Redox Effector Factor-1 Regulates the Activity of Thyroid Transcription Factor 1 by Controlling the Redox State of the N Transcriptional Activation Domain
2002; Elsevier BV; Volume: 277; Issue: 17 Linguagem: Inglês
10.1074/jbc.m200582200
ISSN1083-351X
AutoresGianluca Tell, Alex Pines, Igor Paron, Angela D’Elia, Alessia Bisca, Mark R. Kelley, Giorgio Manzini, Giuseppe Damante,
Tópico(s)Animal Genetics and Reproduction
ResumoThyroid transcription factor 1 (TTF-1) is a homeodomain-containing transcriptional regulator responsible for the activation of thyroid- and lung-specific genes. It has been demonstrated that its DNA binding activity is redox-regulated in vitro through the formation of dimers and oligomeric species. In this paper, we demonstrate that the redox regulation mainly involves a Cys residue (Cys87), which resides out of the DNA binding domain, belonging to the N-transactivation domain. In fact, the oxidized form of a truncated TTF-1 (containing the N-transactivation domain and the DNA-binding domain, here called TTF-1N-HD) looses specific DNA binding activity. Since most of the oxidized TTF-1N-HD is in a monomeric form, these data indicate that the redox state of Cys87 may control the DNA-binding function of the homeodomain, suggesting that Cys87 could play an important role in determining the correct folding of the homeodomain. By using gel retardation and transient transfection assays, we demonstrate that the redox effector factor-1 (Ref-1) mediates the redox effects on TTF-1N-HD binding and that it is able to modulate the TTF-1 transcriptional activity. Glutathione S-transferase pull-down experiments demonstrate the occurrence of interaction between Ref-1 and TTF-1N-HD. Having previously demonstrated that Ref-1 is able to modulate the transcriptional activity of another thyroid-specific transcription factor (Pax-8), our data suggest that Ref-1 plays a central role in the regulation of thyroid cells. Thyroid transcription factor 1 (TTF-1) is a homeodomain-containing transcriptional regulator responsible for the activation of thyroid- and lung-specific genes. It has been demonstrated that its DNA binding activity is redox-regulated in vitro through the formation of dimers and oligomeric species. In this paper, we demonstrate that the redox regulation mainly involves a Cys residue (Cys87), which resides out of the DNA binding domain, belonging to the N-transactivation domain. In fact, the oxidized form of a truncated TTF-1 (containing the N-transactivation domain and the DNA-binding domain, here called TTF-1N-HD) looses specific DNA binding activity. Since most of the oxidized TTF-1N-HD is in a monomeric form, these data indicate that the redox state of Cys87 may control the DNA-binding function of the homeodomain, suggesting that Cys87 could play an important role in determining the correct folding of the homeodomain. By using gel retardation and transient transfection assays, we demonstrate that the redox effector factor-1 (Ref-1) mediates the redox effects on TTF-1N-HD binding and that it is able to modulate the TTF-1 transcriptional activity. Glutathione S-transferase pull-down experiments demonstrate the occurrence of interaction between Ref-1 and TTF-1N-HD. Having previously demonstrated that Ref-1 is able to modulate the transcriptional activity of another thyroid-specific transcription factor (Pax-8), our data suggest that Ref-1 plays a central role in the regulation of thyroid cells. Thyroid transcription factor 1 (TTF-1, 1The abbreviations used are: TTF-1thyroid transcription factor 1TgthyroglobulinTSHthyrotropinRef-1redox effector factor 1rRef-1recombinant Ref-1EMSAelectrophoretic mobility shift assayHDhomeodomainTFtranscription factorTADtransactivation domainHIFhypoxia-inducible factorCMVcytomegalovirusCATchloramphenicol acetyltransferaseGSTglutathione S-transferaseDTTdithiothreitol1The abbreviations used are: TTF-1thyroid transcription factor 1TgthyroglobulinTSHthyrotropinRef-1redox effector factor 1rRef-1recombinant Ref-1EMSAelectrophoretic mobility shift assayHDhomeodomainTFtranscription factorTADtransactivation domainHIFhypoxia-inducible factorCMVcytomegalovirusCATchloramphenicol acetyltransferaseGSTglutathione S-transferaseDTTdithiothreitol also named Nkx2.1) is a tissue-specific transcription factor (TF) that controls the expression of some thyroid- and lung-specific genes (1.Damante G. Tell G. Di Lauro R. Prog. Nucleic Acids Res. 2000; 66: 307-356Crossref Google Scholar). However, it is not well ascertained how TTF-1 is able to activate the transcription of thyroid-specific genes (such as those of thyroglobulin and thyroperoxidase) only in the follicular thyroid cell (2.Francis-Lang H. Price M. Polycarpou-Schwarz M. Di Lauro R. Mol. Cell. Biol. 1992; 12: 576-588Crossref PubMed Scopus (208) Google Scholar) and the transcription of lung-specific genes (such as those encoding for surfactant proteins) exclusively in epithelial lung cells (3.Bruno M.D. Bohinski R.J. Huelsman K.M. Whitsett J.A. Korfhagen T.R. J. Biol. Chem. 1995; 270: 6531-6536Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Thus, unknown regulatory mechanisms must control the TTF-1 transcriptional function. TTF-1, similarly to the large majority of eukaryotic promoter-specific TFs, displays a modular nature. In fact, the two basic molecular functions (i.e. specific DNA binding and transcriptional activation) are performed by distinct domains. The DNA-binding function is brought on by the homeodomain (HD) that recognizes, with high affinity, DNA sequences containing the 5′-CAAG-3′ core motif (4.Damante G. Fabbro D. Pellizzari L. Civitareale D. Guazzi S. Polycarpou-Schwarz M. Cauci S. Quadrifoglio F. Formisano S. Di Lauro R. Nucleic Acids Res. 1994; 22: 3075-3083Crossref PubMed Scopus (101) Google Scholar). Moreover, TTF-1 exhibits two independent transcriptional activation domains, located at the N-terminal (N domain) and at the C-terminal (C domain) regions with respect to the HD (Fig. 1 and Ref. 5.De Felice M. Damante G. Zannini M. Francis-Lang H. Di Lauro R. J. Biol. Chem. 1995; 270: 26649-26656Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). It has been previously demonstrated that the N domain plays a leading role in the activation of the transcriptional machinery, being able to squelch both its own transcriptional activity and the C domain one (5.De Felice M. Damante G. Zannini M. Francis-Lang H. Di Lauro R. J. Biol. Chem. 1995; 270: 26649-26656Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Moreover, the N domain directly interacts with the TATA-binding protein TBP (6.Tell G. Perrone L. Fabbro D. Pellizzari L. Pucillo C. De Felice M. Acquaviva R. Formisano S. Damante G. Biochem. J. 1998; 329: 395-403Crossref PubMed Scopus (32) Google Scholar). Therefore, regulatory mechanisms, acting upon the N domain, could control the activity of the whole molecule. thyroid transcription factor 1 thyroglobulin thyrotropin redox effector factor 1 recombinant Ref-1 electrophoretic mobility shift assay homeodomain transcription factor transactivation domain hypoxia-inducible factor cytomegalovirus chloramphenicol acetyltransferase glutathione S-transferase dithiothreitol thyroid transcription factor 1 thyroglobulin thyrotropin redox effector factor 1 recombinant Ref-1 electrophoretic mobility shift assay homeodomain transcription factor transactivation domain hypoxia-inducible factor cytomegalovirus chloramphenicol acetyltransferase glutathione S-transferase dithiothreitol The modulation of a TF activity is achieved by different means: (i) at a translational level, through the regulation of the expression of the TF itself, and (ii) at a post-translational level, through modifications such as phosphorylation and glycosylation, occurring at specific residues of the TF. During the last few years, another kind of post-translational regulation has become increasingly evident (i.e. redox regulation). This control is exerted through the modulation of the oxidation/reduction state of the thiol groups of cysteine residues usually present in the DNA-binding domain of TFs themselves. It has been previously demonstrated that the DNA binding activity and the dimerization ability of TTF-1 are redox-regulatedin vitro (7.Arnone M.I. Zannini M. Di Lauro R. J. Biol. Chem. 1995; 270: 12048-12055Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). It was shown that a reducing environment is required, in vitro, for a proper DNA-binding activity and that oxidation promotes (i) the formation of disulfide bond(s) between two specific cysteine residues (87 and 363) located outside the homeodomain and (ii) the formation of higher order oligomers of the protein itself. Ref-1 has been identified as a protein capable both of apurinic/apyrimidinic endonuclease DNA repair activity and nuclear redox activity, being able to induce the DNA binding activity of AP-1, NF-κB, Myb, members of the ATF/cAMP-response element-binding protein family, hypoxia-inducible factor (HIF-1α) (8.Xanthoudakis S. Miao G. Wang F. Pan Y.C. Curran T. EMBO J. 1992; 11: 3323-3335Crossref PubMed Scopus (815) Google Scholar), and Pax proteins (9.Tell G. Scaloni A. Pellizzari L. Formisano S. Pucillo C. Damante G. J. Biol. Chem. 1998; 273: 25062-25072Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Moreover, recent developments have pointed out a primary role for Ref-1 in pathways of activation of p53, through redox mechanism (10.Jayaraman L. Murthy K.G.K. Zhu C. Curran T. Xanthoudakis S. Prives C. Genes Dev. 1997; 11: 558-570Crossref PubMed Scopus (440) Google Scholar), together with a direct interaction with p53 itself in vivo(11.Meira L.B. Cheo D.L. Hammer R.E. Burns D.K. Reis A. Friedberg E.C. Nat. Genet. 1997; 17: 145Crossref PubMed Scopus (53) Google Scholar). As for its apurinic/apyrimidinic endonuclease activity, Ref-1 is better known by the acronym APE, which accounts for the role it plays in repairing of DNA damage. This process is due to reactive oxygen species, such as superoxide anion (O2⨪), H2O2, and the hydroxyl radical (⋅OH), which are by-products of respiration. Ref-1 protein expression is selectively induced by nontoxic levels of a reactive oxygen species variety. This is thought to be due to a translational induction, being inhibited by treating cells with cycloheximide (12.Ramana C.V. Boldogh I. Izumi T. Mitra S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5061-5066Crossref PubMed Scopus (375) Google Scholar). Moreover, we have recently demonstrated that reactive oxygen species are able to induce Ref-1 nuclear translocation in B-cells (13.Tell G. Zecca A. Pellizzari L. Spessotto P. Colombatti A. Kelley M.R. Damante G. Pucillo C. Nucleic Acids Res. 2000; 28: 1099-1105Crossref PubMed Scopus (99) Google Scholar) as well as in thyroid cells (14.Tell G. Pellizzari L. Pucillo C. Puglisi F. Cesselli D. Kelley M.R. Di Loreto C. Damante G. J. Mol. Endocrinol. 2000; 24: 383-390Crossref PubMed Scopus (57) Google Scholar). It is largely known that, in thyroid cells, the production of reactive oxygen species occurs after thyrotropin (TSH) stimulation and plays a key role during thyroid hormone synthesis (15.Kimura T. Okajima F. Sho K. Kobayashi I. Kondo Y. Endocrinology. 1995; 136: 116-123Crossref PubMed Scopus (58) Google Scholar, 16.Bjorkman U. Ekholm R. Endocrinology. 1992; 130: 393-399Crossref PubMed Scopus (66) Google Scholar, 17.Kimura T. Okajima F. Kikuchi T. Kuwabara A. Tomura H. Sho K. Kobayashi I. Kondo Y. Am. J. Physiol. 1997; 273: E639-E643PubMed Google Scholar, 18.Leseney A.M. Deme D. Legue O. Ohayon R. Chanson P. Sales J.P. Pires de Carvalho D. Dupuy C. Virion A. Biochimie (Paris). 1999; 81: 373-380Crossref PubMed Scopus (55) Google Scholar). We have concordantly demonstrated that cytoplasm to nucleus translocation of Ref-1 occurs in thyroid cells upon TSH stimulation. These findings suggest that the Ref-1-mediated mechanism may constitute a major switch by which TSH controls thyroid cells. This view is supported by the observation that Ref-1 controls the DNA-binding function of Pax-8, which is another thyroid-specific TF. Pax-8 recognizes the DNA by means of a conserved DNA-binding domain called the paired domain. A conserved Cys residue at position 37 of the Pax-8 paired domain is responsible for the redox regulation of the DNA binding activity (9.Tell G. Scaloni A. Pellizzari L. Formisano S. Pucillo C. Damante G. J. Biol. Chem. 1998; 273: 25062-25072Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The Cys37 residue is required to be in a reduced state in order to allow the structural transition of the Prd domain required for a proper DNA binding. This feature is accomplished, in vivo, through Ref-1, which is able to induce the Pax-8-driven transactivation potential of the thyroid-specific thyroglobulin promoter, as we have demonstrated by co-transfection assays in HeLa cells (19.Tell G. Pellizzari L. Cimarosti D. Pucillo C. Damante G. Biochem. Biophys. Res. Commun. 1998; 252: 178-183Crossref PubMed Scopus (84) Google Scholar). In this paper, we show that the redox control of DNA binding activity demonstrated for TTF-1 (7.Arnone M.I. Zannini M. Di Lauro R. J. Biol. Chem. 1995; 270: 12048-12055Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) is exerted by a unique Cys residue (Cys87), which represents the “redox sensor” of the molecule. Differently from Pax-8 and other TF studied up to now, this Cys residue, involved in the redox control, resides outside the DNA binding domain, mapping in the N transcriptional activation domain of TTF-1. Moreover, the redox regulation demonstrated in vitrois performed, in vivo, by Ref-1. Together with those previously reported by us and other authors (20.Kambe F. Nomura Y. Okamoto T. Seo H. Mol. Endocrinol. 1996; 10: 801-812PubMed Google Scholar), our data suggest a master role for Ref-1 in the control of the thyroid cell physiology. Oligodeoxynucleotides were synthesized with an automated Applied Biosystems DNA synthesizer, model 380B, according to standard procedures and purified by fast protein liquid chromatography using a Mono-Q column (Amersham Biosciences) eluted with an ammonium bicarbonate gradient. The purity of oligonucleotides was controlled on a 20% polyacrylamide, 7 m urea gel electrophoresis. DNA encoding for recombinant TTF-1 N-domain protein was obtained by PCR using the primer TH1 (5′-GGCGCGGATCCATGTCGATGAGTCCAAAGCACACG-3′) and primer TH2 (5′-CCGCGGGATCCCTTGTCCTTCGCCTGGCGCTTCAT-3′) and, as template, the plasmid CMV-TTF1 (5.De Felice M. Damante G. Zannini M. Francis-Lang H. Di Lauro R. J. Biol. Chem. 1995; 270: 26649-26656Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The PCR product was BamHI-digested and cloned into the bacterial expression vector pQE12 (Qiagen). Plasmid pTACAT3 contains the wild-type Tg promoter linked to the chloramphenicol acetyltransferase (CAT) gene, and it is described in Ref. 21.Sinclair A.J. Lonigro R. Civitareale D. Di Lauro R. Eur. J. Biochem. 1990; 193: 311-318Crossref PubMed Scopus (95) Google Scholar. Plasmid with the promoter C5E1b as well as plasmids expressing proteins TTF-1 and Δ14 were described elsewhere (5.De Felice M. Damante G. Zannini M. Francis-Lang H. Di Lauro R. J. Biol. Chem. 1995; 270: 26649-26656Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Plasmids CMV-CAT, RSV-CAT, and Ki-Ras-CAT containing the CMV, RSV, and Ki-Ras promoters linked to the CAT gene (19.Tell G. Pellizzari L. Cimarosti D. Pucillo C. Damante G. Biochem. Biophys. Res. Commun. 1998; 252: 178-183Crossref PubMed Scopus (84) Google Scholar, 22.Sassone-Corsi P. Verma I.M. Nature. 1987; 326: 507-510Crossref PubMed Scopus (71) Google Scholar, 23.Hoffman E.K. Trusko S.P. Freeman N. George D.L. Mol. Cell. Biol. 1987; 7: 2592-2596Crossref PubMed Scopus (56) Google Scholar) together with the plasmid PGL-2 (Promega), containing the promoter and enhancer sequences of SV40 linked to the luciferase gene (LUC), were used in cotransfection studies with Ref-1-expressing plasmid. The mutant C87S of the Δ14 construct (C87SΔ14) and of the recombinant TTF-1N-HD cloned in pQE12 plasmid were created by the QuickChange site-directed mutagenesis kit (Stratagene), using as template the Δ14 or the pQE12TTF-1N-HD constructs and the following oligonucleotides: C87Sa (5′-GCC GTG GGG GGC TAC TCT AAC GGC AAC CTG GGC-3′) and C87Sb (5′-GCC CAG GTT GCC GTT AGA GTA GCC CCC CAC GGC-3′). The introduced mutation was verified by nucleotide sequencing of the entire constructs. Recombinant Ref-1 His-tagged expressing plasmid pDS56Ref-1 was kindly provided by Dr. T. Curran (St. Jude Children's Research Hospital, Memphis, TN) together with the eukaryotic expression vector CMV-Ref-1. The cDNA coding for amino acids 1–230 of rat TTF-1 was cloned into the vector pQE12 (Qiagen) in frame with the coding region of six histidine residues. Thus, the expressed protein (TTF-1N-HD) contains an extra hexahistidine sequence at the C terminus, allowing for protein purification by nickel/nitrilotriacetic acid affinity chromatography. The TTF-1N-HD wild type protein (named TTF-1N-HDWT) and its C87S mutant (named TTF-1N-HDC87S) were expressed in M15 Escherichia coli cells. Overnight cultures were inoculated into Luria-Bertani (LB) medium supplemented with 50 μg/ml Ampicillin and grown at 37 °C toA 600 0.6–0.7, and then they were induced by 1 mm isopropyl-1-thio-β-d-galactopyranoside for 3 h. At the end of the induction phase, bacteria expressing recombinant TTF-1N-HDWT or TTF-1N-HDC87S proteins were pelleted and resuspended in 10 ml of lysis buffer A (20 mm Tris, pH 8.0, 250 mm NaCl, 0.1% Tween 20, 1 mmβ-mercaptoethanol, 0.1 mm phenylmethylsulfonyl fluoride, 0.8 mm imidazol) for each gram of bacterial pellet and centrifuged at 10,000 × g for 20 min at 10 °C. The supernatants were loaded onto a nickel/nitrilotriacetic acid column, equilibrated with buffer A, and washed with 10 volumes of buffer A. The protein was eluted with buffer B (20 mm Tris, pH 8.0, 250 mm NaCl, 0.1% Tween 20, 1 mmβ-mercaptoethanol, 0.1 mm phenylmethylsulfonyl fluoride, 500 mm imidazol). Sample concentrations were determined either spectrophotometrically (using ε278 23,470m−1·cm−1 calculated as described previously (24.Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5010) Google Scholar)) or by Bradford colorimetric assay (25.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211925) Google Scholar). The purified proteins gave a single band on an overloaded SDS-PAGE. Fractions containing purified proteins were dialyzed against water and then stored at −80 °C. Recombinant Ref-1 protein (rRef-1) was obtained as a hexahistidine tag fusion protein from overexpression in E. coli and then purified by nickel-chelate chromatography from bacterial extracts and treated as previously described (26.Abate C. Patel L. Rauscher F.J. Curran T. Science. 1990; 249: 1157-1161Crossref PubMed Scopus (1373) Google Scholar). GlutathioneS-transferase (GST)-Ref-1 protein was obtained as previously described (27.Huq I. Wilson T.M. Kelley M.R. Deutsch W.A. Mutat. Res. 1995; 337: 191-199Crossref PubMed Scopus (7) Google Scholar). Double-stranded oligodeoxynucleotides, labeled at the 5′-end with 32P, were used as probes in gel retardation assays. The C14 oligonucleotide is a 14-mer whose upper strand is 5′-CAGTCAAGTGTTCT-3′ (28.Civitareale D. Lonigro R. Sinclair J. Di Lauro R. EMBO J. 1989; 9: 2537-2542Crossref Scopus (320) Google Scholar). The gel retardation assay was performed by incubating protein and DNA in a buffer containing 20 mm Tris-HCl, pH 7.6, 75 mmKCl, 0.25 μg/ml bovine serum albumin with or without calf thymus DNA (50 μg/ml) as reported in the figure legends, 10% glycerol for 30 min at room temperature. Protein-bound DNA and free DNA were separated on a native polyacrylamide gel run in 0.5× TBE for 1.5 h at 4 °C. The gel was dried and then exposed to an x-ray film at −80 °C. When required, in vitro protein oxidation was obtained by prolonged air exposure or by diamide treatment. The monomeric binding of TTF-1N-HDWT and the C87S mutant on the C14 sequence were determined by UV cross-link analysis, following the procedure of Molnar et al. (29.Molnar G. O'Leary N. Pardee A.B. Bradley D.W. Nucleic Acids Res. 1995; 23: 3318-3326Crossref PubMed Scopus (14) Google Scholar). Briefly, proteins and DNA were incubated as described for EMSAs but without glycerol. After 30 min, aliquots were either loaded onto a native polyacrylamide gel to detect the protein-DNA complexes or subjected to UV cross-linking analysis (300 nm, 50 W) for 10 min. After UV exposure, 4× Laemmli sample buffer with or without β-mercaptoethanol was added, and samples were loaded onto a 12% SDS-PAGE. Following electrophoresis, the gel was dried and exposed to autoradiography. Prestained protein molecular markers were from MBI Fermentas. HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, glutamine, and antibiotics. For transient transfection assays, cells were plated, 12 h before transfection, at 0.3 × 106 cells/100-mm culture dish. Transfections were carried out using a calcium phosphate method (30.Graham F.L. van der Eb A.J. Virology. 1973; 52: 456-467Crossref PubMed Scopus (6463) Google Scholar). The following amounts of plasmids were transfected: pTACAT3 (3 μg), C5E1b (3 μg), CMV-β-galactosidase (2 μg), RSV-CAT (3 μg), Ki-Ras-CAT (3 μg), PGL-2 (3 μg), CMV-CAT (3 μg). CAT was measured by an ELISA method (Roche Molecular Biochemicals). A CMV-β-galactosidase expression plasmid was used as an internal control for transfection efficiency, according the protocol provided by the manufacturer (Roche Molecular Biochemicals). LUC activities were measured by a chemiluminescence procedure (31.Francis-Lang H. Zannini M. De Felice M. Berlingieri M.T. Fusco A. Di Lauro R. Mol. Cell. Biol. 1992; 12: 5793-5800Crossref PubMed Scopus (102) Google Scholar). For pull-down experiments, Ref-1 was expressed as GST fusion protein. For the interaction between the purified TTF-1N-HD and GST-Ref-1, 100 ng of the former and 500 ng of the latter were mixed for 30 min at room temperature, coprecipitated by 30 μl of GST-agarose (Amersham Biosciences), washed three times with PBS in the presence of 0.1% Nonidet P-40, and eluted by 50 μl of 10 mm GSH in PBS. Eluted samples were subjected to 12% SDS-PAGE and immunoblot with α-His (Amersham Biosciences) or α-TTF-1 antibodies or to EMSA analysis with the radiolabeled oligonucleotide C14. The blots were developed using the ECL chemiluminescence method (Amersham Biosciences). It has been previously demonstrated that a reducing environment is required for a proper DNA-binding activity by the TTF-1 whole molecule (7.Arnone M.I. Zannini M. Di Lauro R. J. Biol. Chem. 1995; 270: 12048-12055Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Two of the four Cys residues of TTF-1 (Cys87 and Cys363) had turned out to be the targets of the redox control, which is mainly exerted, in vitro, through the modulation of the oligomerization state of TTF-1. The authors proposed that a role for this kind of regulation may occur in vivo, hypothesizing the presence of an unknown co-factor for the control of the redox state of TTF-1. However, our previous data demonstrated that Cys87, on its own, is sufficient for controlling the dimerization state of TTF-1, therefore suggesting a master role for this residue in the TTF-1 redox control (6.Tell G. Perrone L. Fabbro D. Pellizzari L. Pucillo C. De Felice M. Acquaviva R. Formisano S. Damante G. Biochem. J. 1998; 329: 395-403Crossref PubMed Scopus (32) Google Scholar). To test if the reducing environment may affect the DNA binding function of TTF-1 HD, through the modulation of the redox state of Cys87, we tested the ability of a deleted form of TTF-1, TTF-1N-HD (see Fig. 1), containing only the N domain and the HD, to specifically recognize the high affinity DNA-binding site (oligonucleotide C14). The specific TTF-1N-HD binding activity to the site C14, assessed by EMSA and in the presence of a specific competitor DNA from calf thymus, was abolished after the addition of the oxidizing agent diamide (1,1′-azobis(N-dimetilformamide)) (see Fig. 2 A, lane 3 versus lane 2). This effect was observed at a concentration as low as 0.1 mmdiamide and was completely reversible by the addition of an excess of the reducing agent dithiothreitol (DTT) (Fig. 2 A,lane 4), thus underlying a possible functional role in vivo. As has been previously demonstrated by Arnoneet al. (7.Arnone M.I. Zannini M. Di Lauro R. J. Biol. Chem. 1995; 270: 12048-12055Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), the loss of DNA binding activity is not directly attributable to a loss of binding by the HD, because it does not contain Cys residues and it is not sensible to modulation of redox conditions (Fig. 2 B). To assess whether the DNA binding inhibition seen for TTF-1N-HD could be related to the dimerization state of the protein, we performed SDS-PAGE analysis of the samples used in the EMSA experiments described above. The monomeric state of the TTF-1N-HD protein was dependent on redox conditions. The protein was totally present as a single monomeric form only under reducing conditions (data not shown). Following a treatment with 5 mm diamide, dimeric species were present. However, it should be noticed that a considerable amount (about 80% of the total protein) of monomeric protein is still available in the oxidizing environment (data not shown). Therefore, dimerization does not account for the complete loss of specific DNA binding activity demonstrated in Fig. 2. To characterize in a better way the reasons why the oxidized form of TTF-1N-HD was unable to recognize the C14 sequence, the specific binding activity of this protein was evaluated in the presence of decreasing amounts of competitor DNA (genomic calf thymus DNA) and in reducing and oxidizing conditions (Fig. 3 A). As is evident fromlanes 5–7 of Fig. 3 A, the oxidized form of TTF-1N-HD was still able to interact with the oligonucleotide C14 sequence. However, the affinity of this interaction was lower if compared with that observed when the protein was present in the reduced form (Fig. 3 A, lanes 2–4). From the relative densitometric evaluation of the retarded bands present inA, and then reported in B, the loss of specific DNA binding by the oxidized protein is strikingly evident in the presence of genomic DNA. Absolute quantitation of the retarded bands in oxidizing conditions revealed that the amount of the reduction in DNA binding affinity can be estimated at 3 orders of magnitude (Fig. 3 C). These results suggest that the weakening of the TTF-1N-HD/oligonucleotide C14 interaction by oxidation would be due to (i) absolute loss of binding activity or (ii) inability to discriminate between different DNA sequences with following subtraction of protein involved in nonspecific bonds. Ref-1 is the major cofactor involved in redox regulation of several TFs, and it also plays a primary role in thyroid cells (14.Tell G. Pellizzari L. Pucillo C. Puglisi F. Cesselli D. Kelley M.R. Di Loreto C. Damante G. J. Mol. Endocrinol. 2000; 24: 383-390Crossref PubMed Scopus (57) Google Scholar). To test whether Ref-1 is also able to control the DNA binding activity of the isolated TTF-1N-HD, we performed an EMSA analysis with the oxidate form of the TTF-1N-HD protein and the rRef-1 protein. As we have previously demonstrated, the oxidized form of the TTF-1N-HD was unable to show any kind of specific DNA-binding activity (Fig. 4 A, lane 4). However, the presence of Ref-1 was sufficient per se to reconstitute the complete binding activity (lane 5) as in the presence of DTT (lane 3). The role of Ref-1 is not directed to the HD, since, as demonstrated in Fig. 4 B, the addition of rRef-1 to the sample does not affect the HD DNA binding affinity (lane 5). To test the specificity of the redox control played by Ref-1 over TTF-1N-HDWT, we performed EMSA analysis of the oxidized form of TTF-1N-HDWT in the presence of three unrelated proteins: cytochromec (Fig. 4 C, lane 6), bovine serum albumin (lane 7), and ovalbumin (lane 8). As is evident from Fig. 4 C, the three proteins are not able to rescue the DNA binding activity of the oxidized TTF-1N-HDWT with respect to Ref-1 (lanes 4 and 5). Ref-1 has been shown to be the redox regulator of different TFs (8.Xanthoudakis S. Miao G. Wang F. Pan Y.C. Curran T. EMBO J. 1992; 11: 3323-3335Crossref PubMed Scopus (815) Google Scholar, 9.Tell G. Scaloni A. Pellizzari L. Formisano S. Pucillo C. Damante G. J. Biol. Chem. 1998; 273: 25062-25072Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 10.Jayaraman L. Murthy K.G.K. Zhu C. Curran T. Xanthoudakis S. Prives C. Genes Dev. 1997; 11: 558-570Crossref PubMed Scopus (440) Google Scholar, 32.Walker L.J. Robson C.N. Black E. Gillespie D. Hickson I.D. Mol. Cell. Biol. 1993; 13: 5370-5376Crossref PubMed Scopus (259) Google Scholar). However, there has been direct evidence that documents a physical interaction between the two proteins (33.Gaiddon C. Moorthy N.C. Prives C. EMBO J. 1999; 18: 5609-5621Crossref PubMed Scopus (206) Google Scholar) only in the case of p53. To test this possibility in the case of TTF-1, we used a GST pull-down approach. After expression and purification of Ref-1 as GST fusion protein (27.Huq I. Wilson T.M. Kelley M.R. Deutsch W.A. Mutat. Res. 1995; 337: 191-199Crossref PubMed Scopus (7) Google Scholar), we applied GST pull-down for testing the interaction with TTF-1N-HD (see “Experimental Procedures” for details). As is evident from Fig. 5, TTF-1N-HD specifically interacts with GST-Ref-1 but not with GST alone, demonstrating the occurrence of the interaction between the two proteins. To test if the stimulatory effect of Ref-1 on the TTF-1 N-domain activity could have relevance in vivo (Fig. 6), a cell transfection approach was used. The thyroglobulin promoter (Tg) is not functional when transfected in HeLa cells, and its activity can be reconstituted by the forced expression of the construct Δ14, which encodes for the partial TTF-1 protein containing the transactivating N-domain and the DNA binding dom
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