The Synergistic Activity of Thyroid Transcription Factor 1 and Pax 8 Relies on the Promoter/Enhancer Interplay
Stefania Miccadei,
Rossana De Leo,
Enrico Zammarchi,
Pier Giorgio Natali and
Donato Civitareale
Laboratory of Immunology (S.M., R.D.L., P.G.N., D.C.), Regina Elena Cancer Institute, Rome 00158, Italy; Department of Pediatrics (R.D.L., E.Z.), Meyer Hospital, University of Florence, Florence 50132, Italy; and Institute of Neurobiology and Molecular Medicine (D.C.), Consiglio Nazionale delle Ricerche, Rome 00137, Italy
Address all correspondence and requests for reprints to: Dr. Donato Civitareale, Laboratory of Immunology, Regina Elena Cancer Institute, Via delle Messi dOro 156, Rome, Italy 00158. E-mail: dciv{at}yahoo.com.
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ABSTRACT
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The transcription factors, thyroid transcription factor 1 (TTF-1) and Pax 8, play a pivotal role in the transcriptional regulation of the thyroid differentiation marker genes and in the differentiation of the thyroid follicular cells. They have a very restricted tissue distribution, and the thyrocyte is the only cell type with the simultaneous expression of these factors. Here we show that TTF-1 and Pax 8 cooperatively activate their target genes and that their synergistic activity requires the cross-talk between enhancer and gene promoter. We have characterized the cis and trans requirements of the TTF1/Pax 8 synergistic activity on the thyroperoxidase gene. We show that their synergy is also important for thyroglobulin gene transcription.
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INTRODUCTION
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DIFFERENTIATED THYROID follicular cells are characterized by the ability to synthesize the thyroid hormones, the iodothyronines T3 and T4. They derive from the degradation of a very large precursor, thyroglobulin (Tg), that is iodinated, on tyrosine residues, by thyroperoxidase (TPO), a thyroid-specific enzyme (1). The iodinated Tg is stored in the lumen of the thyroid follicles and upon hormonal stimuli, mainly exerted by TSH, it is reabsorbed in the thyrocytes and degraded, and the final products, T3 and T4, are secreted in the bloodstream (2). Thus, Tg, TPO, and the TSH receptor as well as the sodium/iodide symporter, able to concentrate the iodide in the thyrocytes, are the differentiation markers of the thyroid follicular cells (3). The tissue-specific expression of these genes is regulated at the transcriptional level, and the studies on their transcriptional regulatory regions have shown that their thyroid-specific expression relies on the activity of a set of transcription factors (4). Among them a critical role is played by thyroid transcription factor 1 [TTF-1, also termed thyroid-specific enhancer-binding protein (T/EBP) and NKX 2.1 (5)] and Pax 8 (6). The former is a homeodomain containing transcription factor that binds to and activates all the known regulatory sequences of the thyroid-specific genes (4). Additionally, TTF-1 is expressed in lung and also during the embryonic life in the diencephalon (7, 8). In lung TTF-1 activates the transcription of the genes encoding the surfactant protein (SP) A, SP-B, SP-C, and the Clara secretory protein (9). TTF-1-/- mice die in utero and, in addition to brain malformations, show defects in the development of both thyroid and lung (10). Pax 8 binds the DNA via its paired domain, and in the thyrocytes it activates the expression of all the thyroid differentiation marker genes except the TSH receptor gene (11, 12, 13, 14, 15). Pax 8 is expressed in adult mouse thyrocytes and in some kidney cells (6). Null mice for Pax 8 gene have smaller thyroids with normal calcitonin-producing parafollicular C cells but no follicular cells (16). Therefore, in the absence of the T4-producing cells, these mice suffer severe hypothyroidism. Moreover, mutations in the Pax 8 gene have been identified in humans affected by congenital hypothyroidism (17). Hence, both TTF-1 and Pax 8 regulate the development and the maintenance of the thyroid differentiated state. Their expression is restricted to a very limited number of tissues, but their simultaneous expression is characteristic only of the thyroid follicular cells. Therefore, it has been proposed that thyroid-specific gene expression relies on the combined activity of these factors (18). Although this hypothesis is suggestive, no direct experimental evidence of molecular interaction of the two factors has been obtained thus far. To identify cooperative activity between TTF-1 and Pax 8, we have studied the transcriptional regulation of the TPO gene, which is controlled by both promoter and enhancer sequences. The rat TPO gene promoter has been extensively studied in its cis and trans interactions by Francis-Lang et al. (11), whereas Kikkawa et al. (19) have identified and characterized the TPO enhancer 5.5 kb upstream of the human gene. The architecture of both promoter and enhancer of the TPO gene is schematically reported in Fig. 2A
. TTF-1 binds three times to the TPO promoter and once to the TPO enhancer, whereas Pax 8 binds both regulatory elements once (20, 21). Interestingly, the Pax 8 binding site overlaps with the TTF-1 binding site on the TPO gene promoter and similarly on the TPO enhancer. It has been shown that Pax 8 and TTF-1 binding to the C site of the TPO gene promoter is mutually exclusive in vitro (20). In this study we show, for the first time, that the combined activity of both the TPO gene promoter and the TPO enhancer drives a strong synergic activity between TTF-1 and Pax 8. We have characterized the cis and trans requirements of this cooperation. Furthermore, we show that the synergy between TTF-1 and Pax 8 is also important for the Tg gene transcription. Similarly to the TPO gene, in the Tg gene transcription, the cooperative activity between TTF-1 and Pax 8 also requires the enhancer/promoter cross-talk.

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Figure 2. Titration Curves
Hela cells were transfected with the TPOEn-TPO-Luc plasmid. In all the reported points the values of SD were negligible. A, The titration curve of the Pax 8 or of TTF-1-encoding plasmid was performed transfecting 2, 4, 10, 20, and 50 ng of the corresponding expression vector as indicated. The values of the fold of activation are reported as well. B, The cooperation tests were performed at fixed concentrations of Pax 8 or of TTF-1-encoding plasmid, and titration experiments of the other expression vectors were performed using 2, 4, 10, and 20 ng as indicated. The CI values are reported as well.
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RESULTS
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To study the role of TTF-1 and Pax 8 in transcriptional trans-activation, we used the strong activity shown by these factors in nonthyroid HeLa cells. As shown previously (20) and in Fig. 1A
, the cotransfection of a plasmid expressing the reporter gene, under the control of the TPO promoter (TPO-Luc), together with the TTF-1-encoding plasmid, or with the Pax 8-encoding plasmid, results in an activation of reporter gene expression. The contemporary expression of TTF-1 and Pax 8 decreases the TPO gene promoter activity. Similarly, using the construct with the reporter gene under the transcriptional control of the TPO enhancer and of the simian virus 40 (SV40) promoter, TPOEn-SV40-Luc plasmid, the simultaneous presence of the two transcription factors does not increase the transcription of the luciferase gene better than they do when expressed alone (Fig. 1B
). Hence, TTF-1 and Pax 8 are not able to cooperate on both the TPO promoter and the TPO enhancer. Conversely, using the plasmid with the reporter gene under the control of both promoter and enhancer of the TPO gene (TPOEn-TPO-Luc), the combined activity of TTF-1 and Pax 8 dramatically increases luciferase gene transcription well over the sum of their activity when tested alone (Fig. 1C
). Hence, TTF-1 and Pax 8 do cooperate, and their synergy requires TPO enhancer/promoter interplay. To measure the cooperation between TTF-1 and Pax 8 we introduce the cooperative index (CI). To obtain CI, the fold of activation of the reporter gene expression, measured by the combined activity of TTF-1 and Pax 8, is divided by the sum of the fold of activation obtained when TTF-1 and Pax 8 are overexpressed alone. In the experiments shown in Fig. 1
, the CI of the TPO promoter or of the TPOEn-SV40 construct is 0.2 and 0.3, respectively, whereas the CI of the TPOEn-TPO construct is 4.0. It is worth mentioning that the experiments shown in Fig. 1C
have been performed at subsaturating concentrations of both Pax 8 and TTF-1, as shown in Fig. 2A
. In Fig. 2B
, we show how the CI changes in titration experiments of one transcription factor at constant concentration of the other factor.

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Figure 1. TTF-1 Cooperates with Pax 8
HeLa cells were transiently transfected with TPO-Luc plasmid (panel A), with TPOEn-SV40-Luc plasmid (panel B), and with TPOEn-TPO-Luc plasmid (panel C) and were cotransfected with the vectors encoding the indicated factors as described in Materials and Methods. For each panel, the relative luciferase activity of the cells transfected only with the control vector was normalized to 1 and the other activities were expressed relative to this. The value of fold activation is reported as well as the SDs deviations. The means of the raw luciferase/ß-galactosidase data for TPO-Luc, TPOEn-SV40-Luc, and TPOEn-TPO-Luc in the control experiments were 7,000/150; 40,000/150; and 10,000/200, respectively. We refer to the experiments reported in this figure as test of cooperation; thus the test of cooperation involves four transfection experiments: the control with the reporter vector only, the control plus Pax 8 expression, the control plus TTF-1 expression, and the control plus TTF-1 and Pax 8 expression. As reported above, the experiments shown in panel C were performed in HeLa cells; we have repeated the same cooperation tests in COS-7 and SAOS-2 cell lines and the CI values were 4.2 and 3.5, respectively.
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As stated above and shown schematically in Fig. 3A
, TTF-1 binds three times to the TPO promoter (A, B, and C sites) and once to the TPO enhancer. Similarly Pax 8 binds both promoter and enhancer of the TPO gene, and in both cases its binding site overlaps with a TTF-1 binding site. To determine the cis elements critical for the cooperative activity between TTF-1 and Pax 8, we have measured the CI of several mutants of the TPO gene promoter and the TPO enhancer (Fig. 3B
). The TPO promoter A, Bmm, Bmr, Em, and Pm mutants have been previously described (11, 20), and their binding features are illustrated in Fig. 3B
. TPO promoter mutants Pm and Em mostly affect the TTF-1 and Pax 8 synergy (Fig. 3B
). Both are centered on the C site of the TPO promoter. As mentioned above, this is where the TTF-1 and Pax 8 binding sites are largely overlapping, and it has been shown that their binding is mutually exclusive, at least in vitro. The Pm mutation slightly reduces TTF-1 binding and completely abolishes the binding of Pax 8; the Em mutation does not affect Pax 8 binding but prevents TTF-1 binding (11). The TPOEn-TPOPm-Luc plasmid has a CI of 1.1; hence the Pm mutation, affecting the Pax 8 binding, drastically reduces the TTF-1/Pax 8 synergy. The CI of the TPOEn-TPOEm-Luc plasmid is lowered to 1.5, demonstrating that TTF-1 binding to the C site of the TPO promoter is very important for its synergy with Pax 8. The other two TTF-1 binding sites, A and B within the TPO promoter, do not play a relevant role in the cooperation with Pax 8. Noticeably the TTF-1/Pax 8 synergy, although reduced, is still detectable in the SV40 enhancer-TPO-Luc construct. In this plasmid, luciferase gene expression is under the transcriptional control of the TPO gene promoter and of the SV40 enhancer. In a control experiment we have verified that the activity of the SV40 enhancer is not induced by TTF-1 or by Pax 8 (data not shown). The SV40 enhancer is well characterized, and it has been reported that it is regulated by Sp1, nuclear factor-
B, activator protein 1, Octamer, and transcriptional enhancer factors 1 and 2 (25, 26, 27). Therefore, one can imagine that a transcription factor or a transcription complex, expressed in HeLa cells and able to bind both the TPO and the SV40 enhancers, could mediate the TTF-1/Pax 8 synergy. According to Kikkawa et al. (19), the E1 site is the region of the TPO enhancer that binds a ubiquitous transcription factor (Fig. 3A
). However, as shown in Fig. 3B
, mutations in the E1 region, in the construct TPOEn.E1-TPO-Luc, do not affect the TTF-1/Pax 8 synergy.

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Figure 3. Characterization of the cis Elements Involved in the TTF-1/Pax 8 Synergy
A, Schematic representation of the cis/trans interactions on the TPO gene promoter (11 ) and on the TPO enhancer (19 ). The nuclear factors termed UF (ubiquitous factor) and Thy-sp (thyroid-specific) are unidentified activities interacting in the E1 and E4 region of the TPO enhancer, respectively. Thy-sp could be TTF-1 (19 ). B, Mutational analysis of the cis elements in the TPOEn-TPO-Luc construct involved in the TTF-1/Pax 8 synergy. Each construct has been transfected in HeLa cells to test its ability to support the TTF-1 and Pax 8 synergy in tests of cooperation. The structure of the reported constructs is shown as well as their CI. The SDs of the CI are less than or equal to ±0.3. As a control experiment, we have tested that both TTF-1 and Pax 8 do not activate the luciferase gene expression in the pGL3 control vector (Promega Corp.) where the reporter gene is under the transcriptional control of the SV40 promoter and of the SV40 enhancer (data not shown).
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The results in Fig. 3B
demonstrate two major points: the first is that TTF-1 and Pax 8 have to bind to the C site of the TPO gene promoter to cooperate, and the second is that as far as the TTF-1/Pax 8 synergy is concerned, the SV40 enhancer can take the place of the TPO enhancer.
To characterize the domains of TTF-1 and Pax 8 involved in this synergy, we have used deletion mutants of the two transcription factors. In Fig. 4
, A and B, experiments with the deletion mutants of Pax 8 and TTF-1, respectively, are shown. Mouse Pax 8 is characterized by the paired domain, located between the amino acids 9 and 136, and an activation domain at its carboxy terminus (6, 28). In Fig. 4A
, we show that the Pax 8 trans-activation domain is crucial for the synergy with TTF-1, although it can be replaced by the VP16 trans-activation domain. Thus, Pax 8 deleted of its activation domain, Pax 8
333457, does not cooperate with TTF-1 but VP16.Pax 8
333457 does. Further deletion of the Pax 8 sequences, up to amino acid 233, does not change its ability to cooperate with TTF-1. Deletion of the sequences in the Pax 8 DNA binding domain (VP16.Pax 8
87457 mutant) does not allow Pax 8 to bind DNA and therefore to cooperate with TTF-1. Two trans-activation domains have been characterized in TTF-1 and are separated by the homeodomain as depicted in Fig. 3B
(29). Deletion of the activation domain either at the amino terminus (TTF-1
3) or at the carboxy terminus (TTF-1
14) does not affect the ability of TTF-1 to induce the activity of the target gene promoters (29), as both TTF-1
3 and TTF-1
14 cooperate with Pax 8 (Fig. 4B
).

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Figure 4. Analysis of Pax 8 and TTF-1 Domains Involved in the Cooperation
Tests of cooperation were performed with the expression vectors encoding the wild-type and the indicated deletion mutants of Pax 8 (panel A) and of TTF-1 (panel B). The schematic representation of the expressed proteins is reported together with their CI. The gray box represents the DNA binding domains, the paired domain in Pax 8 and the homeodomain in TTF-1. The black box represents the VP16 activation domain. The SDs of the CI are less than or equal to ±0.3.
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In the TPOEn-TPO-Luc construct, reporter gene expression is under the control of both the minimal rat TPO gene promoter (11) and of two copies of the 250 bp of the human TPO enhancer (19). To confirm our results, demonstrating TTF-1/Pax 8 synergy, we have tested the construct where the reporter gene is under the control of the 6.3-kb human genomic fragment, which includes the TPO gene promoter and enhancer. Similarly, we have tested a construct with the reporter gene under the transcriptional control of both regulatory elements of the human Tg gene, promoter and enhancer, 1.4-kb Tg (30). The results show that with both constructs TTF-1 and Pax 8 activate the reporter gene transcription cooperatively (Fig. 5
). In a control experiment with the reporter gene under the control of the minimal rat Tg gene promoter (31), Tg-Luc, the CI was 0.7 (data not shown). Hence, synergy between the two thyroid-specific transcription factors is also important in the transcription of the Tg gene and, like TPO gene transcription, it requires the combined activity of enhancer and gene promoter.

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Figure 5. The TTF-1/Pax 8 Synergy Is Supported by the Constructs with the Genomic Regulative Sequence of the Human TPO Gene and of the Human Tg Gene
Tests of cooperation were performed with the 6.3-kb TPO and with the 1.4-kb Tg plasmids. For each panel, the relative activity of the reporter protein in the cells transfected only with the control vector was normalized to 1 and the other activities were expressed relative to this. The value of fold activation is reported as well as the SDs. The means of the raw CAT/ß-galactosidase data for 6.3-kb TPO and 1.4-kb Tg in the control experiments were 280/180 and 850/220, respectively.
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DISCUSSION
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In this study we have presented the first experimental evidence suggesting that TTF-1 and Pax 8 can cooperate in the induction of thyroid-specific gene expression. The biological question that this finding addresses is the regulation of tissue-specific gene expression in thyrocytes. It has been reported that cell type specification is ultimately mediated through the activity of cell type-specific or restricted transcription factors (32). Furthermore, cell type-specific gene expression is often regulated by the combined activity of several transcription factors with a restricted tissue distribution (33). A very interesting example came from the studies on the pituitary cell lineage determination. It is orchestrated by the combinatorial actions of a repertoire of transcription factors, which, through combined and overlapping expression and functional interactions, lead to the differentiation of distinct cell types (34). Pituitary-specific transcription factor 1 (Pit-1) plays a crucial role in pituitary cells determination, but the regulation of the cell-type-specific gene expression is achieved through its functional synergy with different partners. Pit-1/Ets-1 synergy is critical for lactotrope-specific gene expression whereas Pit-1/GATA-2 synergy is required for thyrotrope-specific gene expression (35, 36). The identification of the synergy between TTF-1 and Pax 8 fits well into this framework. Although many other regulatory events are required to achieve cell type specification in thyrocytes, the combinatorial activity of TTF-1 and Pax 8 can explain why the expression of the thyroid differentiation markers, Tg and TPO, is cell type restricted. As reported above, TTF-1 plays a crucial role in cell lineage determination in both cell types of the thyroid gland, the thyrocytes, and the C cells, as well as in the lung (4, 9, 37). It has been reported that in the lung TTF-1 cooperates with several transcription factors including cardiac muscle-specific homeobox protein, RAR, Sp1, and Sp3 (38, 39, 40). The same point can be made for Pax 8. It has been reported that during embryonic kidney development, Pax 8 cooperates with the transcription factor lim-1 (41). Thus, both TTF-1 and Pax 8 can induce cell type-specific gene expression depending on the cellular environment where they are expressed and on their ability to cooperate with other transcription factors. We suggest that the simultaneous expression of Pax 8 and TTF-1, which takes places only in the thyrocytes, triggers the synergistic activation of the thyroid differentiation marker genes.
A remarkable aspect of the TTF-1/Pax 8 synergy emerged from the experiments with the TPO gene promoter mutants (Fig. 3B
). We have shown that the A and B sites of the promoter are not relevant for the synergy and that, to cooperate, both thyroid factors must bind to the C site of the TPO gene promoter. This issue raises the question whether or not TTF-1 and Pax 8 interact directly. Because no data are available in the literature on this point, we envision a TTF-1/Pax 8 synergy entailing an indirect interaction where the two activators do not contact each other directly, but simultaneously bind to a third factor or to different sites within a single transcriptional complex.
A feature of the synergy between TTF-1 and Pax 8 is that it relies on the enhancer/promoter interplay. Although this phenomenon has been studied more extensively in Drosophila cells (42), several examples have also been reported in vertebrates. The vast majority concerns the regulation of tissue-specific gene expression. The synergy between Ig enhancers and promoters (43) has been described, as well as the promoter/enhancer cooperation in the muscle-specific desmin gene (44), in the liver-specific albumin gene (45), and in the globin genes in B cells (46). Interestingly, our results show that, similarly to the TPO enhancer (TPOEn), the SV40 enhancer can induce TTF-1/Pax 8 synergy. Even though the SV40 enhancer is able to implement TTF-1/Pax 8 synergy, the CI value difference between the TPOEn-TPO-Luc construct and the SV40-TPO-Luc plasmid suggests a specific cross-talk between the TPO promoter and the TPO enhancer.
It is worth noting that, in some selected cases, the involvement of the transcriptional coactivator cAMP response element binding protein-binding protein (CBP)/p300 as mediator of the activity of distal enhancers has been demonstrated. The synergistic activation of transcription on the interferon
gene requires the formation of the enhanceosome and the recruitment of CBP/p300 (47). A similar role for CBP has been described in enhancer/promoter interplay of the T cell receptor ß-chain gene (48). We have recently reported the requirement of p300 for Pax 8 activation of the TPO promoter and we have provided evidence for the formation of a complex between the two factors (22). In addition, it has been reported that TTF-1 cooperates with CBP in activation of the SP-B gene expression in type II epithelial cells of the lung (49). Thus, both TTF-1 and Pax 8 can utilize CBP/p300 as coactivator. Hence, it is likely to envisage that the parallel interaction of TTF-1 and Pax 8 with p300 could be an important feature of their synergy.
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MATERIALS AND METHODS
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Plasmids and Cell Transfection
The TPO-Luc and TPOEn-SV40-Luc plasmids, also termed TPO-pGL3 and p2XEn-WT, respectively, have been previously described (21, 22). The TPOEn-TPO-Luc plasmid has been constructed inserting the SacIPvuII fragment from p420TPOL (11) in TPOEn-SV40-Luc (21) digested with SacI and NcoI; the NcoI site was blunted with Klenow enzyme. Similarly, the TPO promoter mutants, described in Ref. 11 , were excised with SacI and PvuII and inserted in the TPOEn-SV40-Luc plasmid digested with SacI and NcoI; the NcoI site was blunted with Klenow enzyme. The plasmids with the TPO promoter mutants were kindly provided by R. Di Lauro (Naples, Italy). We have constructed the TPO.A-luc plasmid with the A mutation in the TPO promoter (11), inserting in the pGL3 vector (Promega Corp., Madison, WI), digested with SacI and with XhoI; the PCR fragment was obtained according to the protocol published by Ho et al. (23). The oligos used to obtain the two fragments of the mutated TPO promoter were: oligo A 5'-caaaggtgccacgacctgaaagcattcttggctgcctg-3' with oligo B 5'-gttctcgagctcggcgggaaaggt-3' and oligo C 5'-gaatgctttcaggtcgtggcacctttgttctgaccagcc-3' with oligo D 5'-gtgaatctcgagtactttctggagacttggttacccaccatataaatggactccatgc-3'. The TPO-Luc plasmid was used as template. The entire mutated promoter was obtained as a PCR fragment with the oligos A and D. As control of the TPO.A-Luc construct we have constructed the TPO.WT-luc plasmid where the luciferase expression is under the transcriptional control of the wild-type rat TPO gene promoter. This was obtained as a PCR fragment using the oligos A and D with the TPO-Luc plasmid as template. It was digested with SacI and XhoI and ligated in the pGL3 vector restricted with the same enzymes. We have controlled that the TPO promoter in the TPO.WT-Luc plasmid is activated, in transfection experiments in HeLa cells, by TTF-1 and/or Pax 8 similarly to the TPO promoter in the TPO-Luc plasmid. We have obtained the TPOEn-TPO.A and the TPOEn-TPO.WT plasmids inserting the fragment SalIBamHI from the TPOEn-TPO-Luc vector in the TPO.A-Luc and TPO.WT-Luc plasmids, respectively. We have controlled that the CI, described in Results, of the new construct TPOEn-TPO.WT-Luc is 4.0, similar to the TPOEn-TPO-Luc plasmid. To construct the TPOEn-TPO.A-Bmm vector, we have first constructed the TPO.A-Bmm plasmid. It was obtained by inserting the fragment SacIBanI, from the TPO.A-Luc, and the fragment BanI-HindIII, from the TPO.Bmm-Luc plasmid, into the TPO-Luc vector digested with SacI and HindIII. The fragment SacIXbaI from TPO.A-Bmm was ligated to the fragment SacIXbaI from the TPO En-TPO-Luc plasmid, resulting in the TPOEn-TPO.A-Bmm construct. The TPOEn.PstI-TPO-Luc plasmid was obtained by digesting the TPOEn.TPO-Luc plasmid with PstI and religating the deleted vector. The TPO enhancer mutated in the E1 site was obtained with the following procedure. The oligo E 5'-tgaaagtcgacgggattccatcttttattctaatgcatacttgatctggttgtattctgaatcctccttacaggaatgtgatgtggactgacacgt-3' was annealed with the oligo F 5'-ctgctctatgaagtgtgaagaatccctggttccattcaagagccttctccg-tgatacgcgtagacgtgtcagtccacatc-3' and filled in with Klenow enzyme. This fragment was digested with SalI and ligated in the TPOEn-TPO-Luc vector digested with PvuII and SalI, resulting in the TPOEn.E1*-TPO-Luc vector. The fragment PvuI-SmaI from this vector was ligated to the same plasmid digested with PvuI and SalI, with the SalI end blunted with Klenow enzyme, resulting in the TPOEn.E1-TPO-Luc plasmid. Using the same procedure we have constructed the TPOEn.E2+3-TPO-Luc vector; in this case the oligo E was annealed with the oligo G 5'-ctgagagcgtcctgtgtccatatgtagtagtccattcaagagccttctagtagtttccaaattacgtgtcagtccacatc-3'. The resulting double-stranded oligo EG was filled in with Klenow, digested with SalI, and cloned in the TPOEn-TPO-Luc vector digested with PvuII and SalI, resulting in the TPOEn.E2+3*-TPO-Luc vector. The fragment PvuISmaI from this vector was ligated to the same plasmid digested with PvuI and SalI, with the SalI end blunted with Klenow enzyme, resulting in the TPOEn.E2+3-TPO-Luc plasmid. The SV40-TPO-Luc plasmid was constructed inserting the fragment SacISphI from TPO-Luc in the pGL3 Control vector (Promega Corp.) digested with the same enzymes. The Pax 8
333457 mutant was obtained by removing the DNA fragment of about 300 nucleotides, PstIXbaI, from the Pax 8 expression vector (20), Both termini were blunted with T4 DNA polymerase and ligated with T4 DNA ligase. The VP16.Pax 8 construct was obtained by inserting the fragments EcoRIXhoI and XhoIBamHI from pBlu-Pax 8 (21) in the pV16 plasmid (CLONTECH Laboratories, Inc., Palo Alto, CA) digested with EcoRI and BamHI. The vector encoding for the VP16.Pax 8
333457 was obtained by digesting the VP16.Pax 8 construct with PstI, removing the 300-bp fragment, and ligating the two other fragments. The vector encoding for the VP16.Pax 8
233457 was obtained by digesting the VP16.Pax 8 construct with XhoI and XbaI, after which the termini were filled in with Klenow enzyme and ligated. The vector encoding for the VP16.Pax 8
162457 was obtained by digesting the VP16.Pax 8 construct with PvuII and XbaI, after which the XbaI terminus was blunted with Klenow enzyme and the termini were blunt ligated. The vector encoding for the VP16.Pax 8
87457 was obtained by digesting the VP16.Pax 8 construct with BstXI and XbaI, after which the termini were blunted with T4 DNA Polymerase and ligated. The vectors encoding the TTF-1 deletion mutants
3 and
14 (29) were kindly provided by R. Di Lauro, Naples, Italy. The TTF-1 HD expression vector was obtained by inserting the HindIIIPstI fragment from the pTTF-1
3 plasmid (the PstI terminus was blunted with T4 DNA Polymerase), into pcDNA 3 (Invitrogen, San Diego, CA) digested with HindIII and EcoRV. The 6.3-kb TPO and the 1.4-kb Tg plasmids were a generous gift from S. Kimura (Bethesda, MD) and D. Christophe (Brussels, Belgium), respectively. All the new constructs were controlled by restriction analysis and/or by direct sequencing using the T7 Sequencing kit (Pharmacia Biotech, Piscataway, NJ).
HeLa cells were transfected using Effectene reagent (QIAGEN, Chatsworth, CA) following the protocol suggested by the manufacturer. The following amount of the indicated plasmids were transfected: 0.4 µg of the gene reporter plasmids, 4 ng of the Pax 8, wild type or mutant, expressing vector; 2 ng of TTF-1, wild-type or mutant, expressing vector. The efficiency of transfection was assayed with 10 ng of pCMV-ß-galactosidase plasmid, and 48 h after the transfections cell extracts were prepared. The luciferase activity was measured according to the Luciferase Assay System (Promega Corp.), and ß-galactosidase assay was performed with the chlorophenol red-ß-galactopyranoside substrate as previously reported (24). The chloramphenicol acetyltransferase (CAT) assays, in the extracts of cells transfected with the 6.3-kb TPO and 1.4-kb Tg plasmids, were performed with the CAT kit (Roche, Indianapolis, IN). The values obtained for luciferase or CAT assays were corrected for transfection efficiency with the ß-galactosidase assay. Transfection experiments were done in duplicate or in triplicate and repeated at least three times. For each experiment we report the mean of three independent experiments, and the SDs are shown. The statistical analysis, performed utilizing the raw data, of all transfection experiments resulted in P < 0.05. For each experiment, the t test (the probability associated with the t test) was calculated vs. the respective control group.
Band Shift and Western Blot
Nuclear extracts from transfected cells were prepared according to Suzuki et al. (37), and the band-shift assays were performed as previously described (13) using the double-strand oligonucleotide C with both the TTF-1 and Pax 8 binding sites from the Tg promoter (Ref. 31 and Fig. 6
). The same nuclear extracts were boiled in Laemli buffer and resolved in 10% SDS-PAGE. After electroblotting onto nitrocellulose membrane, VP16-Pax 8 fusion proteins were localized with a specific VP16 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) using an ECL kit (Amersham Pharmacia Biotech, Arlington Heights, IL; Fig. 6
).

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Figure 6. Transfection Data and Controls
In panels A, B, and C are reported the values of fold of activation obtained in the tests of cooperation that have been used to calculate the CIs presented in Figs. 3B and 4, A and B , respectively. In panel D are reported the band-shift experiments, lanes 110, and the Western blot experiments, lanes 1114, performed to control the expression level of Pax 8 and TTF-1, wild-type and mutants, exogenously expressed in the transfection experiments presented in this study as follows. Lanes 1 and 10, With nuclear extract from HeLa cells transfected with the plasmid encoding Pax 8 wild type. Lanes 2 and 5, With nuclear extract from HeLa cells transfected with the plasmid encoding TTF-1 wild type. Lane 3, Nuclear extract from HeLa cells expressing both Pax 8 wild type and TTF-1 wild type. Lane 4, Nuclear proteins extracted from HeLa cells transfected with the empty expression vector. Lanes 6, 7, and 8, With proteins from HeLa cells transfected with the vector encoding TTF-1 3, TTF-1 14, and TTF-1 HD, respectively. Lanes 9, 11, 12, 13, and 14, With nuclear proteins from HeLa cells transfected with the vector encoding Pax 8 333457, VP16.Pax 8 333457, VP16.Pax 8 233-457, VP16.Pax 8 162-457, and VP16.Pax 8 87-547, respectively.
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ACKNOWLEDGMENTS
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We thank Helen Francis-Lang, Gennaro Ciliberto, and Thomas Wagner for critical reading of the manuscript. We sincerely thank our colleagues for providing us with critical reagents. Their names appear in Materials and Methods.
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FOOTNOTES
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The authors dedicate this work to the memory of Franco Tatò.
Abbreviations: CAT, Chloramphenicol acetyltransferase; CBP, cAMP response element binding protein-binding protein; CI, cooperative index; Pit-1, pituitary-specific transcription factor 1; SP, surfactant protein; SV40, simian virus 40; Tg, thyroglobulin; TPO, thyroperoxidase; TPOEn, TPO enhancer; TTF-1, thyroid transcription factor 1.
Received for publication August 27, 2001.
Accepted for publication December 6, 2001.
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