Thyroid Transcription Factor 1 Rescues PAX8/p300 Synergism Impaired by a Natural PAX8 Paired Domain Mutation with Dominant Negative Activity
Helmut Grasberger,
Usanee Ringkananont,
Paule LeFrancois,
Marc Abramowicz,
Gilbert Vassart and
Samuel Refetoff
Departments of Medicine (H.G., U.R., S.R.), Pediatrics (S.R.), and Committee on Genetics (S.R.), The University of Chicago, Chicago, Illinois 60637; Centre Hospitalier de la Region Annecienne (P.L.), 74011 Annecy, France; Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucleaire (M.A., G.V.) and Department of Genetics, Campus Erasme (G.V.), Universite Libre de Bruxelles, B1070 Brussels, Belgium
Address all correspondence and requests for reprints to: Samuel Refetoff, The University of Chicago, MC3090, 5841 South Maryland Avenue, Chicago, Illinois 60637. E-mail: refetoff{at}uchicago.edu.
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ABSTRACT
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Mutations in the paired domain transcription factor PAX8 are a rare cause of congenital hypothyroidism due to thyroid dysgenesis. We identified a novel and unique PAX8 mutation segregating in seven affected members of a three-generations family. The mutation replaces an invariant serine residue within helix 2 of the paired DNA-binding domain for phenylalanine. The mutant protein (PAX8-S48F) does not induce the thyroglobulin promoter in nonthyroid cells, but displays almost half of wild-type PAX8 activity in thyroid cells. PAX8-S48F shows no defect in expression, nuclear targeting, or DNA binding and retains the ability to synergize with thyroid transcription factor 1 (TTF-1, NKX2.1). However, we found that in nonthyroid cells, the acetylation-independent synergism with the general transcriptional adaptor p300 is completely abrogated, suggesting that PAX8-S48F may be unable to efficiently recruit p300. Reconstitution experiments in nonthyroid cells reveal that TTF-1 can partially rescue PAX8-S48F/p300 synergism and thus reproduce the situation in thyroid cells. These functional characteristics result in a dominant negative effect of PAX8-S48F on coexpressed wild-type PAX8 activity, which is not observed in paired domain mutations with DNA binding defect. Our results describe the first dominant negative missense mutation in a paired domain and provide evidence for a crucial role of the p300 coactivator in mediating the functional synergism between PAX8 and TTF-1 in thyroid-specific gene expression.
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INTRODUCTION
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CONGENITAL HYPOTHYROIDISM (CH), affecting about one in 3000 newborns, is the most common inborn endocrine disorder, frequently caused by developmental defects of the thyroid gland (thyroid dysgenesis). However, mutations in the genes known to be crucial for thyroid gland development were found to account for only a small proportion of sporadic and familial cases of thyroid dysgenesis (reviewed in Ref. 1). These genes encode the TSH receptor (2, 3) and three transcription factors: the homeodomain-containing factor TTF-1 (thyroid transcription factor 1) (4, 5), the forkhead domain-containing protein TTF-2 (thyroid transcription factor 2) (6), and PAX8 (7).
As other proteins of the Pax family, PAX8 binds to DNA via a conserved 128-amino acid paired domain. PAX8 is required both for morphogenesis of the thyroid gland (8) and maintenance of the thyrocyte cell type (9). PAX8 is essential for the thyrocyte-specific promoter activation of the thyroperoxidase (TPO), thyroglobulin (TG), and sodium/iodide symporter genes (10, 11). Whereas deletion of one Pax8 allele in mice merely causes a higher prevalence of elevated plasma TSH compared with wild-type (WT) littermates (12), heterozygous loss of function mutations in humans have been associated with CH due to dysgenesis. So far, six different PAX8 mutations, two sporadic and four familial, have been reported (7, 13, 14, 15). All these are localized to the paired domain, and of the five tested (7, 14, 15), all had a severe DNA binding defect in common, suggesting that CH is secondary to PAX8 haploinsufficiency.
Whereas the central role of PAX8 in thyroid organogenesis and thyroid-specific gene expression has been firmly established, the molecular mechanisms involved in activation of PAX8 target genes are less well characterized. It has been demonstrated recently that a physical interaction of PAX8 with TTF-1 underlies the synergistic effect of both factors on TG promoter activation (16), providing a molecular basis for the specific expression of TG in thyrocytes, the only tissue in which both factors are coexpressed. Apart from TTF-1, functional cooperation and in vitro interaction with PAX8 has been described for the general coactivator p300 (17). p300 has been found to interact with components of the basal transcriptional machinery and several sequence-specific transcription factors (reviewed in Ref. 18), including TTF-1 (19).
Here, we report the identification and functional characterization of a novel PAX8 mutation, S48F, segregating in seven affected members of a family with CH. Although the mutated residue is located within the paired domain, the mutant protein does not display a loss in DNA binding affinity, but rather a specific defect in transactivation. In nonthyroid cells, in contrast to thyroid cells, the mutant protein is completely inactive and fails to synergize with p300, but not TTF-1, in activation of the TG promoter. Coexpression of TTF-1 partially rescues the PAX8-S48F/p300 synergism. Furthermore, when coexpressed with the WT protein, PAX8-S48F displays a unique dominant negative effect on WT PAX8 activity.
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RESULTS
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Clinical Findings and Detection of a Novel PAX8 Gene Mutation
Seven members of a nonconsanguineous family were found to be hypothyroid with apparent autosomal-dominant mode of inheritance (Fig. 1A
). Of note is a striking variability in the initial clinical presentation. For example, patient III-5 was identified by neonatal screening and had no thyroidal 123I uptake on scintigraphy, consistent with apparent athyreosis. In contrast, her affected brother (patient III-4) had a normal TSH value upon neonatal screening and was found only to have elevated serum TSH level with normal free T4 at the age of 5 yr, in the course of the diagnostic work-up of the family. Overall, three subjects had serum TSH values above 100 mU/liter with low, free T4 concentrations and were identified at birth, whereas the remaining four had serum TSH values ranging from 13.9 to 50 mU/liter and were identified in the course of family screening, later in life (for details see Materials and Methods). Linkage analysis showed haplotype sharing of the affected family members for microsatellite markers covering the PAX8 locus on chromosome 2q13 (Fig. 1A
). Sequence analysis disclosed that the patients carried a heterozygous C-to-T transition in exon 3, resulting in the substitution of an invariant serine at position 48 within
-helix 2 of the PAX8 paired domain by phenylalanine (PAX8-S48F) (Fig. 1
, B and C).
PAX8-S48F Does Not Transactivate a PAX8-Responsive Promoter in Nonthyroid Cells
To evaluate the functional significance of the S48F mutation, we tested the ability of the mutant protein to activate a cotransfected reporter gene under control of the human TG promoter. Cotransfection of WT PAX8 expression vector into HeLa cells produces up to 4-fold stimulation of luciferase activity compared with cotransfection with empty vector, whereas coexpression of the PAX8-S48F mutant did not result in reporter activation (Fig. 2A
). Under the same conditions, WT PAX8 elicited a moderate (
70%) stimulation of the human TPO proximal promoter, but again PAX8-S48F failed to induce a significant response (Fig. 2B
). Western blotting of whole-cell protein extracts of HeLa cells, transfected with expression vectors for either WT PAX8 or PAX8-S48F, revealed no difference in synthesis efficiency or electrophoretic mobility between the WT and the mutant PAX8 proteins (Fig. 2C
), indicating that the S48F mutation does not cause destabilization of the mutant protein.

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Fig. 2. Transactivation of the PAX8-S48F Mutant Protein
Forty nanograms of either WT PAX8 (WT), PAX8-S48F (S48F), or empty (pcDNA) expression vectors were cotransfected along with 0.5 µg reporter construct containing a firefly luciferase gene driven by (A) either the human TG promoter (Tg-Luc) or (B) the human TPO promoter (Tpo-Luc), respectively, into HeLa cells. Firefly luciferase activities were normalized to the Renilla luciferase activity derived from cotransfected pRL-Tk internal control plasmid and represented relative to the activity obtained by cotransfection of empty vector. Results are means ± 1 SD from five independent experiments, each performed in triplicate transfections. C, Equal amounts of cell extracts from one of the transfection experiments described above were analyzed by Western blotting using a polyclonal antibody to PAX8. n.s., Nonspecific; pcDNA, empty vector; WB, Western blotting.
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PAX8-S48F Has No Defect in DNA Binding Activity
The location of the mutated residue within the paired domain suggested that the S48F mutation may produce a defect in DNA binding, similar to previously reported mutations in the PAX8 paired domain (7, 14, 15). DNA binding was assessed using a PAX8 response element of the rat Tpo promoter (CT-element) (20) as a probe in band shift assays. Specificity of binding was established by competition with a cold binding site. As shown in Fig. 3A
, PAX8-S48F bound to the probe in a complex with the same electrophoretic mobility as the WT PAX8 protein. Surprisingly, in four independent experiments, the signal intensity of the complex containing PAX8-S48F was consistently stronger than that produced by the WT protein. There was no relevant difference in the amount of mutant vs. WT protein used in the band shift reactions as confirmed by parallel in vitro-translation reactions that were labeled with [35S]methionine and analyzed by phosphor imaging (Fig. 3B
). After correction for relative protein expression, the amount of probe bound in equilibrium to PAX8-S48F was approximately 1.5 times that bound to WT PAX8 (1.54 ± 0.38; P < 0.03). In dissociation analysis with increasing concentrations of specific competitor, binding of the probe to WT and S48F mutant PAX8 proteins was competed with comparable efficiency (Fig. 3C
). Enhanced DNA binding of the mutant protein was also observed when nuclear extracts of HeLa cells, transiently transfected with either the WT or mutant expression constructs, were employed in the band shift reactions (data not shown).

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Fig. 3. DNA Binding of Mutant PAX8-S48F
A, The DNA binding abilities of in vitro-translated WT and S48F mutant PAX8 proteins were evaluated using the PAX8 response element of the rat Tpo promoter (64/41 region) (20 ) by EMSAs. The band shift shown is representative of four independent experiments giving similar results. Background corrected volumetric counts of the shifted probe signals were obtained by phosphor imaging (WTbound and MUTbound, respectively). B, WT PAX8 and PAX8-S48F mutant proteins were synthesized in vitro as above but in the presence of [35S]methionine. Equal amounts of labeled reactions were separated by PAGE and analyzed by phosphor imaging (WTprot and MUTprot, respectively). C, For dissociation analysis, constant amounts of protein and probe were incubated in the presence of increasing concentrations of unlabeled CT oligonucleotide (comp; 2x, 5x, 10x, 30x, 100x, and 300x, respectively, the probe concentration). pcDNA, Empty vector.
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The S48F Mutation Does Not Compromise Subcellular Targeting
The folded PAX8 paired domain functions as a complex nuclear localization signal, which cannot be reduced to a smaller amino acid sequence without loss of nuclear targeting capacity (21). Hence, inability of PAX8-S48F to activate a PAX8-responsive promoter, despite unimpaired DNA binding in vitro, could be due to diminished transport of PAX8-S48F into the nucleus. To test this possibility, we generated constructs expressing either WT or mutant PAX8 as fusions with enhanced green fluorescent protein (EGFP). In contrast to EGFP, which is diffusely distributed throughout the cell, including the cytoplasm (Fig. 4
), the WT PAX8-EGFP fusion protein is efficiently targeted into the nucleus. The PAX8-S48F-EGFP fusion protein is transported equally well into the nucleus, and its nuclear distribution shows no obvious difference compared with WT PAX8-EGFP (Fig. 4
).

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Fig. 4. Subcellular Targeting of WT PAX8 and Mutant PAX8-S48F
EGFP fused to the C terminus of WT PAX8 or PAX8-S48F was expressed in HeLa cells (green signal). The localization of TTF-1 (expressed from cotransfected pRc/CMV-TTF-1) was visualized by indirect immunofluorescence with a monoclonal anti-TTF-1 antibody followed by Alexa Fluor 568 conjugated secondary antibody (red signal). DNA was counterstained with Hoechst 33342 (blue signal). Both EGFP-fusion proteins are efficiently targeted to the nucleus and show no obvious difference in their intranuclear distribution.
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PAX8-S48F Fails to Synergize with p300 but Not TTF-1
The general transcriptional coactivator p300 has been shown to be crucial for PAX8 activity on the rat Tpo promoter (17). p300 plays a pivotal role in the activity of many transcription factors, either by bridging sequence-specific DNA-binding factors with elements of the basal transcriptional machinery, such as transcription factor IIB and polymerase 2, or by its intrinsic histone acetyltransferase (HAT) activity. It is reasonable to postulate that the inability of PAX8-S48F to recruit p300 and assemble the transcriptional coactivation complex could explain the loss of its transactivation potential. As previously shown using the Tpo promoter (17), we found that with the TG promoter, overexpression of p300 results in a dose-dependent potentiation of WT PAX8 activity (normalized for the induction of the thymidine kinase (Tk) promoter; Fig. 5A
). In contrast, p300 is unable to enhance the activity of PAX8-S48F. Note that in HeLa cells and without cotransfected WT PAX8, the TG promoter is, in fact, less p300 responsive than the Tk promoter of the internal control (inset, Fig. 5A
), but is specifically sensitized and, thus, rendered more p300 responsive than the Tk promoter by the presence of WT PAX8.

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Fig. 5. Cooperation of PAX8-S48F with TTF-1 but Not p300 in Transactivation of the TG Promoter
HeLa cells were cotransfected with constant amount of either WT (black bars) or S48F mutant (gray bars) PAX8 expression vectors along with increasing amounts of p300 (panel A) or TTF-1 (panel B) expression constructs. For each transfection, the total amount of DNA was kept constant by adding empty vector (pcDNA) as outlined below the graph (numbers indicate micrograms of DNA transfected per well). Data were normalized for Renilla luciferase activity driven from pRL-Tk internal control vector. In panel A, the effect of p300 coexpression on Renilla luciferase activity is illustrated in the inset (transfection with 0.32 µg empty pcDNA or p300 vector, respectively). WT PAX8 specifically sensitizes the TG promoter, as compared with the thymidine kinase promoter, to the action of p300, whereas PAX8-S48F does not change the p300 responsiveness of the TG promoter. In contrast, PAX8-S48F retains the ability to synergize with TTF-1 to coactivate the TG promoter.
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Contrary to the action of p300, TTF-1 is able to synergize with coexpressed PAX8-S48F (4.0-fold activation over basal TG activity by TTF-1/PAX8-S48F combination, compared with no significant activation by either factor alone; Fig. 5B
). Yet, overexpression of TTF-1 can only partially overcome the functional defect of PAX8-S48F when compared with WT PAX8 (4.0-fold vs. 20.4-fold).
Loss of transactivation of the PAX8-S48F mutant in nonthyroid cells is consistent with the observed loss of cooperation with the general coactivator p300. Whereas p300 has been shown to physically interact with the PAX8 C terminus in vitro, the mechanism of the PAX8/p300 cooperation has not been characterized. Recruitment of p300 by PAX8 may induce localized chromatin remodeling via its intrinsic HAT activity, resulting in enhancement of PAX8-mediated transcription. To test whether the synergism requires HAT activity, the effect of p300 on PAX8-mediated TG promoter activation in HeLa cells was studied in the presence and absence of specific inhibitors of either histone deacetylases (trichostatin A) (22) or acetyltransferases (anacardic acid) (23). To measure PAX8-dependent promoter stimulation within each drug treatment group, luciferase activity was first normalized by the Renilla luciferase activity driven by the internal control vector pRL-Tk, and then expressed relative to the cotransfection with empty vector (pcDNA in Fig. 6
). Although both drugs had pronounced effects on the absolute TG and Tk reporter activities (see inset in Fig. 6A
), inhibition of HAT activity did not abolish, but rather augmented, the PAX8/p300 synergy, whereas inhibition of deacetylases had the opposite effect. Thus, acetylation is not a prerequisite of the PAX8/p300 synergism. These findings favor the idea that p300 enhances PAX8 activity by bridging PAX8 to the transcriptional machinery and/or other cofactors, rather than by HAT-induced chromatin remodeling. It is to be noted that these findings are not limited to a specific cell line, because independence of the PAX8/p300 synergistic effect from acetylation is also observed in HEK293 cells (Fig. 6B
).

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Fig. 6. The PAX8/p300 Synergism Is Independent of Acetyltransferase Activity
Effect of the acetyltransferase inhibitor anacardic acid (AA) and the histone deacetylase inhibitor trichostatin A (TSA) on the PAX8/p300 synergism in activation of the TG promoter. A, HeLa cells transfected with the indicated constructs were incubated for 12 h with TSA, AA, or solvent before harvest for luciferase assay. The effect of these drugs on the Renilla luciferase activity driven by the thymidine kinase promoter of the internal control vector (pRL-Tk) is shown in the inset. To compare the activation attributable to the PAX8/p300 synergism, data were normalized within each drug treatment group for Renilla activity and then expressed relative to the activity in cells cotransfected with empty vector. Despite enhancing basal TG promoter activity, acetylation is not required for the PAX8/p300 synergism. B, Effect of AA on the PAX8/p300 synergism in HEK293 cells. Data were normalized as in panel A. HEK, Human embryonic kidney; pcDNA, empty vector.
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TTF-1 Partially Rescues the PAX8-S48F/p300 Synergism
To further study the interplay of p300 and TTF-1 with the WT and mutant PAX8 proteins, respectively, we employed HEK293 cells, because these cells are deficient in endogenous p300 due to the expression of the adenovirus E1A protein (24), which sequestrates p300 in the cytosol. Although, in the absence of p300 coexpression, the activity of exogenous PAX8 in this cell line is approximately 5 times lower (1.6-fold stimulation over basal) as compared with other nonthyroid cells (e.g. HeLa, NIH3T3), it shows a robust synergism with coexpressed p300 [3.8 fold; recruitment index (RI) = 2.5 (Fig. 7A
)]. This corroborates the role of the PAX8/p300 synergism in PAX8-mediated TG promoter activation. Remarkably, the results of these experiments also demonstrate a synergistic effect when both p300 and TTF-1 were coexpressed together with WT PAX8 (13.6-fold activation over basal TG promoter activity), compared with the combination of PAX8 with either p300 (3.8-fold) or TTF-1 (5.2-fold) alone (Fig. 7A
). Because TTF-1 and PAX8 are able to physically interact with each other (16) as well as p300 (17, 19), it is possible that this synergism involves the direct cooperation of all three factors. In contrast, and consistent with the results obtained in HeLa cells, PAX8-S48F shows no synergism with p300 in the absence of cotransfected TTF-1 (RI = 1; see inset in Fig. 7A
). Whereas TTF-1, in the absence of cotransfected PAX8 (WT and mutant), does not synergize with p300 (1.5-fold activation of the TG promoter by cotransfection of p300 with TTF-1 vs. 1.5-fold activation by p300 alone and no activation by TTF-1 alone), cotransfection of TTF-1 is able to rescue the synergistic effect of p300 on PAX8-S48F-mediated transactivation (6.4-fold activation; RI = 4.6). These data are compatible with the idea that the S48F mutation disrupts the direct interaction between the PAX8 and p300, yet retains the ability to interact with TTF-1, which allows recruitment of p300 to the proximal promoter via binding to TTF-1. Such a PAX8-S48F/TTF-1/p300 complex would be less stable than the native complex, comprising WT PAX8, and could thus compensate only partially for a defect in direct p300 recruitment by PAX8-S48F.

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Fig. 7. Partial Rescue of PAX8-S48F/p300 Synergy in the Presence of TTF-1
A, HEK293 cells were cotransfected with Tg-Luc reporter, the pRL-Tk internal control vector, and the indicated amounts (in micrograms) of expression vectors or empty vector (pcDNA). The total amount of transfected DNA was equalized by empty vector. Transfected cells were harvested in 48 h for dual luciferase assays, and the average normalized activity is presented relative to the cotransfection with only empty vector (set to 1). Calculation of the RIs for the interaction between WT or S48F mutant (S48F) PAX8 with p300, with or without coexpressed TTF-1, is depicted in the table. The RI is calculated by dividing the activity (over basal Tg-Luc activation) obtained by PAX8/p300 coexpression by the sum of the activities obtained by overexpression of PAX8 and p300 alone. An RI value greater than 1 indicates cooperation of PAX8 and p300 in the activation of the TG promoter. B, Transient transfection assays in FRTL-5 rat epithelial thyroid cells. The data represent the means ± 1 SD of four (panel A) or three (panel B) independent experiments performed in triplicate transfections. HEK, Human embryonic kidney.
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The S48F Mutation Is Hypomorphic in Thyrocytes
Partial rescue of PAX8-S48F activity by exogenous TTF-1 in the nonthyroid HEK293 cell line prompted us to test the activity of PAX8-S48F in the more physiologically relevant cellular environment of a rat epithelial thyroid cell line (FRTL-5), which expresses endogenous PAX8 and TTF-1 (25). In agreement with previous results (25), transient overexpression of WT PAX8 produces a significant, up to approximately 2-fold, stimulation of the cotransfected TG reporter (1.78 ± 0.17; P < 0.02) (Fig. 7B
). In marked contrast to the transfections in nonthyroid cells, expression of PAX8-S48F was also able to elicit significant stimulation of the reporter in the absence of TTF-1 cotransfection (1.35 ± 0.11; P < 0.005). However, the activity of PAX8-S48F under these conditions was still significantly lower than that of WT PAX8 (P < 0.05). Compared with WT PAX8, the relative activity of PAX8-S48F in thyroid cells closely resembles that obtained in the TTF-1/p300 reconstitution experiments in HEK293 cells (45% of WT PAX8 activity in FRTL-5 vs. 43% in HEK293 cotransfected with TTF-1/p300).
PAX8-S48F Is a Novel Paired Domain Mutation with Dominant Negative Activity
Because the S48F mutation apparently alters cofactor interaction but not nuclear targeting or DNA binding, it is tempting to speculate that in the heterozygous state, PAX8-S48F competes with WT PAX8 for DNA binding. Hence, a dominant negative mechanism might contribute to the phenotypic expression of the mutated allele. To examine the importance of the ratio of mutant to WT PAX8 proteins for transcriptional activation, we cotransfected a constant amount of WT PAX8 expression plasmid and increasing amounts of mutant PAX8 expression plasmid together with the TG reporter construct into HeLa cells and measured the combined transcriptional activation potential of WT and mutant PAX8. Two previously characterized PAX8 mutants, Q40P (14) and L62R (7), both stably expressed but essentially lacking DNA binding activity, were used as controls. As shown in Fig. 8
, coexpression of PAX8-S48F diminishes the activity of WT PAX8 (by 82% at a 1:1 WT-to-mutant ratio), whereas coexpression of PAX8-Q40P or PAX8-L62R does not significantly affect the activity of WT PAX8. These data imply that in the heterozygous state, the S48F mutant can efficiently compete with WT PAX8 for binding sites in target genes and thus affect WT PAX8 activity in a dominant negative fashion.

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Fig. 8. Transdominant Negative Effect of the PAX8-S48F Protein on WT PAX8 Activity
Transient cotransfection assays were performed in HeLa cells using 0.04 µg of expression plasmid encoding PAX8, 0.5 µg of the Tg-Luc reporter, and two different doses (0.04 and 0.08 µg) of expression plasmids encoding the S48F, Q40P (14 ), or L62R (7 ) PAX8 mutants. The PAX8-S48F mutant protein shows a dominant negative effect on WT PAX8 activity. In contrast, the two PAX8 mutants with DNA binding defect do not impair WT PAX8 activity. Of note, a gene dosage effect was excluded by the response of Tg-Luc to the transfection of increasing amounts (0.04, 0.08, and 0.12 µg) of WT PAX8 expression vector alone.
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DISCUSSION
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We have identified and functionally characterized a novel mutation, S48F, in the PAX8 gene, found in the heterozygous state in seven members of a family with CH of variable severity. Although S48F is located in the paired domain, as are the previously reported PAX8 missense mutations, its functional properties turned out to be unique. It is the first described missense mutation within the PAX8 paired domain, which abrogates the transactivation function but not DNA binding.
The reason for significantly increased DNA binding in the band shift reactions is not clear but might involve a difference in folding of the mutant protein during the DNA binding-induced conformational change (26). Furthermore, reduced recruitment of cofactors, as implied by the reduced cooperation with p300, may, for sterical reasons, promote better association of PAX8-S48F with DNA. This concept is supported by the fact that increased DNA binding affinity has been described as a common feature of C terminus-truncated PAX6 proteins (27) lacking the transactivation domain and, thus, the major interface for cofactor recruitment.
In contrast to previously reported paired domain mutations, which lack DNA binding, PAX8-S48F can efficiently antagonize WT PAX8 activity by competition for DNA-binding sites (Fig. 8
). Because PAX8 binds DNA as a monomer, such a strong dominant negative effect was unexpected but can be accounted for by the higher DNA-binding activity of the mutant compared with WT PAX8 (Fig. 3
). Dominant negative mutant PAX proteins have been described so far only for truncations that remove the transactivation domain yet retain the DNA binding paired domain (27). Hence, the S48F mutation represents a new type of paired domain mutation in human disorders.
Based on the crystal structure models of paired domain-DNA complexes (28, 29), serine 48 is located in
-helix 2 of the paired domain (Fig. 1C
) and is not predicted to form direct DNA contacts. Studies on PAX6 do suggest, however, that, at least in the DNA-bound state, helix 2 interacts with the region immediately N-terminal to the homeodomain-like (30, 31) region conserved in all Pax family proteins. Thus, mutations in helix 2 of the paired domain might well affect folding and conformation of C-terminal regions of PAX8, including regions known to be crucial for transactivation activity (21), without necessarily abrogating DNA binding. This idea would be in line with the observation that PAX8-S48F fails to synergize with p300, a coactivator that interacts with the C-terminal PAX8 transactivation domain in the two-hybrid system (17). However, it should be noted that for two reasons, the aforementioned studies do not exclude the possibility of a physical contact between helix 2 of the DNA-bound PAX8 paired domain and p300 in vivo. First, the PAX8 paired domain undergoes a dramatic rearrangement of its secondary structure upon DNA binding (32), a conformational change not expected to occur in the context of a Gal4-PAX8 fusion protein tethered to DNA via its Gal4 DNA-binding domain. Second, the failure of PAX8 constructs containing the paired domain to interact with p300 in the two-hybrid system may be secondary to a folding defect of these PAX8 proteins. At least in our hands, fusions of either the Gal4 DNA-binding domain or green fluorescent protein N-terminal to the PAX8 paired domain produced misfolded proteins, as evidenced by their sequestration in aggrosomes (H. Grasberger, unpublished observation).
p300 has been shown previously to be essential in mediating PAX8 activation of the Tpo promoter (17). Furthermore, adenovirus E1A, which is known to sequestrate and thus inactivate p300, blocks thyrocyte differentiation (33) and abrogates PAX8 activity on the Tpo promoter (17). The results presented here extend the role of a functional PAX8/p300 cooperation to the TG promoter, suggesting that p300 is an essential element of PAX8 activity beyond the context of the Tpo promoter. The crucial role of p300 in PAX8 activity is also evident in the low activity of exogenous PAX8 in the p300-deficient HEK293 cell line, which can be restored to the level observed in other nonthyroid cells by overexpression of p300 (Fig. 7A
).
Two important features of p300 could be involved in mediating PAX8 activity: its ability to act as a bridging factor to other coactivators and the basal transcriptional machinery, and its HAT activity. The results of our experiments show that inhibition of HAT activity does not abrogate, but rather increases, the PAX8/p300 synergism (i.e. the PAX8-dependent component of TG promoter activation by p300), whereas the inhibition of deacetylases has the opposing effect (Fig. 6
). These results favor the hypothesis that p300 potentiates PAX8 activity by providing a physical link to the transcriptional machinery rather than by employing its HAT activity (Fig. 9A
). In the case of the S48F mutant, formation of the p300-containing coactivator complex is likely disrupted, consistent with absent activity in nonthyroid cells (Fig. 9C
). The negative impact of acetylation on the PAX8/p300 synergy is an intriguing observation and may reflect the acetylation of nonhistone factors, e.g. of p300 itself or Pax8. If factor acetylation follows a slower kinetics compared with histone acetylation, it could play a role in turning off, and thus limiting, Pax8-dependent transcription. Indeed, opposing effects of p300 on the activity of a sequence-specific transcription factor due to acetylation of both histones and nonhistone factors would not be without precedent (34, 35, 36).

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Fig. 9. Hypothetical Model of p300 as Mediator of the PAX8/TTF-1 Synergism and the Proposed Mechanism for the Functional Defect in the PAX8-S48F Mutant
A, Model for PAX8/p300 synergy in the absence of TTF-1. Recruitment of p300 by DNA-bound PAX8 promotes the formation of a preinitiation complex. B, Model of synergistic activation of a thyroid-specific target gene (TG) in the presence of both TTF-1 and PAX8, in which proximal promoter-bound PAX8 in conjunction with TTF-1 recruits p300 to form a stable complex that enhances the assembly of the transcriptional preinitiation complex, resulting in robust activation of transcription. C, Model of impaired TG promoter activation by PAX8-S48F in the absence of TTF-1, in which the mutant protein fails to recruit p300 directly and, thus, lacks synergism with p300. D, In cells expressing both PAX8-S48F and TTF-1 (i.e. in the patients thyrocytes), the mutant protein may interact with TTF-1, which, in turn, can recruit p300 into a less stable complex, resulting in partial rescue of the functional defect. TFIID, transcription factor IID; RNApol-II, RNA polymerase II.
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In contrast to the complete loss of cooperation with p300, PAX8-S48F was still able to synergize with TTF-1 on the TG promoter, as indicated by the significantly higher stimulation of reporter gene expression in cells expressing both PAX8-S48F and TTF-1, as compared with the stimulation with TTF-1 alone (Fig. 5B
). However, the cooperation with TTF-1 cannot completely rescue the activity of the S48F mutant to the level obtained with WT PAX8.
Both, TTF-1 (19) and PAX8 (17), utilize p300 as coactivator, suggesting that p300 may serve as mediator of the PAX8/TTF-1 synergism. Indeed, our study provides, for the first time, experimental evidence that p300 is crucial for the well-described PAX8/TTF-1 synergism (9, 37, 38). Because p300 is present in limiting amounts within cells (39, 40, 41), consumption of p300 explains the previously noted phenomenon (16, 37) that the PAX8/TTF-1 synergy is most robust at low cellular concentrations of both transactivators, but less apparent when either one of them or both are expressed at high levels (compare Fig. 5B
). Although it remains to be shown whether binding of p300 stabilizes the PAX8/TTF-1 interaction, our findings can be reconciled with the idea of a PAX8/TTF-1/p300 complex, the stability of which is determined by interactions between all three factors (Fig. 9B
). According to this model, the partial rescue of PAX8-S48F activity by TTF-1 (Fig. 7A
) would be due to the formation of a PAX8-S48F/TTF-1/p300 complex that lacks stabilization by a direct PAX8-S48F/p300 interaction (Fig. 9D
). In thyroid cells, endogenous TTF-1 appears to be responsible for the rescue of PAX8-S48F activity to almost half the activity of WT PAX8 and, thus, to the same extent as observed by reconstitution with TTF-1 and p300 in the nonthyroid HEK293 cell line. Unfortunately, for technical reasons, we were as yet unable to directly demonstrate the formation of such complex using the two-hybrid system, sequential coimmunoprecipitation, and band shift assays with or without cross-linking. Apart from the weak or transient nature of such interactions, a reason for our failure to demonstrate a ternary complex could be that the conformational change induced by DNA binding of PAX8 and TTF-1 is required for these interactions to occur.
The PAX8/TTF-1 synergism involves the so-called "region C" of the proximal Tg promoter (16), which contains the PAX8 response element overlapping with one of the TTF-1-binding sites (10). Given that TTF-1 does not functionally interact with p300 in the absence of PAX8 (Fig. 7A
) and DNA binding-deficient PAX8 mutants are unable to synergize with TTF-1 (15), the synergistic effect seems to require binding of at least PAX8 to the cis-element in the proximal promoter. Direct binding of PAX8 and TTF-1 to this region is mutually exclusive in vitro, and no complex indicative of simultaneous binding has been observed in band shift assays (Ref. 10 and our unpublished observations). Although these data do not completely exclude the possibility that PAX8 recruits TTF-1 via protein-protein interaction without TTF-1 binding to DNA, it favors the idea that TTF-1 interacting with PAX8 is itself bound to DNA at an upstream TTF-1 site, which may be promoter dependent either in the proximal TG promoter (e.g. the B site, but apparently not the A site, of the Tg promoter) (17) or in the upstream enhancer of the TPO promoter (37). We believe that, in vivo, efficient interaction of TTF-1 with PAX8 is likely to require other factors, such as p300, that together form a larger complex, and DNA binding of both transcription factors may well be a prerequisite for the protein-protein interactions to take place, owing to the conformational change induced by DNA binding (26).
How do these functional data relate to the situation in our patients thyroid glands? It can be concluded with certainty that, contrary to previously described paired domain mutations with loss of function due to a defect in DNA binding, the activity of PAX8-S48F in thyrocytes is modulated by the expression level of other limiting factors, as shown here for p300 and TTF-1. Because PAX8-S48F activity is partially rescued by TTF-1, net PAX8 activity in our patients thyrocytes may well be similar to the patients with pure loss-of-function/haploinsufficiency mutations, despite the transdominant negative properties of PAX8-S48F. This is supported by our results from cotransfection experiments in thyrocytes (Fig. 7B
) and would be consistent with the rather mild clinical phenotype in the carriers of the S48F mutation (i.e. only one of seven with morphological evidence of thyroid dysgenesis). Yet one has to assume that, depending on the promoter-specific interactions with cofactors, PAX8-S48F differentially affects other PAX8 target genes. We hypothesize that similar PAX8 mutations with a defect in cofactor interaction may cause CH not fitting a typical dysgenesis phenotype and that screening of PAX8 in bona fide dysgenesis cases is likely biased toward the detection of complete loss-of-function mutations.
In conclusion, the characterization of a natural PAX8 mutation revealed a novel molecular mechanism, i.e. a specific defect in coactivator recruitment, in PAX8-associated CH. Our analysis of the synergistic action of PAX8 with TTF-1 and p300 raises the possibility that, at least on the TG promoter, these factors form a complex the stability of which is determined by mutual interactions. A defect in one of these interactions, as implied by PAX8-S48F, may still allow formation of a complex, albeit of reduced stability and hence less competence, to transduce the activation signal to the basal transcriptional machinery. Serine 48 in helix 2 of the paired domain is an invariant residue despite no direct DNA contacts, as inferred from crystal structure models (28, 29). Because p300 recruitment has been described for other PAX proteins (42, 43) and may well be a basic feature common to the action of all PAX proteins, helix 2 of the paired domain can be envisaged to play an evolutionary conserved role in the p300 recruitment to PAX proteins.
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MATERIALS AND METHODS
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Human Study Subjects (RTSH-33 Family)
Patient III-1, the proposita, born after an uneventful pregnancy, had an unremarkable clinical examination at birth, but screening for neonatal hypothyroidism at 6 d of age revealed a blood TSH of 129 mU/liter (normal, <20). At 3 wk of age, serum TSH was confirmed to be elevated at 203 mU/liter (normal, 0.44.0) in the presence of low free T4 of 0.6 ng/dl (normal, 0.81.7). Both the patient and her mother had no detectable TPO or TG antibodies. A 99mTc scintigraphy showed a thyroid gland normal in shape and position. Treatment with L-T4 was initiated, resulting in normal physical and mental development. Patient III-2, brother of the proposita, was born after an uneventful pregnancy and had no clinical signs or symptoms of hypothyroidism when, at the age of 5 d, his plasma TSH was found to be 191 mU/liter with a free T4 of 0.9 ng/dl. His 99mTc scintigraphy showed a gland of normal shape and in normal position. Patient III-5, a cousin of the proposita, was also found to have CH in the frame of the neonatal screening program (blood TSH of 575 mU/liter). Her free T4 was very low at 0.4 ng/dl. On scintigraphy, no thyroidal or ectopic uptake of 123I was detectable. Her father, patient II-4, has been treated with L-T4 since the age of 5 yr when he was initially found to be hypothyroid. Patient II-1 was reported to have mild hypothyroidism by routine blood testing at age 37 and has been on continuous L-T4 therapy (100 µg/d). When additional family members were screened, patient III-4, a cousin of the proposita, who had no signs or symptoms of hypothyroidism, was found to have elevated TSH of 24.1 mU/liter with normal free T4 of 1.0 ng/dl at the age of 5 yr. The grandmother of the proposita, patient I-1, was found to have moderate thyroid failure with elevated TSH of 50 mU/liter and low free T4 of 0.6 ng/dl at the age of 67 yr. Individuals I-1', I-4', II-2, and II-3 were clinically euthyroid and found to have TSH levels within the normal range in the absence of L-T4 therapy.
Linkage Analysis and DNA Sequencing
After obtaining informed consent to participate in the study, blood was collected from all available family members, and DNA was extracted from peripheral leukocytes. Microsatellite markers encompassing the genetic interval of PAX8 (D2S293, D2S340, D2S121, D2S410) were amplified using fluorescently labeled primer pairs (Invitrogen, Carlsbad, CA), and the allele sizes were determined by electrophoresis on polyacrylamide sequencing gels (ABI 377 DNA sequencer; Applied Biosystems, Foster City, CA). For DNA sequence analysis, all PAX8 coding exons, including exon-intron junctions, were amplified with primers complementary to the flanking introns (7). After treatment with exonuclease I and shrimp alkaline phosphatase (Amersham Biosciences, Piscataway, NJ), the PCR products were employed as templates in the cycle-sequencing reactions using d-rhodamine chemistry (Applied Biosystems) and analyzed on an ABI 377 DNA sequencer.
Plasmids
The PAX8 expression vector has been described previously (13). The S48F and Q40P mutations were introduced by site-directed mutagenesis (QuikChange; Stratagene, La Jolla, CA) using sense primers 5'-GTGTAAGGCCCTGCGACATCTTTCGCCAGCTCCGCGTCAG-3' (for PAX8-S48F) and 5'-CGTAGACCTGGCCCACCCGGGTGTAAGGCCCTGC-3' (for PAX8-Q40P; mutated nucleotides underlined). To generate fusion proteins with EGFP, the coding sequences of WT PAX8 and PAX8-S48F were amplified using forward primer 5'-CCCAAGCTTGGTACCATGCCT-3' and reverse primer 5'-ATGGATCCACAGATGGTCAAAGGCCGTG-3' and then cloned in frame into pEGFP-N2 (CLONTECH Laboratories, Inc., Palo Alto, CA) via the introduced HindIII and BamHI restriction sites.
To create Tpo-Luc, a 411-bp fragment comprising the human TPO promoter was amplified using forward primer 5'-ATCTCGAGCTGCACCCAACCCAATC-3' and reverse primer 5'-ATAAGCTTCACGGCTGTAACTCTTCTGA-3' and cloned via the introduced XhoI and HindIII restriction sites into pGL3basic (Promega Corp., Madison, WI). All constructs were confirmed by sequencing. The TTF-1 expression plasmid pRc/CMV-TTF-1 (44), the reporter vector Tg-Luc (4), pCMV-HA-p300 (45), and the PAX8-L62R (7) expression vector have been described previously.
EMSA
Nuclear extracts of transfected HeLa cells were prepared essentially as described previously (46), and protein concentrations were determined by the bicinchoninic acid method (Pierce Biotechnology, Rockford, IL) with BSA standard curves. Relative amounts of WT and mutant PAX8 proteins in nuclear or whole-cell extracts were estimated by densitometry of Western blots, using an antiserum raised against recombinant canine Pax (47) as primary antibodies and enhanced chemiluminescence (Amersham Biosciences) for detection.
In vitro translated proteins were synthesized in a coupled transcription/translation rabbit reticulocyte system (Promega). As a control for synthesis efficiency, separate reactions in the presence of [35S]methionine were performed in parallel and analyzed by SDS-PAGE followed by phosphor imaging.
The band shift probe was prepared by 32P-end labeling of two PAGE-purified complementary oligonuclotides corresponding to the 64/41 region of the rat Tpo promoter (5'-CTGTCTAAGCTTGAGTGGGCATCA-3') (20) using T4 polynucleotide kinase (New England Biolabs, Beverly, MA). Duplex DNA assembled by annealing of the labeled complementary oligonucleotides was purified on G50 spin columns (Roche Applied Science, Indianapolis, IN). The specific competitor double-stranded DNA was prepared by annealing of the unlabeled oligonucleotides. Binding reactions contained 0.5 pmol probe, 2 µg nuclear protein extract or 2 µl reticulocyte lysate, 0.5 µg denatured salmon sperm DNA, and 0.5 µg poly(dI-dC) (Amersham Biosciences) in a total volume of 20 µl. All binding reactions were carried out in 25 mM Tris (pH 7.9), 6.25 mM MgCl2, 0.5 mM EDTA, 50 mM KCl, 0.5 mM dithiothreitol, and 10% glycerol. For competition experiments, excess unlabeled competitor DNA was included in the binding reactions. After incubation for 20 min at room temperature, the samples were loaded onto a 5.5% polyacrylamide gel (32:1 acrylamide-bisacrylamide) and separated at 10 V/cm in 0.5x TBE (45 mM Tris, 45 mM borate, 1 mM EDTA). Dried gels were analyzed by phosphor imaging (Storm 860 Optical Scanner; Molecular Dynamics, Sunnyvale, CA) using ImageQuant software (Molecular Dynamics).
Indirect Immunofluorescence
To delineate the subcellular localization of TTF-1 relative to PAX8, Hela cells cotransfected with pRc/CMV-TTF-1 and one of the EGFP fusion constructs (WT PAX8 or PAX8-S48F) were rinsed twice in PBS and fixed for 20 min in 4% paraformaldehyde/PBS followed by permeabilization with 0.2% Triton X-100/PBS for 5 min. After washing with PBS, cells were blocked in 3% BSA/1% goat serum/PBS. Antibody incubations were performed with a monoclonal anti-TTF-1 antibody (clone 8G7G3/1; NeoMarkers/Lab Vision Corp., Fremont, CA) at 1:1,000, followed by Alexa Fluor 568-conjugated antimouse IgG (Molecular Probes, Eugene, OR). The DNA was counterstained with Hoechst 33342 fluorochrome (Molecular Probes). After rinsing, slides were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Microscopic images were taken on a Nikon Eclipse E800 microscope (Nikon, Melville, NY).
Cell Culture and in Vitro Functional Assays
HEK293 and HeLa cells were maintained in DMEM supplemented with 2 mM L-glutamine, 4.5 g/liter D-glucose, 50 U/ml penicillin, 50 µg/ml streptomycin, and 10% fetal bovine serum under humidified 5% CO2/95% air at 37 C.
FRTL-5 cells were grown in Hams F12K medium containing 2 mM L-glutamine (ATCC, Manassas, VA) adjusted to contain 1.5 g/liter sodium bicarbonate and supplemented with 5% fetal bovine serum and a six-hormone mixture (6H) comprising bovine TSH (10 mU/ml), insulin (10 µg/ml), hydrocortisone (10 nM), transferrin (5 µg/ml), somatostatin (10 ng/ml), and glycyl-L-histidyl-L-lysine acetate (10 ng/ml) (all from Sigma, St. Louis, MO).
For reporter assays, HEK293 or HeLa cells, grown in 12-well plates to 7080% confluence, were cotransfected (i.e.
5 x 105 cells per well) with 0.5 µg reporter plasmid, 4 ng pRL-Tk internal control vector (Promega), and various doses of effector plasmids/empty vector using FuGENE 6 reagent (Roche Applied Science). FRTL-5 cells were transfected for 9 h in 2% calf serum/OptiMEM-1 (Invitrogen) with 0.9 µg reporter, 0.1 µg pRL-Tk, and a total of 0.64 µg of the various effector plasmids using Lipofectamine 2000 (Invitrogen). Cells were harvested 3648 h later and analyzed sequentially for firefly and Renilla luciferase activities (Dual-Luciferase Reporter Assay System, Promega). In some experiments, 0.4 µM trichostatin A (3.3 mM stock in dimethylsulfoxide; Sigma) or 100 µM anacardic acid (100 mM stock in dimethylsulfoxide; ALEXIS Biochemicals/Axxora, San Diego, CA) was added 24 h post transfection, and the cells were harvested after an additional 12 h.
The ratios between the measured firefly and Renilla luciferase activities were expressed relative to the ratios obtained in cells transfected with reporter and empty expression vector (pcDNA3) only. Unless indicated otherwise, the means ± 1 SD of three independent experiments performed in triplicate transfections are given. To calculate the RI (also called cooperative index) (17, 37) from the average activities normalized as above, the specific (i.e. factor attributable) activities (
basal) were first calculated by subtraction of the normalized basal reporter activity (= 1). For two given factors, A and B, the RI was then calculated by dividing the specific activity obtained by coexpression of A and B by the sum of the specific activities obtained by expression of factors A and B alone. A RI value greater than 1 indicates synergism of factor A with factor B.
Statistical Analysis
For statistical comparisons, the probability associated with Students paired t test was calculated, with significance at P < 0.05.
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ACKNOWLEDGMENTS
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We thank all family members for their participation in this study; Graeme Bell for sharing laboratory equipment; Leslie De Groot for providing FRTL-5 cells; Fredric Wondisford for providing the p300 plasmid; Parvis Minoo for providing the 12A2 clone of TTF-1 cDNA; Massimo Tonacchera for the gift of the PAX8-L62R expression vector; and Daniel Christophe for a gift of anti-PAX8 antibody.
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FOOTNOTES
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This work was supported in part by National Institutes of Health Grants DK17050, DK20595, and RR00055 (to S.R.) and grants from the Belgian "Fonds National de la Recherche Scientifique Médicale" and "Fondation Erasme" (to G.V.).
First Published Online February 17, 2005
Abbreviations: CH, Congenital hypothyroidism; EGFP, enhanced green fluorescent protein; HAT, histone acetyltransferase; RI, recruitment index; Tk, thymidine kinase; TG, thyroglobulin; TPO, thyroperoxidase; TTF-1, thyroid transcription factor 1; WT, wild type
Received for publication October 20, 2004.
Accepted for publication February 10, 2005.
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