficiently repressed basal transcription from a TRE-linked minimal promoter containing only the TATA box (23) and (ii) both TR- and TR- interact directly with the initiation factor TFIIB (48, 49) .

In this report, we investigated the initiation complex assembly process as a potential target of transcriptional silencing by unliganded TR-. We observed that in a cell-free reconstituted transcription system, the addition of TR- during PIC assembly led to the formation of a transcriptionally inactive complex. Ligand binding to TR reversed this transcriptional repression. We found that hormone-free TR- or TR--RXR heterodimer can interact stably with TFIIBTBPTATA complex, an early intermediate during PIC assembly. Binding of TR- or TR--RXR to the TFIIBTBPTATA complex was greatly reduced in the presence of thyroid hormone. Based on these results, we propose a hypothesis that transcriptional silencing by TR- may involve stable but nonproductive interactions between the unliganded receptor and an assembling core promoter complex such as TFIIBTBPTATA. Hormone binding to TR reverses silencing by reducing such abortive interactions and allowing subsequent assembly of a functional PIC at the promoter of the thyroid hormone-responsive gene.


MATERIALS AND METHODS

Plasmids

The construction of transcriptional templates pLovTATA and pAdML200 has been described previously (37, 38) . The test template TRETATA was constructed by introducing two copies of a palindromic TRE (TREpal) oligonucleotide (5`-GATCCTCAGGTCATGACCTGA-3`) into the BglII site of plasmid pLovTATA. The construction of bacterial expression vectors containing human TBP and human TFIIB cDNAs have been described before (50, 51) .

Antibodies

Polyclonal antibodies against human TBP and human TFIIB were purchased from Upstate Biotechnology, Lake Placid, NY. Polyclonal antibody against TR- was obtained from Affinity Bioreagents, Neshanic Station, NJ.

Bacterial Expression and Purification of Receptors

A full-length cDNA of human TR- (with 5`- NdeI and 3`- BamHI ends) or human RXR (with 5`- NdeI and 3`- BglII ends) was engineered between NdeI and BamHI sites of the bacterial expression vector pET15b (Novagen, Inc., Madison, WI). These constructs were transformed into the bacterial strain BL21(DE3)plysS. The recombinants were grown in enriched medium (500 ml) containing Terrific broth and antibiotics such as ampicillin and chloramphenicol. The bacteria were grown to an optical density (at 600 nm) of approximately 1.0 and then induced by 1 mM isopropyl-1-thio--D-galactopyranoside. After 2 h of induction the bacteria were harvested and stored frozen at -70 °C.

We employed nickel-affinity chromatography (50) to purify human TR- or human RXR tagged with six histidine residues at the N-terminal end. The bacterial pellet was thawed and resuspended in a buffer containing 20 mM Tris-HCl, pH 7.9, 500 mM NaCl, 10 mM mercaptoethanol, 10% glycerol, and a mixture of protease inhibitors such as phenylmethylsulfonyl fluoride, leupeptin, aprotinin and pepstatin. The bacteria were lysed by repeated, mild sonication and the soluble supernatant was collected following centrifugation at 20,000 g for 15 min. The supernatant was adjusted to 5 mM imidazole and applied to a nickel-nitrilotriacetic acid affinity column (Qiagen, CA). The affinity chromatography was performed according to the procedure of Hoffmann and Roeder (50) . The receptor was eluted with a buffer containing 150 mM imidazole and 5 µM zinc chloride. The peak fractions were pooled, bovine serum albumin was added as a carrier protein to a final concentration of 0.2 mg/ml and the resulting fractions were stored at -70 °C. Control protein fractions were generated from bacteria transformed with pET15b by following the same protocol. The bacterially expressed TR- displayed ligand binding affinity ( K= 1 nM) similar to that of the native receptor isolated from tissue sources. The nickel-affinity purified TR- or RXR preparations were typically 50-70% pure as estimated by SDS-PAGE. Human TFIIB and TBP were purified by following the published procedures (50, 51) .

Cell-free Transcription Assay

The cell-free transcription conditions and isolation of P-labeled transcripts have been descibed previously (38) .

Electrophoretic Mobility Shift Assay (EMSA)

The EMSAs were performed as described previously (37, 44) .

Construction of TR- Mutants

The TR- mutants TRN, TRND, and TRC lacking N-terminal 80, N-terminal 145 and C-terminal 196 amino acids, respectively, were constructed by polymerase chain reaction-assisted and oligonucleotide-directed mutagenesis. The resulting DNA fragments containing NdeI and BamHI ends were subcloned into pET15b. The mutant proteins were expressed in bacteria and purified by nickel-nitrilotriacetic acid affinity chromatography as described above.


RESULTS

Ligand-free TR- Silences Basal Level of Transcription from a TRE-linked Promoter in Cell-free Transcription Extracts

To investigate the mechanism of TR-mediated transcriptional regulation of target gene promoters, we devised a cell-free gene expression system using bacterially expressed human TR- as a source of receptor protein. We employed a synthetic target gene template TRETATA containing two copies of TREpal linked to a minimal TATA promoter and a G-free cassette as a reporter sequence. Transcription of this template was carried out in HeLa cell nuclear extracts which served as a source of RNA polymerase II and several other general transcription factors that are required for basal level of RNA synthesis. The nuclear extracts did not contain any detectable endogenous TR. When the transcription reaction was performed in the absence of any exogenously added TR, a significant level of basal transcription was observed from the test promoter. Addition of increasing amounts of purified TR- resulted in a progressive inhibition of basal transcription from the TRE-linked promoter (Fig. 1 A). This TR--induced transcriptional repression was promoter-specific since transcription from an internal control adenovirus major-late promoter remained unaffected by the addition of TR. Quantitation of the transcripts by densitometric analysis revealed that as much as 80% of basal level of RNA synthesis was repressed upon addition of 25 nM TR- to the transcription reaction (Fig. 1 B). Similar inhibition of RNA synthesis from a test template containing two copies of a direct repeat TRE (AGGTCACAGGAGGTCA) was observed also upon addition of exogenous TR- (data not shown). Our results, therefore, indicate that in cell-free nuclear extracts unliganded TR- exhibits transcriptional silencing properties similar to that displayed by the receptor in transient transfection experiments (23, 24, 25, 26) .


Figure 1: Silencing of basal transcription by unliganded TR-. Panel A, increasing amounts of TR- (0.2, 0.6, 1.0 pmol) were added to an in vitro transcription reaction containing HeLa nuclear extract and a TREpal-linked reporter template, TRETATA. The transcription reactions (volume, 30 µl each) were carried out and the transcripts were analyzed as described previously (38). The filled and the hollow arrowheads indicate transcripts from the TREpal promoter and the internal control AdML promoter, respectively. Panel B, quantitations of the RNA signals generated by the test template were performed by densitometry followed by normalization with respect to the internal control AdML signals. The plotted results represent an average of three different experiments. In each of three experiments, the value for basal transcription (- TR) was set to 100% and the corresponding values for test (+ TR) measurements were obtained. The error bars represent the standard deviation of the mean. Panel C, transcription reactions containing either TRE-linked TRETATA ( lanes 1 and 2) or TRE-less pLovTATA ( lanes 3, 4, and 5) templates were performed with or without exogenously added TR- ( lane 2, 0.5 pmol; lane 4, 0.5 pmol; and lane 5, 0.75 pmol).



To examine the functional role of TRE in transcriptional repression by TR-, we used a template pLovTATA, which exactly resembled the test template except for a lack of TRE sequences. As shown in Fig. 1 C, basal transcription from the TRE-less promoter was not affected significantly by the addition of TR- (compare lanes 3, 4, and 5), whereas greater than 60% inhibition of basal transcription from the TRE-linked test promoter was observed upon addition of the receptor (compare lanes 1 and 2). These results demonstrate that the TR-mediated silencing is entirely dependent on the presence of TRE(s) in the target promoter.

We considered the possibility that exogenously added TR may inhibit basal transcription passively by displacing from the TRE sites an endogenous transcriptional activator present in HeLa nuclear extracts. We observed, however, that the basal levels of transcription from equimolar amounts of test templates containing two copies of TRE or lacking TREs were essentially equivalent (Fig. 1 C, compare lanes 1 and 3). If a transcriptional activator bound to the TRE, one would expect relatively enhanced level of RNA synthesis from the template containing TREs. This observation strongly disfavors the idea that a transcriptional activator may bind to the AGGTCA motifs of a TRE in HeLa nuclear extracts.

Ligand Binding Reverses TR--mediated Silencing of Basal Transcription

Previous studies showed that the gene regulatory activity of TR is greatly influenced by the binding of hormonal ligand to the receptor (23, 24, 25, 26, 27, 28) . We therefore, examined the effect of Triac, a thyroid hormone analog, on the transcriptional silencing activity of TR- in the cell-free transcription assay. As shown in Fig. 2, A and B, while hormone-free TR- inhibited basal transcription ( lane 2), incubation of TR- with 1 µM Triac prior to its addition to the transcription reaction abolished receptor-mediated transcriptional repression ( lane 3). Addition of Triac did not affect transcriptional activity of the control AdML promoter (compare lanes 2 and 3) indicating that the hormonal effect on RNA synthesis was indeed mediated through TR-. These results demonstrate clearly that TR- ceases to function as a repressor of target gene transcription upon hormone binding.


Figure 2: TR--induced silencing is reversed by addition of hormone. Panel A, TR- (1 pmol) was preincubated with or without 10 M Triac before addition to the cell-free transcription reaction. Panel B, the plotted results represent an average of three independent experiments. The RNA transcripts generated from the test template were quantitated by densitometry and normalized with respect to the AdML transcripts. In each experiment the value for basal transcription (- TR) was adjusted to 100%, and the corresponding values for test measurements were obtained. The error bars denote standard deviation of the mean.



Roles of N-terminal and DNA Binding Domains of TR- in Gene Silencing

Previous studies by Baniahmad et al.(49) indicated that the transcriptional silencing function of TR- lies within the C-terminal ligand binding domain between amino acid residues 168 and 456. The functional role of either the N-terminal region or the DNA binding domain of TR- in receptor-mediated silencing, if any, has not been elucidated. To investigate this, we constructed mutant TR- molecules in which various functional domains were deleted (Fig. 3 A). The mutant receptor proteins were expressed in bacteria, purified, and assayed for their gene regulatory activities in cell-free transcription system (Fig. 3, Panels B and C). These proteins were also analyzed by EMSA for their DNA binding activities in the presence or in the absence of RXR.


Figure 3: Transcriptional activities of TR- mutants. Panel A, linear structures of human TR- and its mutants. Panel B, SDS-PAGE profile of nickel-affinity-purified TR- mutants. The proteins were visualized by Coomassie staining. Lane 1, wild-type TR-; lane 2, TRN; lanes 3 and 4, TRC; lanes 5 and 6, TRND. Panel C, TR- and various deletion mutants (1 pmol of each) were analyzed for silencing activities in cell-free transcription assays. The protein concentrations of mutant receptors were determined by comparing the intensities of stained bands with that of TR- in SDS-PAGE. Quantitations of RNA signals were performed by densitometry followed by normalization with respect to the AdML signals. The value for basal transcription (- TR) was set to 100% and the corresponding test (+ TR) values were obtained. The results of a representative experiment are shown.



The mutant TRN with intact DNA and ligand binding domains but harboring a deletion of N-terminal 82 amino acids, silenced basal transcription as efficiently as the wild-type receptor (Fig. 3 C, lane 4). TRN formed heterodimers with RXR and exhibited high affinity binding to TRE (data not shown). The addition of the hormonal ligand reversed silencing by this mutant (Fig. 3 C, lane 5). These results indicated that the N-terminal 82 amino acids of TR- are not critical for receptor-mediated gene silencing.

In contrast, another receptor mutant TRC containing the N-terminal and the DNA binding domains but, missing the C-terminal 196 amino acids, failed to repress basal transcription from the target promoter (Fig. 3 C, lane 6). TRC did not display any significant DNA binding activity either in the presence or in the absence of RXR (data not shown), presumably due to the loss of the dimerization function (52) . Recent studies by Au-Fliegner et al.(53) suggest that heterodimerization of TR is necessary for its silencing activity. A third mutant TRND with a deletion of the N-terminal 145 amino acids that included the N terminus and most of the DNA binding domain, also had no effect on basal level of RNA synthesis from a TRE-linked promoter in the cell-free transcription assay (Fig. 3 C, lane 8). This mutant, however, retained the entire ligand binding domain (168-456) that exhibits silencing activity when fused to a heterologous DNA binding domain (49) . A deletion of 63 amino acids within the DNA binding domain of TRN therefore, converted an active repressor to TRND that is impaired in both DNA binding and silencing. We conclude from these studies that specific DNA binding is essential for transcriptional silencing by TR-.

TR- Inhibits the Assembly of a Functional Transcription Preinitiation Complex at the Target Promoter

During transcription by RNA polymerase II there are many steps that can be subjected to inhibition by a gene-specific repressor. Active repression may occur at the level of assembly or function of the transcription initiation complex or elongation of the RNA transcripts. We first examined whether the assembly of the PIC is the regulatory point at which TR- exerts its gene repression effects. To test this we added TR- during or after the formation of the PIC at the target promoter and monitored RNA synthesis following the addition of nucleotide triphosphates (Fig. 4). The presence of low levels of Sarkosyl in the reactions blocked reinitiation of transcription and ensured that only one round of RNA synthesis occurred (54) .


Figure 4: TR- inhibits functional assembly of the preinitiation complex. In reaction 1, template DNA and HeLa extract were preincubated at 30 °C for 30 min without ( lane 1) or with ( lanes 2, 3, and 4) TR- and then nucleotide mixture is added to initiate RNA synthesis. In reaction 2, a mixture of HeLa extract and template DNA is preincubated initially for 15 min at 30 °C. TR- was then added ( lanes 6, 7, and 8) and the incubation was continued for 15 more min followed by the addition of nucleotide mixture. The transcription reactions were then carried on for 45 min at 30 °C. Both reactions were performed in the presence of 0.025% Sarkosyl, a non-ionic detergent.



When TR- was present during the incubation of the test template with HeLa nuclear extract in the reaction 1, we observed strong inhibition of basal RNA synthesis (Fig. 4, compare lane 1 with lanes 2, 3, or 4). In contrast, in the reaction 2, when TR- was added following preincubation of the test template with the nuclear extract which generated a PIC at the TATA promoter, no significant repression of basal transcription was noted (Fig. 4, compare lane 5 with lanes 6, 7, or 8). These results suggest that TR-, anchored to a TRE at the target promoter, may actively inhibit one or more steps during the assembly of a functional PIC at the TATA box. Once the PIC is fully assembled, it is refractory to inhibition by TR-.

TR- Interacts Directly with an Intermediate TFIIB-TBP-TATA during Preinitiation Complex Assembly

Our observation that hormone-free TR- prevents the assembly of a PIC at a minimal promoter containing only a TATA box suggests that TR- may function by interacting with components of the basal transcription machinery either directly or indirectly through a mediator protein(s). Recent studies indicated that TR- or TR- bound directly to the general transcription initiation factor TFIIB when the two proteins were combined in vitro(48, 49) . However, these studies did not reveal either the functional significance of this interaction or the identity of transcriptional intermediate(s) in the initiation complex assembly pathway that are targets of inhibition by TR- or TR-. It is also not clear how ligand may modulate the interactions between the receptor and the target intermediate(s) to reverse silencing.

The stepwise assembly of a functional initiation complex has been studied in vitro by an EMSA (43, 44, 46) . The various DNA-protein intermediates that are formed at the TATA promoter due to the sequential additions of general transcription factors and RNA polymerase II can be detected and analyzed by this assay. We employed EMSA to investigate how TR- may interact with the components of the basal transcription machinery to regulate the initiation process. It is known that in the earliest step of initiation complex assembly, the TBP, a component of the multisubunit basal factor TFIID recognizes the TATA box. The resulting complex is, however, unstable under standard EMSA conditions and cannot be easily detected by this assay. TFIIB, which does not bind to the TATA box by itself, interacts with TBP bound at the TATA box to generate the TFIIBTBP complex (40, 41) . This is the earliest intermediate detectable under EMSA conditions. We, therefore, analyzed the interaction of TR- with the TFIIBTBP complex.

As shown in Fig. 5A, incubation of either TFIIB or TBP alone with an oligonucleotide containing the AdML gene TATA box sequence did not result in the formation of any stable DNA-protein complex under our reaction conditions. However, incubation of a combination of TFIIB and TBP proteins with the TATA oligonucleotide resulted in the formation of a retarded DNA-protein complex that we term complex 1 (Fig. 5 A, lane 4). We characterized complex 1 further by employing antibodies directed specifically against TFIIB and TBP, respectively, in EMSA (Fig. 5 B). Our results showed that both antibodies could interact with and supershift complex 1, indicating that complex 1 is an authentic complex of TFIIB and TBP at the TATA box.


Figure 5: Unliganded TR- binds to the TFIIBTBPTATA complex. Panel A, a P-labeled double-stranded oligonucleotide (40 bp long, 0.1 ng) containing sequences from -30 to +10 of the AdML promoter including the TATA box was used as a probe in the EMSA which was performed as described before (44). Bacterially produced human TBP (10 ng), human TFIIB (7 ng), and human TR- (1 pmol) were used in EMSA reactions (10 µl) where indicated. The human TBP and TFIIB proteins were expressed in E. coli and purified by published procedures (50, 51). NS1 and NS2 denote nonspecific complexes. NS1 and NS2 are formed by weak interactions of nonreceptor contaminant proteins with the P-labeled DNA probe and complex 1, respectively. Panel B, preformed complex 1 was incubated with anti-TBP or anti-TFIIB antibodies as indicated in the figure. Panel C, preformed complex 1 was incubated with TR- in the presence of increasing concentrations (0.2, 0.5, 1.0, and 2.0 µl) of an anti-TR- antibody. Panel D, preformed complex 1 was incubated with increasing concentrations (0.5-1.0 µl) of the anti-TR- antibody.



We next examined whether TR- can interact with the TFIIBTBP complex. In EMSA, incubation of the TATA oligonucleotide with purified TR- alone (Fig. 5 A, lane 1) or TR- in combination with either TFIIB or TBP (data not shown) did not generate any specific DNA-protein complex. However, when the TATA oligonucleotide was preincubated with both TFIIB and TBP, and TR- (100 nM) was subsequently added to this reaction, a new, stable complex, that is retarded further in the gel, is formed. We term this higher molecular weight DNA-protein complex as complex 2 (Fig. 5 A, lanes 5-8). We also noted the generation of two nonspecific complexes indicated as NS1 and NS2 (a smeary complex immediately underneath complex 2) in Fig. 5A ( lanes 5-8) and Fig. 5 C ( lane 1). Unrelated bacterial proteins that were retained by the nickel-affinity column upon passage of Escherichia coli extract through it, existed as contaminants in our protein preparations. In control experiments (data not shown), addition of these irrelevant bacterial proteins to complex 1 generated the nonspecific complexes NSI and NS2 but not complex 2. These results indicated strongly that the formation of complex 2 was indeed TR--dependent. Since TR- itself did not bind to the TATA-containing probe, complex 2 appeared to be generated by protein-protein interaction between the receptor and the TFIIBTBP complex.

To confirm further the presence of TR- in complex 2, we studied the effects of adding an antibody specific for TR- on the formation of complex 2 in the EMSA. As shown in Fig. 5C, addition of a specific anti-TR- antibody but not a control antibody (data not shown) greatly reduced the intensity of complex 2 signal indicating that TR- is indeed a component of complex 2. The antibody did not affect the formation of either the NS1 or the NS2 complex, thus demonstrating the specificity of the antibody-antigen reaction. The anti-TR- antibody however, stimulated slightly the formation of complex 1 (Fig. 5 D). Such enhancement of complex 1 was observed also upon addition of an unrelated, control antibody (data not shown), which did not inhibit complex 2 formation. It is therefore, clear that the binding of the anti-TR antibody to the receptor prevented its physical association with complex 1. These results demonstrated that under our EMSA conditions, TR- can interact with the TFIIBTBP complex through direct protein-protein interactions. The presence of a TRE at the target promoter may facilitate stable protein-protein contacts between the TRE-bound receptor and the TFIIBTBP complex at the TATA box under cell-free transcription conditions. Based on our results, we are tempted to propose that the mechanism of transcriptional silencing by TR- may involve the formation of a stable complex of unliganded receptor, TFIIB and TBP at a TRE-linked TATA promoter at an early rate-determining step of initiation complex assembly.

TR--RXR Heterodimers Interact with the TFIIBTBPTATA Complex

RXRs exist ubiquitously in all tissues (16) . Numerous studies indicate that TR monomers or homodimers display only weak binding to TRE and heterodimerization with RXR markedly enhances binding of TR to its response element. It is thus likely that TR-RXR heterodimers may represent the functional form of TR in vivo. We therefore investigated whether the TR--RXR heterodimers can interact with the assembling transcriptional complexes during the initiation event. As shown in Fig. 6 A, lane 1, TR- alone bound weakly to TREpal. In the presence of an equimolar amount of human RXR, TR--RXR heterodimers formed readily and were the predominant species that bound to DNA. When we added increasing concentration of RXR in the presence of excess TREpal, the amount of the heterodimer-TRE complex increased only marginally (Fig. 6 A, lanes 2-6). These results indicated that under our EMSA conditions a 3-fold molar excess of RXR was sufficient to drive all the TR- in the reaction into heterodimeric association with RXR.


Figure 6: TR--RXR heterodimer binds to TFIIBTBPTATA complex. Panel A, TR-RXR heterodimers form readily on TREpal. A 20-bp double-stranded oligonucleotide containing TREpal was end-labeled by P and used as a probe in EMSA. TR-, 0.5 pmol, was combined with increasing amounts of bacterially produced human RXR, 0.5, 1.0, 1.5, 2.5, and 5.0 pmol ( lanes 2-6), in EMSA reactions. The notations TR-TR and TR-RXR point to the positions of migration of the TR homodimer and the TR-RXR heterodimer, respectively. Panel B, binding of TR--RXR heterodimers to complex 1. Complex 1 was incubated with none ( lane 1); RXR, 1 pmol ( lane 2); TR-, 0.5 pmol + RXR, 1 pmol ( lane 3); TR-, 0.5 pmol + RXR, 1.5 pmol ( lane 4); and TR-, 0.5 pmol ( lane 5). The added proteins were preincubated with unlabeled TREpal oligonucleotide (1 ng) before addition to preformed complex 1. Complex 2 and 2` denote TRTFIIBTBPTATA and TRRXRTFIIBTBPTATA, respectively.



We then examined whether these heterodimers were capable of interacting directly with the TFIIBTBP complex. For this purpose, TR- alone or RXR alone or a combination of TR- and RXR in a molar ratio of 1:3 was preincubated in the presence of excess unlabeled TREpal and added to a reaction containing TFIIB, TBP, and TATA element (Fig. 6 B). TR- alone bound to complex 1 and formed complex 2 as expected (Fig. 6 B, lane 5). The addition of equivalent amount of RXR alone to complex 1, however, did not result in the formation of any complex retarded further in the gel. This result indicated that either RXR does not recognize complex 1 or the interaction between RXR and complex 1 is rather weak, so that any RXRTFIIBTBP complex that may form is not stable enough to be detected under the conditions of the EMSA (Fig. 6 B). An enhancement of TFIIBTBP complex formation, however, was reproducibly seen when RXR was added (Fig. 6 B, lanes 1 and 2). The reason for this effect is not clear. The addition of TR--RXR heterodimers to complex 1, on the other hand, generated a retarded complex 2` (Fig. 5 B, lanes 3 and 4). Complex 2` migrated to the same region of the gel as complex 2. When gels were run for longer times, however, complex 2` appeared to migrate slightly slower than complex 2 (data not shown). This slight difference in migration has been reproducibly observed in multiple experiments and is consistent with the difference in migration of homo- and heterodimeric receptor-DNA complexes in mobility shift assays. These results thus indicated that under conditions that ensure TR--RXR heterodimerization, these heterodimers can undergo direct protein-protein interaction with the TFIIBTBP complex at the TATA box. Our results also demonstrated that TR--RXR heterodimers can interact with the TFIIBTBP complex even when bound to the hormone response element (TREpal).

Hormonal Ligand Reduces Interaction of TR- and TR--RXR with TFIIBTBPTATA Complex

Transcriptional activity of TR is modulated by thyroid hormone. Ligand binding is also known to induce an alteration in the receptor conformation (55, 56) . We, therefore, investigated whether binding of the hormonal ligand to TR- influences its interaction with the TFIIBTBP complex. In this experiment, we preincubated TR- with or without 1 µM Triac, added the receptor to complex 1 and monitored the formation of complex 2 by EMSA. Treatment of TFIIB and TBP with Triac did not exhibit any effect on the formation of complex 1 (Fig. 7 A, lanes 1 and 2). The addition of TR- preincubated with Triac however, generated markedly lesser amounts of complex 2 compared to that produced by unliganded TR- (Fig. 7 A, lanes 3 and 4). By our estimate, complex 2 formation declined by greater than 75% upon ligand binding to TR-. If this reduction in complex 2 formation is due to the inability of ligand-bound TR- to bind efficiently to complex 1, one may expect to detect unbound complex 1 in these reactions. Interestingly, as the amount of complex 2 diminished upon ligand binding to TR-, there was no concomitant appearance of complex 1 signal in these reactions (compare lanes 3 and 4). We noted, instead, an increase in the signal of the nonspecific complex NS2 upon ligand treatment. This may reflect the increased binding of irrelevant bacterial proteins to complex 1 as the formation of the specific complex (complex 2) declined. TR--RXR heterodimers treated with Triac also generated significantly lesser amounts of complex 2` than those incubated without ligand (Fig. 7 B, lanes 1 and 2). Ligand, therefore, strongly reduced the interaction between TR- or TR--RXR and the TFIIBTBP complex.


Figure 7: Ligand decreases interaction between complex 1 and TR- or TR--RXR. Panel A, in EMSA reactions, complex 1 was treated without hormone ( lane 1); with hormone ( lane 2); hormone-free TR- ( lane 3); and hormone-treated TR- ( lane 4). A P-labeled TATA oligonucleotide was used as probe. In lanes 5 and 6, complex 2 was initially formed on the TATA box and then treated with 1 µM Triac and analyzed by EMSA. NS2 indicate nonspecific complex. Panel B: TR- treated with (lane 2) or without ( lane 1) Triac (1 µM) was added to an EMSA reaction containing RXR, and complex 1. A P-labeled TATA oligonucleotide was used as probe. In lanes 3 and 4, Complex 2` was first formed and then treated with hormonal ligand as indicated. Panel C, TR- and mutants TRN and TRC (1 pmol of each) were treated with ( lanes 2, 6, and 9) or without ( lanes 1, 5, and 8) Triac and added to EMSA reactions containing TFIIB, TBP, and TATA probe as indicated in the figure.



We next examined the effects of the hormonal ligand on preformed complex 2. For this purpose, we initially generated complex 2 by incubating unliganded TR- with complex 1 and then treated it with or without Triac. As shown in Fig. 7 A ( lanes 5 and 6), the amount of preformed complex 2 was significantly reduced upon incubation with Triac but not with a control buffer. A similar reduction in complex 2` was observed when ligand-free TR-RXR-BTBP complex was preformed and then incubated with Triac (Fig. 7 B, lanes 3 and 4). It is likely that ligand binding induces a conformational change in TR that perturbs its interaction with TFIIB or TBP or both in complex 2 or 2`. Our results are consistent with the observation of Baniahmad et al.(49) that ligand binding reduced the interaction of the C terminus of TR- with TFIIB.

We also analyzed the protein-protein interactions between the TFIIBTBP complex and each of the three TR- truncation mutants that we generated. The N-terminal truncation mutant TRN, which repressed basal transcription, displayed direct binding to complex 1 (Fig. 7 C, lane 5). This interaction occurred presumably through the C-terminal domain of the receptor and generated a specific complex further retarded in the gel. In contrast, ligand-bound TRN, which failed to silence basal transcription, did not bind to complex 1 efficiently (Fig. 7 C, lane 6). These results are consistent with the idea that the silencing function resides in the C terminus of TR- and gene repression is a consequence of stable interaction between this region of the receptor and the assembling complex 1. Ligand binding to the C terminus of the receptor which reduced such interactions, also relieved transcriptional silencing.

In EMSA, the mutant TRND behaved in a manner essentially similar to that of TRN. Unliganded TRND bound to complex 1, while the hormone-bound mutant showed significantly reduced binding (data not shown). As mentioned earlier, this mutant did not bind to DNA and failed to inhibit basal RNA synthesis in cell-free transcription assay. Taken together, these results suggest that although the C terminus of TR- can potentially undergo protein-protein interaction with the basal transcription machinery, binding of the receptor to its recognition site at the target promoter is essential for stabilizing such interactions. Interestingly, the mutant TRC missing 196 C-terminal amino acids in the ligand binding domain also displayed binding to complex 1 (Fig. 7 C, lane 8). This interaction was mediated apparently through the N terminus of the receptor and as expected, was not affected by the addition of ligand (Fig. 7 C, lane 9). This result is consistent with previous reports indicating that the N terminus of TR- can also bind to TFIIB (49) . The functional consequence of such interaction is, however, not clear. Based on our observation that the mutant TRN functions efficiently as a silencer (Fig. 3 C), we propose that the C terminus of TR- rather than its N terminus, in concert with the DNA binding domain, plays a crucial role in gene silencing.


DISCUSSION

We describe here the reconstitution of a cell-free transcription system to investigate the mechanisms of transcriptional silencing of a TRE-linked gene by unliganded TR- and the ligand-induced reversal of this repression. A number of thyroid hormone- and receptor-dependent cell-free transcription systems have been described recently. Fondell et al.(48) employing bacterially produced TR- demonstrated that the hormone-free receptor repressed basal transcription in vitro, while the hormone-bound receptor failed to do so. Suen and Chin (57) have described an in vitro system in which an endogenous mixture of the TR isoforms, and , in rat GH3 pituitary cell extracts stimulated (about 4-fold) transcription from a TRE-linked promoter in the presence of T. However, the receptor pool being endogenous in this transcription system, it is not clear whether the observed hormone-induced activation is indeed over and above the basal level of transcription or simply represent a ligand-dependent release of TR-mediated silencing of target gene transcription. Recently, Lee et al.(58) using baculovirus-expressed rat TR- have reported a modest (2-fold over minus hormone control) ligand-induced stimulation of RNA synthesis from a TRE (malic enzyme)-linked template in B-cell nuclear extracts. It is possible that the apparent lack of TR--dependent RNA synthesis above the basal level in our transcription reactions is due to a paucity in HeLa extracts of putative cofactors that may facilitate such activity. Nevertheless, the HeLa extract-based cell-free transcription system described here (Figs. 1 and 2) is ideal to work out the mechanism of TR-mediated gene silencing and its ligand-induced reversal.

Our studies revealed that the addition of hormone-free human TR- during PIC assembly led to the formation of a transcriptionally inactive complex. In contrast, a fully assembled PIC was refractory to inhibition by TR-. Our observations are reminiscent of earlier studies by Johnson and Krasnow (59) , who showed that the Drosophila even-skipped (EVE) homeodomain protein functioned as a transcriptional repressor by inhibiting an early step in PIC assembly. The assembling complexes became resistant to repression by EVE at a subsequent (undefined) step. Similar results have recently been reported for unliganded TR- by Fondell et al.(48) indicating an inhibitory role of the receptor during the formation of a functional PIC. As the function of a fully assembled complex was not inhibited by TR, we reasoned that the receptor blocked one or more intermediate steps leading to the formation of the PIC.

We identified the TFIIBTBP complex, an early intermediate in the PIC assembly pathway, as a potential target of silencing by TR. Based on the results of our protein-protein interaction experiments, we postulate that the unliganded receptor represses basal transcription by interacting with this intermediate transcriptional complex and altering its function. One can think of at least two possible scenarios by which this interaction may lead to the inhibition of initiation complex assembly. Binding of TR- to the TFIIBTBP complex may lead to an impairment in the recruitment of downstream basal factor(s) such as TFIIF, RNA polymerase II, TFIIE, and TFIIH. Alternatively, TR- may bind to TFIIBTBP, and allow it to recruit some or all of the downstream factors. The resulting complex(es) may, however, enter nonproductive pathway due to an improper configuration.

The precise nature of the molecular contacts between TR- or TR--RXR and the individual basal transcription factors in the context of the TFIIBTBP complex remains to be determined. Using immunoprecipitation technique, Fondell et al.(48) reported that TR- bound to TFIIB. Baniahmad et al.(49) demonstrated that the N terminus of TR- interacted with the C terminus of TFIIB, while the C-terminal ligand binding domain of the receptor recognized the N-terminal region of TFIIB. Our studies using truncation mutants of TR- indicated also that both N and C termini of the unliganded receptor can interact with the TFIIBTBP complex. Hisatake et al.(60) reported that the C-terminal domain of TFIIB is involved in TFIIBTBP complex formation at the TATA box. Studies by Ha et al.(61) revealed that the N terminus of TFIIB interacts with TFIIF. Taken together, these results raise the interesting possibility that the interaction of the C terminus of TR- with the N terminus of TFIIB may affect TFIIF binding and subsequent recruitment of RNA polymerase II. The role of the N terminus of the receptor in transcriptional silencing however, remains unclear. Our observation that a mutant TR- lacking N-terminal 82 amino acids silenced basal transcription efficiently indicates that the interaction of the N terminus of the receptor with the basal transcription complex is not essential for silencing.

In our experiments TFIIBTBP complex represents a simplified version of the more native TFIIBTFIID complex. In the TFIID complex, TBP is associated with a number of polypeptides which are termed TBP-associated factors (39) . TBP-associated factors are essential for activator-induced transcription and TBP can efficiently replace TFIID for basal level of RNA synthesis (39, 62) . It is conceivable that TR- either in unliganded or in ligand-bound state may contact one or more TBP-associated factors in the native TFIIBTFIID complex. One should also consider the possibility that the receptor may contact directly additional components of the basal transcription machinery besides TFIIB, such as RNA polymerase II and regulate the activity of one or more transcriptional complex(es) which follow the TFIIBTFIID complex in the assembly pathway. Future studies investigating these possibilities will shed further light on how TR-, functioning as a hormone-dependent gene regulator, interacts with the native, assembling initiation complexes at the TATA box.

We demonstrate that the specificity of transcriptional silencing by TR is determined by the presence of specific hormone-response elements in the target gene promoter. Our studies showed that (i) unliganded TR- did not inhibit basal transcription from a control TRE-less promoter and (ii) a receptor fragment (145-456) containing the putative repressor domain but lacking the DNA binding function failed to silence basal transcription from a TRE-linked promoter. These results extend an earlier observation by Damm et al.(23) that the silencing effect of TR is TRE-dependent. It is interesting to note that, although the 145-456 fragment of the receptor exhibited protein-protein interaction with TFIIBTBP complex under EMSA conditions, it did not inhibit basal transcription. These results indicate that anchoring of the receptor to a TRE at the target promoter is essential for transcriptional repression. It is likely that in the intact cell or in the cell-free transcriptional extracts, the receptor may function as a gene repressor by making stable protein-protein contacts with complexes such as the TFIIBTBP at the TATA box, only when it is promoter-bound.

Binding of the hormonal ligand elicits a conformational change in TR and dramatically alters its gene-regulatory activity (55, 56) . We therefore, reasoned that the interaction of TR- with the basal transcription machinery may alter upon ligand binding to the receptor. Consistent with this line of reasoning, we observed that ligand binding to TR- drastically reduced its interaction with the TFIIBTBP complex at the TATA box under EMSA conditions. Similar results were obtained with mutant receptors, TRN and TRND. The molecular interactions within the TR-TFIIBTBP complex that are disrupted by ligand binding to the receptor are not entirely clear at present. Previous reports indicated that ligand binding weakened the interaction of a C-terminal peptide of TR- with TFIIB (49) . Our studies also suggest that ligand binding triggers the release of the C terminus of the receptor from the TFIIBTBP complex. The combined results of the cell-free transcription and in vitro protein-protein interaction assays presented here, tempt us to postulate that the mechanism of ligand-dependent reversal of TR-induced gene silencing may involve the dissolution of an abortive association between the C terminus of the TRE-bound receptor and the TFIIBTBP complex. The ligand-induced release of the inhibitory function of the receptor may allow the recruitment of downstream initiation factors and lead to the subsequent assembly of an active transcription initiation complex at the target promoter.

Recent studies suggest the possibility that efficient TR-mediated silencing of basal transcription may require additional cofactors, which may exist in unfractionated HeLa nuclear extracts. Fondell et al.(48) showed that unliganded TR- or TR--RXR heterodimers repressed basal transcription from a TREpal-linked promoter in a cell-free transcription system reconstituted with purified basal transcription factors. The overall repression of basal transcription by TR or TR-RXR in this purified system was, however, remarkably less efficient compared to that in a nuclear extract. Recent reports by Casanova et al.(63) and Baniahmad et al.(64) indicate that coexpression of a ligand binding domain peptide of either chicken TR- or human TR- inhibited transcriptional repression by a chimeric TR- or TR- protein in the absence of hormone during transient transfection in HeLa or CV1 cells. These results are consistent with the scenario that cofactor(s) that enhance TR-mediated gene repression may exist in nuclear extracts.

Our in vitro studies provide a plausible mechanism by which TR, anchored to a nuclear target site, can direct different regulatory events, namely, repression and hormone-dependent activation of a cellular gene. To understand precisely how TR controls the assembly of a functional PIC, further analysis of the composition and structure of the receptor-bound core promoter complexes and assessment of their functional activities in cell-free transcriptional assays will be necessary. Moreover, isolation of potential coactivator or corepressor molecule(s) may allow us to determine the contribution of these modulatory factors in TR-mediated gene regulation.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: The Population Council and Rockefeller University, 1230 York Ave., New York, NY 10021. Tel.: 212-327-8761; Fax: 212-327-7678.

The abbreviations used are: TR, thyroid hormone receptor; TRE, thyroid hormone response element; TBP, TATA binding protein; TF, transcription factor; RXR, retinoid X receptor; GRTH, generalized resistance to thyroid hormone; PAGE, polyacrylamide gel electrophoresis; T, triiodothyronine; PIC, preinitiation complex; TREpal, palindromic thyroid hormone response element; EMSA, electrophoretic mobility shift assay; Triac, [4-(4-hydroxy-3-iodophenoxy)-3,5-diiodophenyl]acetic acid; DB, TFIID-TFIIB complex.


ACKNOWLEDGEMENTS

We thank Drs. Bert O'Malley and Ming-Jer Tsai for providing the plasmids pLovTATA and pAdML200. We are grateful to Drs. Danny Reinberg and Alexander Hoffmann (Roeder Laboratory) for generously providing the cDNA clones for human TFIIB and human TBP, respectively. We also thank Dr. Ronald Evans for the generous gift of the cDNAs for human TR- and human RXR. We thank Dr. C. W. Bardin for his help and support during the entire course of this study. We acknowledge Dr. Indrani Bagchi for critical reading of the manuscript and Dr. M. Jeyakumar for preparing transcription factors.


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