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Endocrinology Vol. 138, No. 11 4705-4712
Copyright © 1997 by The Endocrine Society

ARTICLES

Active Repression by Thyroid Hormone Receptor Splicing Variant {alpha}2 Requires Specific Regulatory Elements in the Context of Native Triiodothyronine-Regulated Gene Promoters

A. Farsetti1, J. Lazar1, M. Phyillaier, R. Lippoldt, A. Pontecorvi and V. M. Nikodem

National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Genetics and Biochemistry Branch (A.F., J.L., M.P., R.L., V.M.N.), Bethesda, Maryland 20892-1766; Institute of Experimental Medicine, Consiglio Nazionale Delle Richerche (A.F.) and Institute of Medical Pathology (A.P.), Catholic University, Rome 00168, Italy

Address all correspondence to: Antonella Farsetti (present address: Institute of Experimental Medicine, CNR, c/o IRE-CRS Molecular Oncogenesis Laboratory, Via delle Messi D’Oro 156, 00158 Roma, Italy). Requests for material should be sent to V.M.N. E-mail: Farsetti{at}dotto.ifo.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Structural requirements for the inhibitory action of thyroid hormone receptor splicing variant {alpha}2 (TR{alpha}2) on T3/TRß1-mediated transactivation were investigated in native promoters of two T3-regulated genes: the brain-specific myelin basic protein (MBP) and the housekeeping malic enzyme (ME). T3/TRß1 transactivation of MBP256-chloramphenicol acetyl transferase (CAT) and ME315-CAT constructs was inhibited and unaffected by TR{alpha}2, respectively. In electrophoretic mobility shift assays, TR{alpha}2 bound MBP-thyroid response element (TRE) as a monomer but failed to interact with ME-TRE. Mutations of ME-TRE allowed TR{alpha}2 binding but not inhibition of T3/TRß1-mediated transactivation. In the context of the MBP promoter, replacement of MBP-TRE with ME-TRE or exchange of MBP TATA-like box with the ME GC-rich region spanning the transcription start site abolished TR{alpha}2 dominant negative action. Simultaneous introduction of both MBP-TRE and MBP TATA-like box in the context of ME promoter, however, triggered TR{alpha}2 inhibition of T3/TRß1 transactivation, indicating that these regulatory elements are necessary, but not individually sufficient, to mediate TR{alpha}2 dominant negative activity. Functional studies at low TR{alpha}2/TRß1 ratios revealed that binding to TRE facilitates TR{alpha}2 dominant negative action while prevention of DNA interaction by altering TR{alpha}2 P-box structure preserved TR{alpha}2 inhibitory effect, although with lower potency. In conclusion, the results suggest that, in native promoters of T3-regulated genes, a dual molecular mechanism, with DNA-binding dependent and DNA-binding independent components, underlies TR{alpha}2 dominant negative activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID hormone action is initiated by the binding of T3 to nuclear thyroid hormone receptors (TRs), which are members of a large superfamily of zing finger transcription factors that also includes receptors for steroids, retinoids, and vitamin D, as well as factors for whom the interacting ligand is still unknown (orphan receptors) (1, 2, 3). The {alpha} and ß c-erbA genes encode the functional TR{alpha}1 and TRß1, respectively. By alternative splicing of the c-erbA {alpha} gene, two TR{alpha} variants, TR{alpha}2 and TR{alpha}3 (4, 5), whose functions are still poorly characterized, are produced. TR{alpha}2 is identical to TR{alpha}1 in the first 370 amino acids but diverges at the carboxyl terminus, exhibiting 122 additional amino acids and interrupting the sequence of the ninth heptad (6, 7, 8), now identified as helix 11 in the crystallographic structure of TR ligand binding domain (9, 10). Because this sequence variation occurs in the ligand binding domain, TR{alpha}2 is unable to bind T3 and has been shown to exert dominant inhibitory action on T3/TRs-dependent transactivation of target genes (11, 12).

Expression of TRs occur in a developmentally specific fashion, particularly in the brain. In the rat, TR{alpha}1 is expressed very early during development, even before fetal thyroid hormones are available (13, 14, 15). TRß1 expression increases about 40-fold during the critical period of brain differentiation (16, 17), in parallel with the increase in both serum and cytosolic T3 levels. The importance of the rise of TRß1 concentration in brain is substantiated by the preferential binding to three T3-dependent genes coding for myelin basic protein (MBP) (18), Purkinje-cell protein 2 (19), and TRH (20). In the rat brain, temporal expression of TR{alpha}2 coincides with that of TR{alpha}1, but its levels are markedly higher (21), suggesting that TR{alpha}2 might be a critical regulator of thyroid hormone action by interfering with T3 effect on the expression of brain-specific genes.

When bound to DNA, TRs exhibit dual regulatory properties on genes harboring specific thyroid response elements (TREs), because of their ability to function either as transcriptional activators, in the presence of T3, or, in the absence of hormone, as repressors of basal transcription (22, 23). A diverse mode of action seems to be required for the nonhormone-binding TR{alpha}2 to inhibit, in a dominant negative fashion, transactivation mediated by ligand-activated TR{alpha}1 and TRß1 (11, 12, 24, 25, 26). TR{alpha}2, in fact, is transcriptionally inactive (11, 12) and does not significantly alter promoter basal activity (24).

The molecular mechanisms underlying the dominant negative activity of TR{alpha}2 are currently under intense investigation. Two different mechanisms have been proposed: the first, described by Katz et al. (27), involves a passive repression, in which TR{alpha}2 blocks TRs action by competing for binding to TREs; the second mechanism has been proposed by Liu et al. (28), who demonstrated that TR{alpha}2 inhibitory effect can occur in the absence of binding to TRE and suggested that protein-protein interactions (i.e. coactivators, general transcription factors (GTFs), etc.) might instead play a crucial role. Indeed, recent studies (29) supported the latter hypothesis by demonstrating that TR{alpha}2, as well as functional TRs, are capable to directly interact with components of the general transcription machinery.

In the present study we report the identification of functional regulatory elements required for TR{alpha}2 dominant negative action in the context of native promoters of two T3-regulated genes, the brain-specific MBP and the housekeeping malic enzyme (ME).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids
Rat TR{alpha}2 (rTR{alpha}2) expression plasmid for transient transfection assays was generated from rTR{alpha}2 complementary DNA (cDNA) (4) by replacing its 5' untranslated region with the Kozak sequence and by inserting the modified cDNA into the HindIII/KpnI sites of the expression vector pcDNA3 (Invitrogen, San Diego, CA). The rTR{alpha}2 expression plasmid for in vitro transcription/translation, encoding a protein containing a FLAG sequence (Eastman Kodak) at the C-terminus, was prepared by digestion of rTR{alpha}2 with EcoO109 and KpnI. The C-terminus was then extended using double-stranded oligonucleotides containing sequences from the EcoO109 site and the FLAG sequence followed by a stop codon. Construction of rTR{alpha}1 and rTRß1 expression plasmids used in transient transfection assays or in vitro transcription/translation and constructs MBPTRE-thymidine kinase (TK)-chloramphenicol acetyl transferase (CAT), MBP256-CAT, and ME315-CAT have been previously described (30, 31). All constructs were verified by DNA sequencing in both orientations using T7 Sequenase Version 2.0 DNA sequencing kit (Amersham Life Sciences, Arlington Heights, IL).

To make MBPTRE-ME315-CAT or ME315CT-CAT, the ME315/ClaI-CAT construct (32) was digested with ClaI, dephosphorylated, gel purified, and ME-TRE replaced by double-stranded oligonucleotides, flanked by ClaI cohesive ends, containing the MBP-TRE (5'-ATCGATAGAACAATGGGACCTCGGCTGAGGACACGGCGATCGAT) or the mutated ME-TRE (5'ATCGATGGCATCCAGGACGTTGGGGTTAGCTGAGGACA-TCGAT; mutation is italicized), respectively.

MBP256GC-CAT was prepared by PCR (GeneAmp; Perkin-Elmer, Norwalk, CT) using as template plasmid MBP256-CAT; forward primer (5'-TCTAGAAGGCCTCGTACAGGCC) [p1] contained a XbaI site and spanned sequences from 256 to 241 of the MBP promoter; reverse primer (5'-CGATCCGTTGTCCTCCCTTCCCGGGGCAT-3') corresponded to MBP sequences from -38 to -60 and was flanked by a BamHI site. The PCR product was digested with XbaI and BamHI, gel purified, and ligated with double-stranded oligonucleotides corresponding to ME promoter (-36/+3) and containing 5'BamHI and 3'XhoI cohesive ends into the XbaI/XhoI sites of the pBLCAT3 vector.

Construct ME315-MBPTATA-CAT, in which the ME GC-rich region spanning the transcription start site was replaced by the MBP TATA-like box, was created by PCR using ME352-CAT (31) and the following primers: forward primer (5'-TCTAGATGCATCAGGCCCCTGGGGCA) [p3], corresponded to ME sequence -315/-305 flanked by a XbaI site and reverse primer (5'-GGATCCGCCGGGGGCGGGCGTGGG) encompassed ME sequence -36/-53 flanked by a BamHI site. The PCR product was double digested with XbaI/BamHI, ligated with double-stranded oligonucleotides corresponding to MBP promoter from -38 to +3 flanked by BamHI/XhoI sites and containing the MBP TATA-like sequence, and finally subcloned into the XbaI/XhoI of pBLCAT3.

METRE-MBP256-CAT construct was obtained by a two-step PCR, using internal primers 5'-GGACGTTCGGGGTTAGGGGAGGACACGGCGGTGACA [p5] and 5'-CCCTAACCCCAACGTCCTCTGGATCGCATCTGCCT [p6]. The italicized sequence corresponds to ME-TRE, which replaced the MBP-TRE sequence, followed by MBP promoter sequences -175/-158 and -193/-210 for p5 (forward) and for p6 (reverse) primers, respectively. Forward external primer corresponded to the upper strand of the MBP promoter from -256 to -241, flanked by a 5'XbaI site [p1]; the reverse external primer corresponded to the lower strand of the MBP promoter sequence from +1 to -14, flanked on its 5' by a XhoI recognition sequence 5'-CTCGAGTGAAGCTCGTCGGAC [p2]. Two separate primary PCR products of MBP promoter were generated by pairing primers p1-p6 and p2-p5. In the secondary PCR, the two purified primary products were used as templates with external primers, p1 and p2. The full-length amplified MBP promoter, now containing the ME-TRE in place of the MBP-TRE, was then gel purified, double digested with XbaI/XhoI, and directionally subcloned into the XbaI/XhoI sites of pBLCAT3.

Construct MBPTRE-MBPTATA-ME315 containing the double swap of MBP-TRE and MBP TATA-like box into the ME promoter, was generated by using MBPTRE-ME315-CAT as a PCR template and the following primers: forward primer, flanked by a XbaI site, was identical to primer p3 and reverse primer encompassed ME promoter sequences from -142 to -120 (5'-AAGCGCTGAGTCACGGCGGCATC [p4]). The PCR product was digested with XbaI and Eco47III (present at position -126 in ME promoter), and the resulting fragment ligated into ME315-MBPTATA-CAT, previously digested with the same restriction endonucleases.

To create the ME315CT-MBPTATA-CAT construct, ME315CT-CAT was used as PCR template together with primers p3 and p4. The resulting PCR product was digested with XbaI and Eco47III and ligated into ME315-MBPTATA, previously digested with the same restriction enzymes.

Electrophoretic mobility shift assay
For electrophoretic mobility shift assays, 1 x 104 cpm purified, double-stranded oligonucleotides, 5' end-labeled with T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and [{gamma}-32P]ATP (Amersham) were incubated with in vitro transcribed/translated TRß1 or TR{alpha}2 (TNT coupled reticulocyte lysate system; Promega, Madison, WI) for 20 min at room temperature. In vitro binding reactions were performed in a final volume of 20 µl in the presence of 10 mM Tris-HCl (pH 7.5), 30 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 0.5 mM MgCl2, 10% (vol/vol) glycerol, and 1 µg poly(dI-dC) (Pharmacia BioTech, Uppsala, Sweden). Specific DNA-protein complexes were confirmed using M2 FLAG antibody (Eastman Kodak). Samples were loaded onto a 6% polyacrylamide gel and electrophoresed for 3 h at 150 V using 0.5x TBE (45 mM Tris borate, 45 mM boric acid, 2 mM EDTA) as running buffer. Oligonucleotides for the analysis of TRs binding were: MBP-TRE (5'-CAGAACAATGGGACCTCGGCTGAGGACACGGCG) and ME-TRE (5'-AGGACGTTGGGGTTAGGGGAGGACA). In MBP-TRE, mutations (italicized) were made by changing G to T and C to A; in ME-TRE, mutations (italicized) were introduced into the spacer region (GG to CT) to create a 3' half similar to that of MBP TRE.

Cell transfections
All transient transfection studies were performed in NIH3T3 cells, maintained in culture in the presence or absence of T3 (10-7 M) and transfected by electroporation with the indicated plasmid constructs, including a cytomegalovirus promoter-ß-galactosidase (gal) expression vector (0.25 µg/dish) to control transfection efficiency, as previously described (32). Data are represented as the mean ± SE of duplicate samples.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TR{alpha}2 inhibits T3/TRß1-dependent transcriptional activation of MBP but not ME native promoters.

To elucidate mechanisms underlying TR{alpha}2 dominant negative activity, we studied its inhibitory effect on native promoters of two T3-regulated genes: the brain-specific MBP and the housekeeping ME (Fig. 1Go). The inhibitory effect of TR{alpha}2 was evaluated in transient transfection assays. NIH3T3 cells, cultured in the presence or absence of T3, were cotransfected with native MBP and ME promoter-CAT reporters (MBP256-CAT and ME315-CAT, respectively) and TRß1, TR{alpha}1, and TR{alpha}2 expression plasmids. In the presence of a 3-fold excess of TR{alpha}2 over TRß1, approximately 75% reduction of T3/TRß1-mediated transactivation of MBP promoter was observed, a 50% decline of T3-mediated induction of CAT activity being already detected at a 1:1 TR{alpha}2/TRß1 ratio (Fig. 2Go). This effect was entirely due to inhibition of T3 induction of CAT activity, because no significant modification of MBP promoter basal activity was observed on addition of TR{alpha}2 (data not shown). TR{alpha}2 inhibition of T3/TR{alpha}1-mediated transactivation of MBP promoter was less pronounced (data not shown), in agreement with previous observations of Koenig et al. (11) and Rentoumis et al. (24) and with our previous results demonstrating preferential transactivation of MBP promoter by TRß1 (18). Contrary to MBP promoter, T3 stimulation of ME promoter in the presence of either TR{alpha}1 or TRß1 was unaffected by transfection of an excess (up to 5-fold) of TR{alpha}2 (Fig. 2Go and data not shown).



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  		Figure 1. Schematic representation of MBP and ME promoters. TRE orientation is depicted by horizontal arrows, MBP TATA-like sequence and ME GC-rich region are indicated. Position of BamHI and XhoI restriction sites, used to generate swap constructs, are indicated by vertical arrows. Numbers refer to nucleotide distance from major transcription start sites in MBP and ME promoters. Diagrams are drawn to scale.

 


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  		Figure 2. Dominant negative activity of TR{alpha}2 on MBP and ME promoters. NIH3T3 cells were cotransfected by electroporation with TRß1 expression vector (5 µg), MBP256-CAT, or ME315-CAT reporter plasmids (5 µg each), cytomegalovirus-ß-gal (250 ng), to control for transfection efficiency, and either TR{alpha}2 expression plasmid or empty vector (5–15 µg). Cells were incubated for 72 h, with or without T3 (10-7 M), harvested, and assayed for CAT and ß-gal activities. Fold T3 induction is calculated as ratio of normalized CAT activity in presence or absence of hormone. Results represent average (± SE) of eight independent experiments, each performed in duplicate.

 
These results indicate that transcriptional activation of the MBP promoter by T3/TRß1 is highly sensitive to inhibition by TR{alpha}2, suggesting the presence, in the context of MBP promoter, of unique element(s) required for TR{alpha}2-mediated repression.

TRE exchange: MBP-TRE is necessary for TR{alpha}2 inhibition of T3/TRß1 dependent transcriptional activation
One of the main differences between MBP and ME gene promoters is the structure of their TREs, which are arranged as an imperfect inverted palindrome and an imperfect direct repeat, respectively (Fig. 1Go). Because previous studies (27, 33), using a variety of TREs inserted in the context of heterologous promoters, suggested that competition for TRE binding is critical in eliciting TR{alpha}2 inhibitory action, we tested whether the structure of MBP-TRE could play a key role in mediating TR{alpha}2 inhibition of T3-dependent transcriptional activation. For this purpose, we generated chimeric reporter constructs with exchange of TREs between the MBP and ME promoters (Fig. 3Go, C and D). Levels of induction for the various chimeric constructs, following T3 stimulation, is reported in Table 1Go. Substitution of ME-TRE with MBP-TRE, in the context of ME promoter (MBPTRE-ME315-CAT), did not result in any inhibition of T3-TRß1-dependent CAT activity by TR{alpha}2. Replacement of the MBP-TRE with ME-TRE in the context of MBP promoter (METRE-MBP256-CAT) resulted in a lower T3 inducibility in comparison to the wild-type promoter (Table 1Go), in agreement with previous results demonstrating a preferential T3-TRß1 transactivation of MBP-TRE. However, this TRE exchange abolished TR{alpha}2 dominant negative effect (Fig. 4Go).



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  		Figure 3. Schematic representation of MBP (cross-hatched boxes) and ME (solid boxes) native and chimeric promoter constructs. TREs, arranged as inverted palindrome or direct repeat, as well as TATA-like sequence and GC-rich region of MBP and ME, respectively, are indicated by vertical arrows. Single (C, D, E, and F) and double (G) swap chimeras as well as constructs with point mutations in ME-TRE (H and I) are depicted. See Materials and Methods for details of generating chimeric constructs.

 

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  		Table 1. T3/TRß1 induction of wild-type and chimeric reporter plasmids

 


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  		Figure 4. TRE exchange. Native MBP256-CAT and chimeric MBPTRE-ME315-CAT, METRE-MBP256-CAT, and ME315CT-CAT reporters (5 µg each) were cotransfected with TRß1 and TR{alpha}2 (1:2 ratio) into NIH3T3 cells as described in Fig. 2Go. Data are expressed as percentage of repression of T3 induction in absence of TR{alpha}2. Results represent average (± SE) of six independent experiments, each performed in duplicate.

 
These results indicate that the inverted palindromic MBP-TRE is necessary but not sufficient to mediate TR{alpha}2 inhibition of T3-TRß1-dependent activation.

TR{alpha}2 binds to MBP-TRE but not to ME-TRE
Katz et al. (27) proposed that TR{alpha}2 inhibitory effect is the result of direct competition with functional TRs for binding to TRE. To ascertain whether TR{alpha}2 is able to bind MBP- and ME-TRE, gel mobility shift assays (GMSAs) were performed using 32P-labeled TREs and TR{alpha}2 and TRß1 generated by in vitro transcription/translation. As shown in Fig. 5AGo, TR{alpha}2 efficiently binds to wild-type MBP-TRE, presumably as a monomer at the 3' half site, because mutation of the two cytidine residues to adenine in the 5' half site of MBP-TRE did not affect TR{alpha}2 interaction (lane 3), whereas mutation of the two guanine residues to thymidine in the 3' half site abolished TR{alpha}2 interaction (lane 2). Our previous results (18) and data shown in Fig. 5AGo (lane 5) indicate that integrity of both half sites is necessary for TRs interaction, because mutations of either the 5' (lane 6) or 3' (lane 7) half sites of MBP-TRE abolished TRß1 DNA binding. On the contrary (Fig. 5BGo, lane 2), TR{alpha}2 failed to interact with wild-type ME-TRE, in agreement with previous reports (28).



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  		Figure 5. DNA binding of TR{alpha}2, TRß1, and TR{alpha}2 FLAG to wild-type and mutants MBP-TRE and ME-TRE. Indicated in vitro translated TRs were incubated with following oligonucleotide-labeled probes, and protein-DNA complexes resolved by EMSA as described in Materials and Methods. A, Radiolabeled wild-type (lanes 1, 5, 8, and 9) and mutant (lanes 2 and 6 = 3' half-site mutant; lanes 3 and 7 = 5' half-site mutant; lane 4 = 3' and 5' half-site mutant) MBP-TREs; lanes 8 and 9 contain mock-translated reticulocyte lysate. B, Wild-type (lanes 1 and 2) and mutant (GG to CT mutation, lanes 3 and 4) ME-TREs; TR{alpha}2 FLAG specifically binds mutant ME-TRE (lane 3, arrowhead), whereas addition of anti-FLAG antibody generates supershifted band (lane 4, arrowhead). C, Comparison of wild-type MBP-TRE and ME-TRE. Identical 3' half sites of both TREs are underlined, and two GG residues mutated to CT in ME-TRE spacer region are evidenced (in boldface).

 
These results indicate that sequences in the 3' half site of MBP-TRE are involved in TR{alpha}2/TRE interaction.

Binding of TR{alpha}2 to a mutated ME-TRE is not sufficient to elicit TR{alpha}2 dominant negative action
Because in the GMSA TR{alpha}2 failed to interact with wild-type ME-TRE, specific mutations were introduced in the attempt to elicit TR{alpha}2 binding without affecting T3-dependent transactivation. Because the 3' half site of both MBP and ME TREs is identical (Fig. 5CGo), mutations were introduced into ME-TRE to target the dissimilar spacer region. A double mutation (G->C, G->T), which rendered the spacer region of ME homologous to that of MBP, resulted in specific binding of TR{alpha}2, tagged with the FLAG, in a GMSA (Fig. 5BGo, lane 3, arrow), which was supershifted following the addition of an anti-FLAG antibody (Fig. 5BGo, lane 4, arrow). Although preserving T3 responsiveness (Table 1Go), insertion of this mutation into ME promoter (ME315CT-CAT) did not confer inhibition of T3/TRß1-dependent transactivation in the presence of a 2-fold TR{alpha}2 excess (Fig. 4Go). Therefore, acquisition of DNA binding did not cause TR{alpha}2 to elicit dominant negative activity.

These results suggest that, in addition to competition for binding to TRE, the dominant negative effect of TR{alpha}2 on T3-regulated promoters may occur through additional mechanisms involving other regulatory elements.

Exchange of the transcription initiation site: a TATA-like sequence is relevant for TR{alpha}2 dominant negative action
All thyroid hormone receptor isoforms, including TR{alpha}2 have been shown to directly interact with components of the general transcription machinery (22, 29, 34, 35). A possible mechanism for TR{alpha}2 dominant negative activity might therefore result from competition with liganded TRß1 for binding to GTFs. To address this point, we analyzed the role of MBP and ME promoter sequences near the transcriptional start site (Fig. 1Go), where the two genes differs substantially, the MBP featuring a TATA-like box, and the ME resembling a TATA-less promoter with the presence of a GC-rich region spanning the major transcription start site. To swap the GC-rich sequence of ME promoter with the MBP TATA-like box, a BamHI site was appropriately inserted into the MBP promoter creating the ME315-MBPTATA-CAT and MBP256-GC-CAT chimeric constructs (Fig. 3Go, E and F). Replacement of the MBP TATA-like box with the corresponding GC-rich region of ME promoter, although reducing the overall T3 responsiveness (Table 1Go), did not cause any further inhibition of T3/TRß1 transactivation following TR{alpha}2 addition (Fig. 6Go). However, TR{alpha}2 did not cause any significant inhibition of the T3/TRß1-induced CAT activity after substituting the GC-rich region of ME promoter with the MBP TATA-like box (ME315- MBPTATA-CAT) (Fig. 6Go).



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  		Figure 6. Exchange of initiation site. Native MBP256-CAT and chimeric constructs ME315-MBPTATA-CAT and MBP256-GC-CAT were transfected using same conditions described in Fig. 4Go. Results represent average (± SE) of eight independent experiments, each performed in duplicate.

 
These results indicate that specific features of the transcription initiation complex binding site are required, although not sufficient per se, to evoke TR{alpha}2 inhibitory action.

Combination of an extended MBP 3' half site and a TATA-like box is required for TR{alpha}2 dominant negative action
The above results suggest that the exchange of a single regulatory element, either the TRE or the region spanning the transcription initiation site, is not sufficient in eliciting TR{alpha}2-mediated repression. Therefore, we simultaneously exchanged ME-TRE and ME GC-rich region with MBP-TRE and MBP-TATA-like box in the context of ME promoter, respectively, thus generating the chimeric MPBTRE-ME315-MBPTATA-CAT construct (Fig. 3GGo). Interestingly, this construct exhibited a T3 responsiveness similar to that of native MBP promoter (Table 1Go). The double swap allowed TR{alpha}2 to elicit its inhibitory action on T3-TRß1 induction of ME promoter (Fig. 7Go). This effect was not due to inhibition of the basal activity of the double swap construct in the absence of T3 (data not shown). Acquisition of binding and dominant negative activity by TR{alpha}2 on ME promoter was also obtained with the chimeric construct ME315CT-MBPTATA-CAT (Fig. 3HGo), characterized by the presence of a ME-TRE, mutated in the spacer region to mimic the 3' half of MBP-TRE, and the MBP TATA-like box (Fig. 5CGo).



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  		Figure 7. Swapping experiment. Native MBP256-CAT and double swap MBPTRE-ME315-MBPTATA-CAT and ME315CT-MBPTATA-CAT constructs were cotransfected with TRß1 and TR{alpha}2 (1:3 ratio) into NIH3T3 cells as described in Fig. 2Go. Data represent average (± SE) of eight independent experiments, each performed in duplicate.

 
These results indicate that the simultaneous presence of an extended TRE and a TATA-like box is a necessary requirement for TR{alpha}2 to exert its dominant negative activity on native promoters of T3-regulated genes.

To further investigate the role of TATA-like sequences as crucial determinants for TR{alpha}2 dominant negative effect, we performed cotransfection assays using the MBP-TRE inserted upstream to the herpes virus thymidine kinase promoter (MBP-TRE-TK-CAT), known to contain a canonical TATA-box element. Increasing concentrations of TR{alpha}2 were able to repress T3/TRß1-mediated transactivation of MBP-TRE-TK-CAT, with approximately 75% and 90% inhibition at a 1:1 and 1:3 TRß1/TR{alpha}2 ratio, respectively (data not shown). These findings support the hypothesis that the presence of a TATA-box, as in TK gene, or a TATA-like element, as in MBP gene, are important requirements for TR{alpha}2 inhibitory effect and suggests that other factor(s), presumably GTFs, may be involved in evoking TR{alpha}2 dominant negative action.

Binding to DNA is an auxiliary mechanism for TR{alpha}2 dominant negative activity
Katz et al. (27) proposed that binding to TRE is the primary mechanism underlying TR{alpha}2 inhibitory effect. In contrast, Liu et al. (28) showed that TR{alpha}2 can elicit trascriptional inhibition in the absence of DNA binding, Because mutation of P-box of the first zinc finger did not affect TR{alpha}2 dominant negative activity. Our data indicate that TR{alpha}2 interaction with TRE is an important requirement for its dominant negative activity. To assess the relevance of TR{alpha}2 binding to DNA in eliciting its dominant negative action, we evaluated the effect of mutation (C73S) of TR{alpha}2 P-box (TR{alpha}2Pmut) on T3/TRß1-mediated transactivation of MBP256-CAT. GMSA, using 32P-labeled MBP-TRE and in vitro transcribed/translated TR{alpha}2Pmut, showed that mutation of the TR{alpha}2 P-box completely disrupted its ability to bind MBP-TRE (Fig. 8Go). However, despite inability to interact with DNA, TR{alpha}2Pmut retained its inhibitory action, although at lower potency (Fig. 9Go). In fact, transfection of equal amounts of TRß1 and wild-type TR{alpha}2 expression plasmids resulted in inhibition of T3/TRß1 transactivation of MBP256-CAT by approximately 50%. In contrast, TR{alpha}2Pmut failed to inhibit T3-stimulated CAT activity at similar TRß1/TR{alpha}2Pmut ratio. However, transfection of increasing concentrations of TR{alpha}2Pmut over TRß1, produced a progressive regain of the inhibition of T3/TRß1 mediated transactivation (Fig. 9Go).



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  		Figure 8. DNA binding of wild-type and P-box mutant TR{alpha}2-(TR{alpha}2Pmut). In vitro translated TRß1 (lane 2), TR{alpha}2 (lane 3), or TR{alpha}2Pmut, containing a mutated (C73S) P-box (lane 5), were incubated in presence of radiolabeled MBP-TRE oligonucleotide, and protein-DNA complexes resolved by EMSA. Lanes 1 and 4 contained mock-translated reticulocyte lysate.

 


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  		Figure 9. Dominant negative activity of TR{alpha}2 vs. TR{alpha}2Pmut. NIH3T3 cells were cotransfected with MBP256-CAT (5 µg), TRß1 expression vector (5 µg), and increasing concentrations of TR{alpha}2 (open bars) or TR{alpha}2Pmut (solid bars) expression vectors up to 1:3 ratio, as indicated. Results are expressed as percentage of uninhibited TRß1 activity and represent average of two independent experiments, each performed in duplicate.

 
These results suggest that requirement for DNA binding in mediating TR{alpha}2 transcriptional inhibition, although important, may be overcome at higher TR{alpha}2 concentrations.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The inhibitory effect of TR{alpha}2 on T3/TRs transcriptional induction has been mainly investigated on artificial or naturally occurring TREs, usually inserted in the context of heterologous promoters, and at high TR{alpha}2/TRs molar ratios (24, 27, 28). In this study, we attempted to define the structural requirements for TR{alpha}2 inhibition of T3-induced transcription using native MBP and ME promoters. The results showed that MBP probably represents one of the most sensitive genes to the dominant negative action of TR{alpha}2, because 50% repression of T3/TRß1 transactivation may be already observed after transfection of TRß1 and TR{alpha}2 at a 1:1 molar ratio (Fig. 2Go). This finding is noteworthy because the highest levels of TR{alpha}2 expression have been typically found in brain (21), and a strong rise of TRß1 intracellular levels has been reported to occur postnatally (16), at time when active brain differentiation and myelinogenesis occur. Contrary to MBP, T3-dependent stimulation of ME promoter is unaffected by TR{alpha}2, even in the presence of a 3-fold molar excess (Fig. 2Go).

We therefore focused the attention on two distinct features of MBP and ME promoters; first, the TRE, arranged as an imperfect inverted palindrome in MBP and as an imperfect direct repeat in ME promoter, and second, the region spanning the transcription start site, which in MBP promoter is characterized by the presence of a TATA-like element, whereas in ME lacks a TATA-box and shows a high GC content.

Because previous studies suggested that competition for DNA binding by TR{alpha}2 vs. functional TRs is critical for its dominant negative activity (27, 36, 37), we analyzed whether the intrinsic structure of the MBP-TRE could play a key role in determining TR{alpha}2 inhibition of T3/TRß1 transcriptional induction. Indeed, in electrophoretic mobility shift assays, specific binding of TR{alpha}2 to MBP-TRE, but not to ME-TRE, supported this hypothesis (Fig. 5Go, A and B). Although replacement of MBP-TRE with ME-TRE abolished TR{alpha}2 dominant negative action on MBP promoter, substitution of ME-TRE with MBP-TRE, in the context of ME promoter, did not result in TR{alpha}2 inhibition of T3-TRß1-dependent transactivation (Fig. 3Go). TRE exchange experiments, therefore, indicate that binding to TRE is necessary but not sufficient per se to allow TR{alpha}2 inhibitory action.

Because the 3' half sites of MBP and ME TREs exhibit a similar nucleotide motif (Fig. 5CGo), point mutations were introduced into ME-TRE to confer TR{alpha}2 binding without affecting T3-dependent activation. In particular, a GG to CT mutation of ME-TRE spacer region (ME315CT-CAT construct, Fig. 3Go) elicited TR{alpha}2 binding without modifying T3 responsiveness (Table 1Go). This mutation rendered the second half of ME-TRE identical to that of MBP-TRE, while creating a new octamer sequence (TGAGGACA), which has been shown to possess higher affinity for TR{alpha}2 binding (37). The absence of direct correlation between TR{alpha}2 DNA binding and dominant negative activity was not unexpected because an alternative, DNA binding-independent mechanism has been proposed by Liu et al. (28) to explain TR{alpha}2 dominant negative properties.

We also observed that the presence of a TATA-like sequence is a necessary requirement for TR{alpha}2 to elicit its inhibitory function on the native MBP promoter, Because replacement of this sequence with the corresponding ME GC-rich region abrogated TR{alpha}2 dominant negative effect. The importance of the TATA-like box is also supported by TR{alpha}2 inhibition of T3/TRß1 transactivation in transient transfection experiments using MBP-TRE inserted into the heterologous TK promoter, which contains a canonical TATA-box (data not shown). However, replacement of the GC-rich element of ME promoter with the MBP TATA-like box did not confer TR{alpha}2 dominant negative activity, again indicating that this element is not solely responsible for TR{alpha}2 repression.

To ascertain whether concurrence of TRE and transcription initiation region of the MBP gene is a major determinant for TR{alpha}2 dominant negative activity, we replaced both TRE and GC-rich region of ME promoter with TRE and TATA-like element of MBP promoter (MBPTRE-ME315-MBPTATA-CAT). Interestingly, the double swap construct triggered TR{alpha}2 inhibition of T3/TRß1 transactivation of ME promoter, about 70% repression being already observed at a 1:2 ratio of TRß1 vs. TR{alpha}2. These findings suggest that the simultaneous presence of both regulatory elements accomplishes an ad hoc situation for TR{alpha}2 dominant negative activity.

To further assess the importance of DNA binding in eliciting TR{alpha}2 dominant negative activity, we introduced a C73S substitution in the DNA binding domain (TR{alpha}2Pmut), a modification that has been previously shown to completely disrupt DNA binding (28). Although approximately 50% repression was already observed after transfection of equal amounts of wild-type TR{alpha}2 and TRß1, a weaker dominant negative activity was observed with TR{alpha}2Pmut, a 3-fold excess being required to achieve similar levels of inhibition (Fig. 9Go). Although the possibility that different intracellular levels of the two proteins may account for this effect, it is reasonable to propose that, under low TR{alpha}2 concentrations, inhibition of T3/TRß1 transactivation is enhanced when TR{alpha}2 can interact with TRE, whereas, in the absence of DNA binding, TR{alpha}2 repression potency is weakened, and higher TR{alpha}2 concentrations are required. In keeping with these results is the dominant negative effect observed, only at high TR{alpha}2/TRs ratios, with a construct containing ME-TRE in the context of the heterologous TK promoter (27, 28). Therefore, it can be assumed that failure to observe inhibition when the MBP TATA-like box was inserted into the ME promoter (ME315-MBPTATA-CAT; Fig. 6Go) was probably due to the low TR{alpha}2 concentrations used (up to 1:5 ratio of TRß1/TR{alpha}2; Fig 6Go and data not shown). Our observations are in agreement with results obtained by Liu et al. (28) but conflict with that of Yang et al. (36), who recently reported concurrent abrogation of TR{alpha}2 DNA binding and dominant negative activity following insertion of a different mutation (C56A) into the first zinc finger of TR{alpha}2 DNA binding domain. The apparent discrepancy between our results and those of Yang et al. may be explained by disruption, to a different extent, of a still unknown protein-protein interaction that may contribute to determine TR{alpha}2 dominant negative activity. These observations support the hypothesis that TR{alpha}2 is an active transcriptional repressor, distinct from a passive repressor that simply compete for DNA binding (38, 39), exhibiting a bipartite mechanism of action with separate DNA binding-dependent and -independent components.

A possible explanation of TR{alpha}2 inhibitory function may involve the interaction with the nuclear receptor preferred heterodimerization partner retinoid X receptors (RXR). However, under our experimental conditions, TR{alpha}2 was not able to heterodimerize with RXR (data not shown) in agreement with report from Yang et al. (36), rather it bound MBP-TRE or mutated ME-TRE as a monomer (Fig. 5Go, A and B). Cotransfection of increasing concentrations of RXR, in fact, was not able to relieve TR{alpha}2 inhibition of T3/TRß1 transactivation, further indicating that squelching by use of RXR for heterodimerization with functional TRs is not responsible for the DNA binding-independent component of TR{alpha}2 dominant negative activity (data not shown and Ref.28).

The requirement of TATA-like sequences for TR{alpha}2 dominant negative action may also suggest the possibility of a competition between TR{alpha}2 and functional TRs for binding to GTFs. Indeed, several studies have reported that all TR isoforms, including TR{alpha}2, may directly interact with components of the basal transcription complex (34, 35, 22, 29). The possible consequence of TR{alpha}2 interaction with GTFs could be a direct inhibition of the assembly of a functional transcription preinitiation complex at the target promoter, a mechanism similar to that recently proposed to explain repression of basal transcription by unliganded TRs. (40, 41).

In conclusion, our results provide evidence in favor of a dual molecular mechanism underlying TR{alpha}2 dominant negative activity in the context of native promoters of T3-regulated genes; the first mechanism is operative under low TRa2 concentrations and takes advantage from direct binding to TRE, the second appears independent from binding to DNA and may involve the interaction with unknown factors, present in limiting amounts and crucial for T3/TRs-dependent transactivation. The identification of these factors would be of great importance in clarifying TRa2 dominant negative activity, and in better understanding the molecular mechanisms that underlay regulation of gene expression by thyroid hormones.


   Footnotes
 
1 These authors contributed equally to this study. Back

Received May 27, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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