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Departments of Medicine (S.Y.W., R.N.C., H.G., J.N., S.R., R.E.W.) and Pediatrics (S.R.), University of Chicago, Chicago Illinois 60645; and Department of Pediatrics (E.S., D.A.S., N.E.Y.), Abant Izzet Baysal University, Duzce Medical School, 14450 Duzce, Turkey
Address all correspondence and requests for reprints to: Roy E. Weiss, M.D., Ph.D., Thyroid Study Unit, Department of Medicine, University of Chicago, 5841 South Maryland Avenue, Mail Code 3090, Chicago, Illinois 60645. E-mail: rweiss{at}medicine.bsd.uchicago.edu.
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Abstract |
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Objective: We report a newborn who presented with severe RTH (Mkar) with serum TSH 1500 mU/liter and free T3 greater than 50 pM (normal 3.1–9.4) and free T4 25.3 pM (normal 12–22). We hypothesized that the RTH was due to reduced ligand binding and/or abnormal interaction with nuclear cofactors.
Design: These were prospective in vivo and in vitro studies.
Setting: The study was conducted at a tertiary care university hospital.
Patients: Patients included a newborn child and two other subjects with RTH.
Intervention: The effect of various TH-lowering agents in the subject with RTH was studied. In vitro studies including EMSA and mammalian two-hybrid assay as well as in vitro transfection studies were conducted.
Main Outcome Measures: Sequencing of the TH receptor (TR)ß and in vitro measurements of receptor-cofactor interaction were measured.
Results: Sequencing of the TRß demonstrated a de novo heterozygous mutation, 1590_1591insT, resulting in a frameshift producing a mutant TRß (mutTR)-ß with a 28-amino acid (aa) nonsense sequence and 2-amino acid carboxyl-terminal extension. The Mkar mutation was evaluated in comparison to three other TRß frameshift mutations in the carboxyl terminus. EMSA demonstrated that the Mkar mutTRß1 had impaired ability to recruit nuclear receptor corepressor but intact association with silencing mediator of retinoid and thyroid receptor (SMRT).
Conclusion: Our data suggest that alterations in codons 436–453 in helix 11 result in significantly diminished association with nuclear receptor corepressor but not SMRT. This novel mutTRß demonstrates nuclear corepressor specificity that results in severe predominantly pituitary RTH due to impaired release of SMRT.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The mechanism of RTH in subjects heterozygous for a mutant TRß (mutTR) is interference with the function of the wild-type TRß, known as a dominant-negative effect (DNE). One mechanism of DNE is defective interaction of the mutTR with a cofactor (2). The severity of resistance depends on the degree of impaired ligand binding and interaction with tissue-specific nuclear cofactors (3).
Most of mutTRßs are able to bind corepressor in the absence of ligand but cannot release corepressor in response to ligand (4, 5, 6, 7, 8, 9, 10, 11). The two main corepressors are nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT). Lack of hormone binding results in impairment of corepressor release and decreased coactivator recruitment. NCoR and SMRT are ubiquitously expressed in vertebrate tissues but interact differently with nuclear receptors. TRß preferentially recruits NCoR instead of SMRT via the N-terminal interaction domains (N3 and N2) (12, 13). SMRT, on the other hand, prefers to bind to the retinoic acid receptor isoforms mediated by S2, the most proximal interaction domain.
We describe a newborn with severe RTH (Mkar) who had a heterozygous mutation in the TRß gene, 1590_1591insT, resulting in a frameshift that produces a nonsense amino acid (aa) sequence 436–463 and a carboxyl terminal extension of TRß1 by 2 aa. In this study, we evaluated three natural and one artificial frameshift mutations in helix 11 and 12 of TRß to delineate the role of this C-terminal region in cofactor interactions. We used EMSAs and mammalian two-hybrid assays to evaluate the ability of TRßs with mutations in helix 11 and 12 to bind to corepressors NCoR and SMRT and coactivator, steroid receptor coactivator (SRC)-1. The functional properties of transactivation, repression, and DNE were evaluated in transient transfection studies. These studies demonstrated that mutations resulting in alterations in the 436- to 453-aa sequence in the C terminus of TRß result in significantly impaired association with NCoR but not SMRT. This results in severe but predominantly pituitary RTH.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Mkar is a Turkish male born at 38 wk to healthy nonconsanguinous parents. At 6 d of age, he had respiratory distress, tachycardia (190 beats/min), diaphoresis, and a gland four times the normal size. Serum TSH was 220 mU/liter (normal range < 20) with free T3 of 50 pmol/liter (3.7–8.6), and free T4 (FT4) of 25.3 pmol/liter (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22), consistent with severe RTH. TRH stimulation test demonstrated an increase in TSH from 405 to 1716 mU/liter at 30 min. Magnetic resonance imaging showed a normal pituitary gland. Echocardiogram was normal and knee x-ray showed an appropriate bone age. Neither parent has a history of thyroid disease and both have normal thyroid function.
Treatment with propylthiouracil decreased the FT4 to 2.45 pmol/liter, resulting in a marked rise in TSH to 1172 mU/liter. (Fig. 1
). L-T3 was then started in an attempt to lower the TSH but had little effect and was stopped after 8 wk. Two weeks treatment with octreotide did not suppress the serum TSH. 3,5,3'-Triiodothyroacetic acid (TRIAC) at escalating doses up to 1.4 mg/d resulted in a decrease in heart rate and diaphoresis and marked decrease in TSH to 2.36 mU/liter (Fig. 1).
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Mdbs was a newborn male initially misdiagnosed as having hyperthyroidism who after a subtotal thyroidectomy had increased TSH of 77 mU/liter, normal total T4, and elevated total T3 of 300 ng/dl. TRH test demonstrated robust response of TSH (60 min maximum 1500 mU/liter). T3 failed to suppress the TRH-stimulated TSH until 200 µg T3 per day was administered for 3 sequential days.
Other human subjects
PV is a previously described male with RTH who presented at age 7 yr with a TSH of 4.2 mU/liter (0.5–4), total T3 of 369 ng/dl (88–162) and FT4 of 4.6 ng/dl (1–1.9) (14). The PV mutation, 1627_1628incC, produces a frame shift resulting in a 16-aa nonsense sequence after the insertion point and extension of the length of the TRß1 protein by 2 aa.
TRß sequencing
DNA was extracted from circulating white blood cells of Mkar and his parents and from skin fibroblasts of Mdbs. Studies carried out in humans were approved by the institutional review boards, and all individuals or parents of minors gave informed consent. PCR was performed on 100 ng of DNA from Mkar to amplify the coding exons 3–10 of the TRß1 gene and exon 1 of TRß2. The products of amplification were sequenced directly using automated fluorescence-based sequencing. Exons 8, 9, and 10 of TRß1 of Mkar’s parents and Mdbs were also amplified and sequenced. The set of primers used to amplify and sequence all coding exons of TRß1 and -ß2 have been previously described (15, 16).
Fibroblast studies
Human skin was obtained by punch biopsy from Mkar after informed consent. This was approved by the institutional review boards. Fibroblasts were grown in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% bovine calf serum (BCS) as previously described in detail (17). Isolation of RNA and preparation of cDNA have been previously described (18). PCR amplification of a 460-bp segment containing the T insertion at codon 436 as well as the wild-type (WT) sequence was carried out using Mkar cDNA and a control cDNA from fibroblasts of a normal subject. The primers used for this PCR were 5'-ATG GAG ATC ATG TCC CTT CGC-3' (forward) and 5'-TCT AAT CCT CGA ACA CTT CCA AGA A-3' (reverse). To generate cDNA, the PCR used 150 ng RNA as the template and 10 pmol each of the primers. The first denaturation was set at 94 C for 3 min, followed by 35 cycles of 1-min denaturation at 94 C, 1 min of annealing at 60 C, and 1 min of extension at 72 C. The final extension was for 5 min at 72 C.
Allele-specific digestion of the PCR products used the restriction enzyme BsrD1 (New England Biolabs, Ipswich, MA). The digested products were run on a 1.5% agarose gel. Mkar BsrD1 digestion produces two fragments of 380 and 80 bp each in the presence of the Mkar mutation.
Construction of plasmids
TRß constructs. The wild-type (WT) TRß1 expression vector and the manner of its generation was previously described (3). This and other expression plasmids were cloned in pcDNAI/amp (Invitrogen).
Plasmids expressing the two mutant TRß1s (Mkar and Mdbs) were constructed by exchange of PCR-amplified DNA fragments containing the mutations with the corresponding WT TRß insert in the pcDNAI/amp plasmid. This was done using the PGEM-T-Easy vectors (Promega Corp., Madison, WI).
Plasmids expressing the other two mutant TRß1s (AM and PV) were constructed by site-directed mutagenesis (Invitrogen GeneTailor site-directed mutagenesis system). The artificial mutant (AM) that contains only the upstream nonsense sequence of 11 aa of Mkar was constructed as follows. A "T" was inserted between nucleotide 1590 and 1591 to duplicate the mutation of Mkar, and the resulting frameshift was repaired by deletion of the C at nucleotide 1626, corresponding to the PV mutation site. The PV mutant was created by inserting a "C" between nucleotide 1627 and 1628 in codon 448, creating a frameshift resulting in a 16-aa nonsense carboxyl terminal sequence.
All constructs were checked for correct DNA insertion and presence of mutation by sequencing.
The preparation of Gal4-TR, VP16-NCoR, and VP-16-SMRT has been previously described (19). Mutant Gal4-TR constructs, Gal4-Mkar and Gal4-PV, were constructed using site-directed mutagenesis (QuikChange site-directed mutagenesis kit, Stratagene, La Jolla, CA) on the Gal4-TR plasmid.
Reporter constructs
Four firefly luciferase (Luc) reporter constructs were used, two positively (Pal x3Luc and F2) and two negatively regulated (TH 
SU-Luc and UAS Tk-Luc) prepared as previously described (19).
EMSAs
EMSAs were carried out as previously described with a 32P-radiolabeled DR+4 probe (20). Glutathione-S-transferase (GST)-fusion proteins GST-SMRT and GST-SRC-1 were prepared as previously described (13). Nuclear receptors [TRß1 WT, Mkar, Mdbs, AM, and PV as well as retinoid X receptor RXR)-] and NCoR were in vitro translated (IVT) in reticulocyte lysate (Promega) using T7 polymerase. An equivalent GST NCoR construct containing all three interacting domains did not generate usable amounts of protein. Therefore, IVT NCoR containing all three interacting domains was used in these experiments. EMSAs used 4 µl of the TRß1 constructs, 4 µl of IVT NCoR, and 2 µl of IVT RXR. Incubations were carried out for 20 min, complexes were resolved on a 5% nondenaturing gel, and films were developed after 12–24 h exposure.
Mammalian two-hybrid assays
Mammalian two-hybrid transient transfections were performed in 293T cells maintained in DMEM supplemented with 10% BCS at 37 C 5% CO2 with lipofectamine (Life Technologies, Inc., Gaithersburg, MD) such that each well contained equal amounts of plasmid DNA. One hundred nanograms each of reporter UAS-TK Luc, the appropriate construct (Gal4-TR, Gal4-Mkar, Gal4-PV, or empty Gal4), and the corepressor (VP16-NCoR, VP16-SMRT, or empty VP16) were added to each well. Three hours after transfection, cells were washed with PBS and exposed to DMEM containing 10% steroid hormone-depleted BCS. Then 21–27 h after transfection, cells were lysed and assayed for luciferase activity. Experiments were performed in triplicate. Data are expressed as fold stimulation ± SEM.
Transient transfection studies
CV-1 cells that lack endogenous TRs were used to evaluate ligand-independent activity and repression. For all other transfections, the human hepatoblastoma cell line (HepG2) was maintained in DMEM supplemented with 10% BCS at 37 C 5% CO2. Before transfection, cells were transferred to 12-well plates in the complete medium. Transfection was performed with TransFast transfection reagent (Promega) at a 1.3:1 charge ratio of TransFast reagent to DNA. The DNA and TransFast were mixed for 10–15 min before addition to cells. After 24 h the medium was changed to one containing 5% of TH-depleted BCS without and with added T3, and cells were incubated for an additional 48 h before harvesting. Firefly luciferase and Renilla-TK luciferase activities were determined sequentially (Promega dual-luciferase reporter assay system). TH-depleted BCS was prepared as previously described (3).
Reporter plasmids regulated positively by TH, PALx3-Luc, and F2x3-Luc and negatively by TH and SU-Luc were transfected into the cells at concentrations of 1 µg/well. Empty pcDNA vectors, WT TRß1, and mutTRß1 constructs were transfected at 25–50 ng/well. To test for a DNE, equal amounts of WT TRß1 and mutTRß1s at 25 ng each per well were cotransfected. An additional transfection using the PALx3-Luc reporter and varying ratios of mutTRß1 to WT TRß1 constructs, from 1:1 (25:25 ng) to 0.0625:1 (1.56:25 ng), was performed.
Experiments were performed in triplicate and expressed as fold induction ± SEM.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Sequencing all coding exons of the TRß1 and TRß2 genes of Mkar revealed the subject to be heterozygous for a novel single nucleotide insertion (1590_1591insT), resulting in the extension of the length of the protein by 2 aa with 28 nonsense aa after the insertion point (Fig. 2). This is a de novo mutation because both parents have a normal thyroid phenotype and absence of the TRß gene mutation identified in the propositus. The mutation results in a new restriction enzyme site for BsrD1. Digestion of the amplified PCR product of exon 10 of TRß confirmed the presence of the mutation in Mkar and absence of the mutation in his parents (data not shown). Both WT and mutant alleles were detected in skin fibroblasts of MKar.
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). Homodimerization and heterodimerization were intact in mutTRs
To determine whether the mutant TRs retained the ability to bind DNA, an EMSA analysis was performed with a 32P-radiolabeled DR+4 probe (Fig. 3A). WT TRß1 and all mutTRß1s demonstrated intact homodimerization. Addition of 100 nM of T3 dissociated WT TRß1 homodimer but had no significant effect on mutTRß1 homodimers due to lack of T3 binding. All mutTRß1s also retained the ability to bind RXR (Fig. 3A).
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When the receptors were studied with IVT NCoR in EMSAs, the WT TRß1 bound to NCoR in the absence of T3 and released NCoR when 100 nM of T3 were added. The Mdbs mutant interacted with NCoR in the absence of T3 as did the WT TRß1 but continued to interact with NCoR when 100 nM of T3 were added. Surprisingly, however, in the presence of IVT NCoR and the absence of T3, the Mkar mutant did not bind NCoR (Fig. 3B).
To determine the region of the carboxyl-terminal domain that is involved in corepressor binding, we studied the interactions of IVT NCoR in the two other mutTRß1s AM and PV also in gel shift assays. AM contains the proximal nonsense aa sequence of Mkar, codons 436–447, whereas PV shares the distal nonsense aa sequence from codon 448 to 463 with Mkar (Fig. 2). AM and PV also did not bind NCoR in the absence or the presence of 100 nM of T3 (Fig. 3B). The absence of N-CoR binding by Mkar, AM, and PV point to an abnormality in the C-terminal domain of TRß localized to the region between codons 436 and 453.
To confirm the absence of NCoR binding in the Mkar mutant, a mammalian two-hybrid assay was used to evaluate interactions between VP-16-bound NCoR and the mutTRß1s, Mkar and PV, both bound to a Gal4 DNA binding domain. Three different doses of NCoR were used: 0.01, 0.05, and 0.1 µg. Luc activity of the mutTRß1s was corrected for basal empty Gal4 activity and expressed as fold interaction. Gal4 Mkar and Gal4 PV were both found to have significantly less interaction with all three doses of the VP16-NCoR construct, compared with Gal 4 WT (P 
0.01), confirming the EMSA findings (Fig. 3C). NCoR failed to bind to the Mkar-RXR heterodimer (Fig. 3D).
Increasing amounts of WT TRß1, when added to a constant amount of Mkar mutant (simulating a possible in vivo situation), increased binding to NCoR as demonstrated (Fig. 4A). When the opposite experiment was performed and increasing amounts of Mkar mutant were added to a constant dose of WT TRß1, binding to NCoR diminished (Fig. 4B). This is consistent with diminished binding of NCoR to the Mkar mutTR.
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Ligand-independent repression and activation of positive and negative TH response elements (TREs) by the mutant receptors was evaluated in transient transfection studies using CV-1 cells. Mean Luc activity in the presence of the PALx3Luc reporter for WT TRß1, Mkar, Mdbs, AM, and PV mutTRß1s were 0.576 ± 0.03, 0.5 ± 0.06, 0.45 ± 0.01, 0.41 ± 0.06, 0.52 ± 0.07, respectively (Fig. 5A). Basal repression by Mkar was not significantly different from WT. Mdbs and AM had significantly greater repression than WT. These findings demonstrate that basal repression on a positively regulated TRE is not impaired despite the EMSA findings of absent NCoR interaction with the mutants Mkar, AM, and PV.
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T3-dependent transactivation by the mutTRß1s was tested at a T3 concentration of 10 nmol/liter. This dose was chosen because it was shown to result in a significant increase or decrease from baseline with positive and negative reporters, respectively, yet still allowed differences to be seen among mutations (3). All the mutTRß1s exhibited severe impairment of T3-induced transactivation with the two positively regulated reporters, PALx3-Luc and F2x3. Relative Luc activity of the WT TRß1 using PALx3Luc in the presence of 10 nM of T3 was 762 ± 111, compared with only 9.5 ± 1.5 with the mutTRß1s. No significant differences were seen among the mutTRß1s. Similarly, the WT TRß1 exhibited 14- to 23-fold greater relative Luc activity using the F2x3 Luc reporter, compared with the mutTRß1s (Fig. 5B).
T3-dependent repression of the SU-Luc reporter, a negatively regulated TRE, was evaluated. Transfection of WT and mutTRß1s in the presence of 10 nM of T3 demonstrated appropriate repression of activity in the WT TRß1 and significantly impaired repression with the mutTRß1s (Fig. 5C). Data were normalized to 100% to compare the degree of repression. Ligand-independent activation was not significantly different between the mutTRß1s and WT (data not shown).
DNE of the mutTRß1s was determined by cotransfecting WT TRß1 with mutTRß1s with the reporter PALx3Luc (Fig. 5D). All mutants exhibited a significant DNE over the WT TRß1. As the ratio of mutant to WT TRß1 decreased, diminishing dominant-negative inhibition of activity was seen (Fig. 5D).
Thus, all the mutant receptors demonstrate a similar in vitro function despite differences in NCoR association. All mutants have severely impaired ligand-dependent transactivation and repression on positively and negatively regulated TREs, respectively, as well as strong DNE.
All mutTRß1s bind SMRT in the absence of T3 and demonstrate impaired ligand-dependent release of SMRT
A second corepressor, SMRT, was then studied as a possible mechanism for the ligand-independent repression and strong DNE seen in the transient transfections. All mutTRß1s were found to interact with SMRT in the absence of T3 and to have impaired release of SMRT when 100 nM of T3 was added (Fig. 6A). The interaction between SMRT and Mkar was confirmed in a mammalian two-hybrid assay using VP-16 SMRT (Fig. 6B).
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Given the presence of SMRT binding and impaired release in the presence of T3, EMSA was then used to evaluate whether or not coactivator recruitment was defective. At a dose of 1.5 µg of GST-SRC-1, the WT TRß1 had normal recruitment and binding of SRC-1, whereas the mutTRß1s, Mkar and Mdbs, were unable to recruit SRC-1 in the presence of 1000 nM of T3 and even 10,000 nM of T3 (Fig. 6C). These results confirm that the C-terminal domain is an area involved in exchange of corepressors and coactivators, depending on the absence or presence of T3.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Our studies demonstrate a novel finding of absent NCoR binding in a naturally occurring mutTRß. The impairment of NCoR interaction with the Mkar mutant was evaluated by both EMSA and mammalian two-hybrid assays. By studying other mutant TRs with altered aa sequences in helix 11 and 12, we were able to localize the area responsible for this abnormality in NCoR binding to codons 436–453 of TRß1. The mutated aa sequence shared by the three mutTRß1s is unable to bind to NCoR. This may be the mechanism by which these extensive frameshift mutations diminish or abolish corepressor binding. These findings have not been described to date in any other case of RTH.
The nuclear receptor corepressor specificity demonstrated by this novel mutation has been observed in another mutTRß1, namely R429Q (13). This naturally occurring mutTRß1 was found to be defective in its ability to homodimerize. That mutation was unable to recruit NCoR as either a homodimer or heterodimer with RXR but had the ability to recruit SMRT as a heterodimer only. Clinically the subject had primarily pituitary manifestations of RTH, as did Mkar with elevated serum TSH, which failed to suppress with high TH levels, causing thyrotoxicosis at the level of peripheral tissues. Furthermore, R429Q is associated with impaired ligand-dependent repression on TRH and TSHß-negative TREs but normal to enhanced function on positive TREs (25). R429Q differs from our mutation in that homodimerization was not defective in Mkar and Mkar had impaired ligand-dependent function on positive and negative TREs.
Our data add to the observation that SMRT, one of the multiple SMRT isoforms recently described, may be an example of how corepressor function is adapted to different cells (26). This case of severe RTH demonstrates nuclear corepressor specificity in TH action and the importance of the C-terminal domain, in particular codons 436–453, in NCoR interaction.
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Acknowledgments |
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Footnotes |
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Disclosure of Potential Conflicts of Interest: S.Y.U., R.N.C., E.S., D.A.S., N.E.Y., H.G., J.N., and R.E.W. have nothing to declare. S.R. is a consultant for Quest.
First Published Online February 7, 2006
1 S.Y.W. and R.N.C. contributed equally to the study and both should be considered as first authors. 
Abbreviations: aa, Amino acid; AM, artificial mutant; BCS, bovine calf serum; DNE, dominant-negative effect; FT4, free T4; GST, glutathione-S-transferase; IVT, in vitro translated; Luc, luciferase; mutTR, mutant TR; NCoR, nuclear receptor corepressor; RTH, resistance to thyroid hormone; RXR, retinoid X receptor; SMRT, silencing mediator of retinoid and thyroid hormone receptor; SRC, steroid receptor coactivator; TH, thyroid hormone; TR, TH receptor; TRE, TH response element; TRIAC, 3,5,3'-triiodothyroacetic acid, also called tiratricol; WT, wild type.
Received November 7, 2005.
Accepted January 31, 2006.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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