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• W2031088051 abstract "The transcription factor thyroid transcription factor-1 (TTF-1) is a homeodomain-containing protein that belongs to the NK2 family of genes involved in organogenesis. TTF-1 is required for normal development of the forebrain, lung, and thyroid. In a search for factors that regulate TTF-1 transcriptional activity, we isolated three genes (T:G mismatch-specific thymine DNA glycosylase (TDG), homeodomain-interacting protein kinase 2 (HIPK2), and Ajuba), whose products can interact with TTF-1 in yeast and in mammalian cells. TDG is an enzyme involved in base excision repair. In the present paper, we show that TDG acts as a strong repressor of TTF-1 transcriptional activity in a dose-dependent manner, while HIPK2 and Ajuba display no effect on TTF-1 activity, at least under the tested conditions. TDG-mediated inhibition occurs specifically on TTF-1-responsive promoters in thyroid and non thyroid cells. TDG associates with TTF-1 in mammalian cells through the TTF-1 carboxyl-terminal activation domain and is independent of the homeodomain. These findings reveal a previously unsuspected role for the repair enzyme TDG as a transcriptional repressor and open new routes toward the understanding of the regulation of TTF-1 transcriptional activity. The transcription factor thyroid transcription factor-1 (TTF-1) is a homeodomain-containing protein that belongs to the NK2 family of genes involved in organogenesis. TTF-1 is required for normal development of the forebrain, lung, and thyroid. In a search for factors that regulate TTF-1 transcriptional activity, we isolated three genes (T:G mismatch-specific thymine DNA glycosylase (TDG), homeodomain-interacting protein kinase 2 (HIPK2), and Ajuba), whose products can interact with TTF-1 in yeast and in mammalian cells. TDG is an enzyme involved in base excision repair. In the present paper, we show that TDG acts as a strong repressor of TTF-1 transcriptional activity in a dose-dependent manner, while HIPK2 and Ajuba display no effect on TTF-1 activity, at least under the tested conditions. TDG-mediated inhibition occurs specifically on TTF-1-responsive promoters in thyroid and non thyroid cells. TDG associates with TTF-1 in mammalian cells through the TTF-1 carboxyl-terminal activation domain and is independent of the homeodomain. These findings reveal a previously unsuspected role for the repair enzyme TDG as a transcriptional repressor and open new routes toward the understanding of the regulation of TTF-1 transcriptional activity. thyroid transcription factor-1 chloramphenicol acetyltransferase signal transducers and activators of transcription protein inhibitor of activated STAT T:G mismatch-specific thymine DNA glycosylase 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside homeodomain-interacting protein kinase polyacrylamide gel electrophoresis ubiquitin-conjugating enzyme The transcription factor thyroid transcription factor-1 (TTF-11; also named Nkx2.1 or T/EBP) belongs to the Nkx2 class of homeodomain-containing proteins (1Guazzi S. Price M. De Felice M. Damante G. Mattei M.G. Di Lauro R. EMBO J. 1990; 9: 3631-3639Crossref PubMed Scopus (470) Google Scholar,2Mizuno K. Gonzalez F.J. Kimura S. Mol. Cell. Biol. 1991; 11: 4927-4933Crossref PubMed Scopus (125) Google Scholar). Nkx proteins regulate regional specification, cell fate determination, and organ morphogenesis during embryonic development. Among the vertebrate NK2 factors, Nkx2.5 is required for proper heart formation (3Biben C. Harvey R.P. Genes Dev. 1997; 11: 1357-1369Crossref PubMed Scopus (271) Google Scholar, 4Lyons I. Parsons L.M. Hartley L. Li R. Andrews J.E. Robb L. Harvey R.P. Genes Dev. 1995; 9: 1654-1666Crossref PubMed Scopus (955) Google Scholar), and Nkx2.2 for development of the spinal cord and the pancreatic islet cells (5Briscoe J. Sussel L. Serup P. Hartigan-O'Connor D. Jessell T.M. Rubenstein J.L. Ericson J. Nature. 1999; 398: 622-627Crossref PubMed Scopus (610) Google Scholar, 6Sussel L. Kalamaras J. Hartigan-O'Connor D.J. Meneses J.J. Pedersen R.A. Rubenstein J.L. German M.S. Development. 1998; 125: 2213-2221Crossref PubMed Google Scholar), while TTF-1/Nkx2.1 is involved in the regulation of thyroid, lung, and ventral forebrain development (7Sussel L. Marin O. Kimura S. Rubenstein J.L. Development. 1999; 126: 3359-3370PubMed Google Scholar,8Kimura S. Hara Y. Pineau T. Fernandez-Salguero P. Fox C.H. Ward J.M. Gonzalez F.J. Genes Dev. 1996; 10: 60-69Crossref PubMed Scopus (1006) Google Scholar). The Nkx2 proteins share high similarity in their homeodomain, a 60-amino acid region involved in DNA binding. The TTF-1 homeodomain was the first one shown to bind DNA elements containing a 5′-CAAG-3′ core sequence, instead of the canonical 5′-TAAT-3′ (1Guazzi S. Price M. De Felice M. Damante G. Mattei M.G. Di Lauro R. EMBO J. 1990; 9: 3631-3639Crossref PubMed Scopus (470) Google Scholar, 9Damante G. Fabbro D. Pellizzari L. Civitareale D. Guazzi S. Polycarpou-Schwartz M. Cauci S. Quadrifoglio F. Formisano S. Di Lauro R. Nucleic Acids Res. 1994; 22: 3075-3083Crossref PubMed Scopus (104) Google Scholar). The Nkx2 proteins contain two other highly conserved regions, the tin domain (or NK decapeptide) and the NK2-specific domain (10Watada H. Mirmira R.G. Kalamaras J. German M.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9443-9448Crossref PubMed Scopus (87) Google Scholar, 11Harvey R.P. Dev. Biol. 1996; 178: 203-216Crossref PubMed Scopus (494) Google Scholar). The shorttin domain of unknown function is found at the N-terminal region of most NK2 proteins, while the NK2-specific domain is located in a C-terminal region separated from the homeodomain by a short amino acid stretch and is thought to mediate protein-protein interaction. During development, TTF-1 is expressed in the thyroid anlage, in restricted areas of the developing brain and in the lung bronchial epithelium (12Lazzaro D. Price M. de Felice M. Di Lauro R. Development. 1991; 113: 1093-1104Crossref PubMed Google Scholar). It is required for proper development of these tissues, since TTF-1 null mice die at birth lacking lung parenchyma and the thyroid, and have severe defects of the ventral area of the forebrain (7Sussel L. Marin O. Kimura S. Rubenstein J.L. Development. 1999; 126: 3359-3370PubMed Google Scholar, 8Kimura S. Hara Y. Pineau T. Fernandez-Salguero P. Fox C.H. Ward J.M. Gonzalez F.J. Genes Dev. 1996; 10: 60-69Crossref PubMed Scopus (1006) Google Scholar). During embryogenesis, TTF-1 is expressed at embryonic days 8.5–9.5 in the pharyngeal floor, a portion of which evaginates and becomes the thyroid diverticulum. At the same time, other transcription factors that are required for thyroid development start to be expressed (13De Felice M. Ovitt C. Biffali E. Rodriguez-Mallon A. Arra C. Anastassiadis K. Macchia P.E. Mattei M.G. Mariano A. Scholer H. Macchia V. Di Lauro R. Nat. Genet. 1998; 19: 395-398Crossref PubMed Scopus (260) Google Scholar, 14Mansouri A. Chowdhury K. Gruss P. Nat. Genet. 1998; 19: 87-90Crossref PubMed Scopus (498) Google Scholar, 15Zannini M. Avantaggiato V. Biffali E. Arnone M.I. Sato K. Pischetola M. Taylor B.A. Phillips S.J. Simeone A. Di Lauro R. EMBO J. 1997; 16: 3185-3197Crossref PubMed Scopus (218) Google Scholar, 16Poleev A. Fickenscher H. Mundlos S. Winterpacht A. Zabel B. Fidler A. Gruss P. Plachov D. Development. 1992; 116: 611-623Crossref PubMed Google Scholar). However, thyroid-specific gene expression is not turned on until embryonic day 15, suggesting that these transcription factors, although expressed early in development, are inactive or require additional factors to drive thyroid-specific gene expression. Besides TTF-1, other Nkx2 family members, Nkx2.5 and -2.8, are expressed very early in the pharynx (17Lints T.J. Parsons L.M. Hartley L. Lyons I. Harvey R.P. Development. 1993; 119: 969PubMed Google Scholar, 18Reecy J.M. Yamada M. Cummings K. Sosic D. Chen C.Y. Eichele G. Olson E.N. Schwartz R.J. Dev. Biol. 1997; 188: 295-311Crossref PubMed Scopus (54) Google Scholar). Nkx2.8 expression is progressively lost in the pharyngeal floor, while Nkx2.5 expression becomes restricted to the thyroid primordium. The role of Nkx2.5 in thyroid development is unknown given that Nkx2.5 null embryos die before thyroid formation, from failure in the heart tube looping process (3Biben C. Harvey R.P. Genes Dev. 1997; 11: 1357-1369Crossref PubMed Scopus (271) Google Scholar, 4Lyons I. Parsons L.M. Hartley L. Li R. Andrews J.E. Robb L. Harvey R.P. Genes Dev. 1995; 9: 1654-1666Crossref PubMed Scopus (955) Google Scholar). In addition to its role in development, TTF-1 is thought to regulate tissue-specific transcription in differentiated thyroid and lung cells. The expression of thyroid-specific genes, such as thyroglobulin, thyroperoxidase, thyrotropin receptor, and Na-I symporter is positively controlled by TTF-1 (19Endo T. Kaneshige M. Nakazato M. Ohmori M. Harii N. Onaya T. Mol. Endocrinol. 1997; 11: 1747-1755PubMed Google Scholar, 20Civitareale D. Castelli M.P. Falasca P. Saiardi A. Mol. Endocrinol. 1993; 7: 1589-1595PubMed Google Scholar, 21Civitareale D. Lonigro R. Sinclair A.J. Di Lauro R. EMBO J. 1989; 8: 2537-2542Crossref PubMed Scopus (325) Google Scholar, 22Ohmori M. Shimura H. Shimura Y. Ikuyama S. Kohn L.D. Endocrinology. 1995; 136: 269-282Crossref PubMed Scopus (38) Google Scholar, 23Francis-Lang H. Price M. Polycarpou-Schwarz M. Di Lauro R. Mol. Cell. Biol. 1992; 12: 576-588Crossref PubMed Scopus (209) Google Scholar). In the lung, TTF-1 positively regulates the expression of surfactant proteins A, B, and C (24Bohinski R.J. Di Lauro R. Whitsett J.A. Mol. Cell. Biol. 1994; 14: 5671-5681Crossref PubMed Scopus (484) Google Scholar, 25Bruno 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 (238) Google Scholar, 26Kelly S.E. Bachurski C.J. Burhans M.S. Glasser S.W. J. Biol. Chem. 1996; 271: 6881-6888Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). The activity of eukaryotic transcription factors can be regulated by various mechanisms, including phosphorylation, acetylation, and interaction with transcriptional modulators. We have previously shown that TTF-1 is phosphorylated on seven serine residues, three of which serve as substrates for the mitogen-activated protein kinase extracellular signal-regulated kinase (27Missero C. Pirro M.T. Di Lauro R. Mol. Cell. Biol. 2000; 20: 2783-2793Crossref PubMed Scopus (53) Google Scholar, 28Zannini M. Acebron A. De Felice M. Arnone M.I. Martin-Perez J. Santisteban P. Di Lauro R. J. Biol. Chem. 1996; 271: 2249-2254Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Expression of oncogenic Ras in thyroid cell results in TTF-1 inactivation and loss of expression of several thyroid-specific genes. Ras inhibitory effect is at least partially mediated in an extracellular signal-regulated kinase-dependent manner (27Missero C. Pirro M.T. Di Lauro R. Mol. Cell. Biol. 2000; 20: 2783-2793Crossref PubMed Scopus (53) Google Scholar, 29Francis-Lang H. Zannini M.S. De Felice M. Berlingieri M.T. Fusco A. Di Lauro R. Mol. Cell. Biol. 1992; 12: 5793-5800Crossref PubMed Scopus (102) Google Scholar). To identify other regulators of TTF-1, we performed a yeast two-hybrid screening using the full-length TTF-1 cDNA fused to the GAL4 DNA binding domain. We found that T:G mismatch-specific thymine DNA glycosylase (TDG), an enzyme involved in repair of methylated DNA (30Wiebauer K. Jiricny J. Nature. 1989; 339: 234-236Crossref PubMed Scopus (152) Google Scholar, 31Neddermann P. Jiricny J. J. Biol. Chem. 1993; 268: 21218-21224Abstract Full Text PDF PubMed Google Scholar, 32Neddermann P. Gallinari P. Lettieri T. Schmid D. Truong O. Hsuan J.J. Wiebauer K. Jiricny J. J. Biol. Chem. 1996; 271: 12767-12774Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar), interacts with TTF-1 and strongly represses its transcriptional activity. TDG binds to the activation domain at the TTF-1 C terminus and suppresses TTF-1-activated transcription in thyroid and nonthyroid cells. All experiments were performed in the yeast reporter MaV203. The cDNA library was synthesized from rat FRTL-5 cell poly(A)+ RNA plasmid by Life Technologies and cloned into the pPC86GAL4AD vector. Screening of the library was performed essentially following instructions for the ProQuest two-hybrid system (Life Technologies) and has been previously described (33Vidal M. Legrain P. Nucleic Acids Res. 1999; 27: 919-929Crossref PubMed Scopus (197) Google Scholar). Briefly, the GAL4 DNA-binding domain/TTF-1 fusion was constructed in pPC97GAL4DB, and proper expression of the fusion protein in yeast was confirmed. Subsequently, yeasts carrying the pPC97GAL4DB-TTF-1 plasmid were transformed with the pPC86AD-cDNA library and plated onto plates lacking histidine in the presence of 3AT (aminotriazole; 10 mm). Approximately 1.2 × 106 individual clones were plated, and about 200 grew on the selective medium. Resistant colonies were grown on a master plate and then replica-plated onto selection plates to determine their ability to induce three independent reporters (HIS3, URA3, andlacZ). Seventy-five independent clones were isolated after this first screening. DNA was isolated from each positive clone and sequenced to identify the inserts. Independent pPC86AD clones were retransformed into yeast and tested for interaction with a fresh TTF-1 clone. Yeast strains expressing GAL4DB-Rb, GAL4AD-E2F, GAL4DB-Fos, GAL4AD-Jun, GAL4DB-DP1, GAL4AD-E2F1, and full-length GAL4 were obtained from M. Vidal. For the yeast two-hybrid screening, the coding sequence of rat TTF-1 was fused in frame with the GAL4DNA binding domain in the pPC97GAL4DB plasmid. TG-Luc, a plasmid in which a luciferase reporter gene is controlled by a 400-bp thyroglobulin promoter, has been previously described (21Civitareale D. Lonigro R. Sinclair A.J. Di Lauro R. EMBO J. 1989; 8: 2537-2542Crossref PubMed Scopus (325) Google Scholar). C5-CAT is a reporter construct in which the chloramphenicol acetyl transferase is under the control of a promoter containing five binding sites for TTF-1 (34Missero C. Cobellis G. De Felice M. Di Lauro R. Mol. Cell. Endocrinol. 1998; 140: 37-43Crossref PubMed Scopus (56) Google Scholar). Ajuba full-length coding sequence was generated by 5′-rapid amplification of cDNA ends polymerase chain reaction (Life Technologies). The coding sequence of the interactor was amplified using Pfu polymerase (Stratagene) and then inserted in frame with the FLAG epitope in pFLAG-CMV2 (Sigma) to create mammalian expression plasmids. pRC-TTF-1, GAL4-TTF-1, TTF-1 deletion mutants, and G5-CAT were previously described (35De 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). Rat thyroid follicular FRTL-5 cells were maintained in Coon's modified Ham's F-12 medium (Sigma) supplemented with 5% calf serum (Life Technologies) and six growth factors including thyrotropin (1 milliunit/ml) and insulin (10 μg/ml) as previously described (28Zannini M. Acebron A. De Felice M. Arnone M.I. Martin-Perez J. Santisteban P. Di Lauro R. J. Biol. Chem. 1996; 271: 2249-2254Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 36Ambesi-Impiombato F.S. Parks L.A.M. Coon H.G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3455-3459Crossref PubMed Scopus (974) Google Scholar). Human COS-7 cells and HeLa were obtained from ATCC and grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Life Technologies, Inc.). Transient transfections in FRTL-5 and HeLa cells were carried out by calcium phosphate-DNA precipitation (34Missero C. Cobellis G. De Felice M. Di Lauro R. Mol. Cell. Endocrinol. 1998; 140: 37-43Crossref PubMed Scopus (56) Google Scholar). Briefly, FRTL-5 cells were plated at a density of 5 × 105/60-mm dish, and 48 h later C5-CAT or Tg-CAT (2.5 μg) reporter plasmids were transfected in the presence of different expression vectors as indicated in the figure legends. Cells were collected 72 h after transfection. HeLa were plated the day before transfection at 8 × 105/60-mm dish. After transfection, cells were incubated for 48 h and then collected. Almost confluent COS-7 cells were transiently transfected using the LipofectAMINE 2000 reagent following the manufacturer's instructions (Life Technologies). 4 μg of DNA was used for a 60-mm dish (5 × 105 cells). Cells were collected 48 h after transfection. FRTL-5 and HeLa cell extracts were lysed in lysis buffer (10 mm HEPES, pH 7.9, 400 mm NaCl, 0.1 mm EGTA, 0.5 mmdithiothreitol, 5% glycerol, 0.5 mm phenylmethylsulfonyl fluoride). Luciferase and chloramphenicol acetyltransferase (CAT) activities were measured as described (34Missero C. Cobellis G. De Felice M. Di Lauro R. Mol. Cell. Endocrinol. 1998; 140: 37-43Crossref PubMed Scopus (56) Google Scholar, 37Brasier A.R. Tate J.E. Habener J.F. BioTechniques. 1989; 7: 1116-1122PubMed Google Scholar). CAT activity was measured by incubation with 5 mm chloramphenicol and 0.1 μCi of [3H]acetyl coenzyme A (1.4 Ci/mmol; 50 μCi/ml). Reactions were performed in the presence of water-insoluble scintillation fluid (Econofluor-2; Packard Bioscience) at 37 °C, and radioactivity released was counted after 5 h. Luciferase activity was measured in the presence of 0.2 mmd-luciferin (Sigma) in a Lumat LB 9501 luminometer (Berthold). Approximately one-tenth of the total volume was employed for each assay. Whole-cell lysates of transiently transfected COS-7 cells were prepared in in lysis buffer (20 mm Tris, pH 7.9, 120 mm KCl, 5 mmMgCl2 0.2% Nonidet P-40, 5 mm EDTA, 10% glycerol, 0.5 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mm sodium vanadate, 50 mm NaF). 300 μg of total cell extracts were immunoprecipitated with anti-FLAG M2 monoclonal antibody conjugated to agarose beads (Sigma). After 2 h, immunocomplexes were washed five times in lysis buffer and resuspended in sample buffer supplemented with 5% β-mercaptoethanol. Proteins were resolved by SDS-PAGE and blotted onto a polyvinylidene difluoride membrane (Millipore). After transferring, nonspecific binding sites were blocked by incubation with 5% nonfat dry milk in PBS, 0.2% Tween 20. Rabbit polyclonal antibody against TTF-1 was used at approximately 1 μg/ml (29Francis-Lang H. Zannini M.S. De Felice M. Berlingieri M.T. Fusco A. Di Lauro R. Mol. Cell. Biol. 1992; 12: 5793-5800Crossref PubMed Scopus (102) Google Scholar). Anti-FLAG M2 monoclonal antibody was obtained from Sigma. Immune complexes were detected by enhanced chemiluminescence as instructed by the manufacturer (Amersham Pharmacia). Rabbit polyclonal antibody against GAL4 (sc-577) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). In a yeast two-hybrid screen of a rat cDNA library, 65 positive clones were isolated, of which 55 clones corresponded to sequences homologous to known human or mouse genes. Of these, 20 were homologous to mouse ubiquitin-conjugating enzyme (UBC9) (38Seufert W. Futcher B. Jentsch S. Nature. 1995; 373: 78-81Crossref PubMed Scopus (426) Google Scholar), 17 to mouse and human protein inhibitor of activated STAT 3 (PIAS3) (39Chung C.D. Liao J. Liu B. Rao X. Jay P. Berta P. Shuai K. Science. 1997; 278: 1803-1805Crossref PubMed Scopus (802) Google Scholar), one to the highly related protein PIAS1 (40Liu B. Liao J. Rao X. Kushner S.A. Chung C.D. Chang D.D. Shuai K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10626-10631Crossref PubMed Scopus (631) Google Scholar), 10 to mouse Ajuba (a LIM domain-containing protein) (41Goyal R.K. Lin P. Kanungo J. Payne A.S. Muslin A.J. Longmore G.D. Mol. Cell. Biol. 1999; 19: 4379-4389Crossref PubMed Google Scholar), four to human and mouse TDG (32Neddermann P. Gallinari P. Lettieri T. Schmid D. Truong O. Hsuan J.J. Wiebauer K. Jiricny J. J. Biol. Chem. 1996; 271: 12767-12774Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar), two to mouse and human homeodomain-interacting protein kinase 2 (HIPK2) (42Kim Y.H. Choi C.Y. Lee S.J. Conti M.A. Kim Y. J. Biol. Chem. 1998; 273: 25875-25879Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar), and one to the transcription factor Hex (43Pellizzari L. D'Elia A. Rustighi A. Manfioletti G. Tell G. Damante G. Nucleic Acids Res. 2000; 28: 2503-2511Crossref PubMed Scopus (59) Google Scholar) (TableI). The longest clone of each gene was entirely sequenced and found to correspond to the rat homologue of the above mentioned genes.Table ITTF-1 interactors in the yeast two-hybrid systemGAL4AD plasmidNumber of clonesStrength of interactionUBC920+++PIAS317+++Ajuba10+++TDG4++HIPK22+PIAS11+++Hex1−Yeast cells (MaV203) transformed with GAL4DBTTF-1 and GAL4AD interactor constructs were assayed as described in Fig. 1. Seventy-five positive clones were identified after the first screening, of which 55 were sequences homologous to known genes. The number of clones found to correspond to each gene is indicated. +++, strong positive interaction; ++, intermediate interaction; +, weak interaction; −, no interaction. Open table in a new tab Yeast cells (MaV203) transformed with GAL4DBTTF-1 and GAL4AD interactor constructs were assayed as described in Fig. 1. Seventy-five positive clones were identified after the first screening, of which 55 were sequences homologous to known genes. The number of clones found to correspond to each gene is indicated. +++, strong positive interaction; ++, intermediate interaction; +, weak interaction; −, no interaction. Interaction between TTF-1 and the isolated clones was confirmed by retransformation in yeast and test using three independent reporters (HIS3, URA3, and lacZ), in the presence of GAL4 DNA binding domain alone or of TTF-1-GAL4 fusion protein. As shown in Fig. 1, the strongest interaction was detected with Ajuba, UBC9, PIAS3, and the related gene PIAS1. TDG interacted also quite strongly, while interaction between TTF-1 and HIPK2 was weak. Other potential interactors such as the Hex gene and the clones Y66,Y120, and Y137, corresponding to unknown genes, scored positive in the context of one promoter but not in the context of the others and were excluded from further analysis. Thus, rat genes corresponding to TDG, UBC9, Ajuba, HIPK2, PIAS1, and PIAS3 displayed the ability to interact with TTF-1 in yeast, while the other clones selected during the first screening, including all of the unknown genes, did not. The TTF-1 interactors were further characterized for their ability to associate with TTF-1 in mammalian cells. Their coding sequences, lacking the first methionine, were fused at the N terminus to the FLAG epitope and cloned into a mammalian expression vector. Individual constructs were co-transfected with an equal amount of a TTF-1 expression vector in COS-7 cells. After 48 h, cells were harvested and lysed under mild conditions, and then immunoprecipitation was performed with anti-FLAG-specific antibody. Total cell extracts and immunoprecipitates were then run on SDS-PAGE gels under the appropriate conditions, and immunoblotting analysis was performed using either anti-FLAG or anti-TTF-1 antibody. Immunoprecipitation of TDG and Ajuba resulted in a readily detectable amount of TTF-1 (Fig.2, A and C). Although HIPK2 immunoprecipitation was not very efficient due to its low levels of expression, a low but significant amount of TTF-1 was detected in HIPK2 immunoprecipitation (Fig. 2 B). TTF-1 association with these proteins was specific, since no association could be detected with FLAG-UBC9 and FLAG-PIAS3, although both proteins were efficiently produced. PIAS1 was not tested in mammalian cells. On the basis of these results, TDG, HIPK2, and Ajuba were considered as potential regulators of TTF-1 activity. TDG was previously reported to interact in yeast two-hybrid assays with c-Jun and with the retinoic acid receptors RAR and RXR and to weakly enhance RAR and RXR transcriptional activity (44Chevray P.M. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5789-5793Crossref PubMed Scopus (479) Google Scholar,45Um S. Harbers M. Benecke A. Pierrat B. Losson R. Chambon P. J. Biol. Chem. 1998; 273: 20728-20736Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). HIPK2 is a nuclear kinase that was reported to interact with NK2 family members (42Kim Y.H. Choi C.Y. Lee S.J. Conti M.A. Kim Y. J. Biol. Chem. 1998; 273: 25875-25879Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Ajuba was recently cloned as an interactor of GRB2 and was found to shuttle between the cytoplasm and the nucleus (41Goyal R.K. Lin P. Kanungo J. Payne A.S. Muslin A.J. Longmore G.D. Mol. Cell. Biol. 1999; 19: 4379-4389Crossref PubMed Google Scholar,46Kanungo J. Pratt S.J. Marie H. Longmore G.D. Mol. Biol. Cell. 2000; 11: 3299-3313Crossref PubMed Scopus (114) Google Scholar). The ability of these proteins to interfere with TTF-1 transcriptional activity was evaluated in thyroid and nonthyroid cells. Cells were transiently transfected with an expression vector for TDG, Ajuba, or HIPK2 in the presence of either a natural TTF-1-responsive promoter (thyroglobulin) or an artificial one (C5), driving the expression of CAT. These promoters display strong basal activity in FRTL-5 cells and are activated specifically by exogenous expression of TTF-1 in nonthyroid cell lines (21Civitareale D. Lonigro R. Sinclair A.J. Di Lauro R. EMBO J. 1989; 8: 2537-2542Crossref PubMed Scopus (325) Google Scholar, 34Missero C. Cobellis G. De Felice M. Di Lauro R. Mol. Cell. Endocrinol. 1998; 140: 37-43Crossref PubMed Scopus (56) Google Scholar). In thyroid cells, TDG expression strongly repressed C5-CAT in a dose-dependent manner with a maximum inhibition of ∼75% (Fig.3 A). The activity of the thyroglobulin promoter was similarly inhibited by TDG expression (Fig.3 B), while TDG had no effect on the CMV-Luc expression plasmid used as control (data not shown). In contrast, exogenous expression of either HIPK2 or Ajuba had no effect on TTF-1 activity as measured on C5 and thyroglobulin promoters (Fig. 3, C andD, and data not shown). To confirm and further extend these results, HeLa cells that do not express TTF-1 were transiently transfected with the TTF-1-responsive construct C5-CAT in the presence or in the absence of exogenous TTF-1. C5-CAT basal transcription was very low in nonthyroid cells, and its activity was strongly induced in the presence of TTF-1 (Fig.4 A). Expression of either Ajuba or HIPK2 had no effect on TTF-1 activity (data not shown), while TDG expression resulted in strong suppression of TTF-1 activity in a dose-dependent manner (Fig. 4 A). Since TDG is expressed ubiquitously (Ref. 45Um S. Harbers M. Benecke A. Pierrat B. Losson R. Chambon P. J. Biol. Chem. 1998; 273: 20728-20736Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar and data not shown), the endogenous protein may be sufficient to elicit an effect in the presence of low TTF-1 concentrations. Under these conditions, the addition of TDG could be inhibitory due to a nonspecific “squelching” effect rather than a biologically relevant function. To test this hypothesis, TTF-1 activity was measured in a wide variety of concentrations. In HeLa cells, TTF-1 could efficiently activate C5-CAT even at very low doses (0.05–0.1 μg), under which conditions TDG expression strongly repressed TTF-1 activity. In the presence of high doses of TTF-1 and low concentration of the TDG vector, TTF-1 transcriptional activity was still strongly inhibited by TDG expression in a dose-dependent manner (Fig. 4 B). Similar suppression of TTF-1 activity was obtained by TDG expression in NIH3T3 fibroblasts at all tested concentrations (data not shown). Thus, taken together, these data suggest that TDG associates with TTF-1 in mammalian cells, where it acts as a specific repressor of TTF-1 transcriptional activity, while Ajuba and HIPK2 are able to associate with TTF-1 but have no apparent effect on its activity at least under the tested conditions. All four TDG clones identified in the two-hybrid screening included the entire coding sequence. The longest TDG clone was 2883 bp long and included 124 nucleotides of 5′-untranslated region, and 1598 nucleotides of 3′-untranslated region. The open reading frame of 1158 base pairs encoded a protein of 386 amino acid residues with a predicted molecular mass of 42,454 Da (data not shown). The amino acid alignment had 88% identity with human TDG and 90% identity with mouse TDGb, while mouse and human sequences shared 86% identity (data not shown). The DNA glycosylase domain comprised amino acids 98–245 of the rat sequence and contained a single substitution Asn to Ser at position 146 of the rat sequence. No other recognizable domain could be identified in the TDG sequence. To map the TTF-1 domains responsible for the interaction with TDG, previously characterized TTF-1 deletion mutants (Fig.5 A) (35De 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) were transiently transfected in COS-7 cells either in the presence or in the absence of FLAG-TDG and immunoprecipitated with anti-FLAG monoclonal antibody. A TTF-1 deletion mutant lacking the entire C-terminal region (Δ14) was unable to associate with TDG, while a deletion of the first 51 amino acids at the N-terminal (Δ1) could efficiently bind to TDG (Fig.5 B). The Δ35 mutant lacking most of the N-terminal domain and part of the C-terminal region (amino acids 221–295), was able to associate with TDG to the same extent of the wild type (Fig.5 B). These data suggest that TDG binds to either the homeodomain and its N-terminal proximal region (amino acids 96–221), or to the C-terminal activation domain (amino acids 295–372). To assess whether the C-terminal domain was sufficient by itself to bind to TDG in the absence of the homeodomain, COS-7 cells were transfected with plasmids encoding for the TTF-1 activation domains fused to the GAL4 DNA binding domain, in the presence or in the absence of FLAG-TDG. Immunoprecipitation experiments using anti-FLAG monoclonal antibody revealed that TDG could associate specifically with the C-terminal activation domain (amino acids 295–372) fused to GAL4, but not with the N-terminal domain (Fig. 5 D). TTF-1 deletion mutants were tested for their ability to activate C5-CAT transcription in the presence and in the absence of TDG. Similarly to the wild type TTF-1, the Δ1 mutant was strongly inhibited by TDG (Fig. 5 C). In contrast, the Δ14 mutant that was unable to associate to TDG was unaffected by TDG expression. Interestingly, the Δ35 mutant that was sufficient to bind to TDG was unaffected by TDG, suggesting that TTF-1 regions that are not involved in binding to TDG are required for TDG-dependent inhibition. Taken together, these data demonstrate that TDG associates in mammalian cells with TTF-1 specifically through a C-terminal region between amino acids 295 and 372, corresponding to the activation domain. The activation domain is by itself necessary and sufficient for TDG binding, while TDG-mediated repression requires a more extended C-terminal region. Tissue-specific gene expression is spatially and temporally regulated. TTF-1 is a critical component of the transcriptional machinery controlling the expression of thyroid- and lung-specific genes, and thus its activity is likely to be finely regulated during embryonic development and in adult life. In an attempt to isolate TTF-1 regulators, we have identified six genes (Ajuba, HIPK2, PIAS1 and -3, TDG, and UBC9), whose product can bind to TTF-1 in yeast. Among these interactors, the T:G mismatch-specific thymine glycosylase TDG displays a strong ability to repress TTF-1-activated transcription. TDG is an enzyme involved in repair of methylated DNA (30Wiebauer K. Jiricny J. Nature. 1989; 339: 234-236Crossref PubMed Scopus (152) Google Scholar, 31Neddermann P. Jiricny J. J. Biol. Chem. 1993; 268: 21218-21224Abstract Full Text PDF PubMed Google Scholar, 32Neddermann P. Gallinari P. Lettieri T. Schmid D. Truong O. Hsuan J.J. Wiebauer K. Jiricny J. J. Biol. Chem. 1996; 271: 12767-12774Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Spontaneous deamination of 5-methylcytosine generates T:G mismatches whose repair is initiated by TDG. In mammalian cells, there are only two known thymine glycosylases, TDG and MBD4 (47Hendrich B. Hardeland U. Ng H.H. Jiricny J. Bird A. Nature. 1999; 401: 301-304Crossref PubMed Scopus (526) Google Scholar), which display no apparent amino acid sequence similarity, although they share the ability to bind methylated CpG and a very similar catalytic specificity in vitro (47Hendrich B. Hardeland U. Ng H.H. Jiricny J. Bird A. Nature. 1999; 401: 301-304Crossref PubMed Scopus (526) Google Scholar, 48Sibghat U. Gallinari P. Xu Y.Z. Goodman M.F. Bloom L.B. Jiricny J. Day III, R.S. Biochemistry. 1996; 35: 12926-12932Crossref PubMed Scopus (82) Google Scholar). DNA repair has recently revealed important links to transcription. For instance, proteins involved in nucleotide excision repair are components of the basal transcription factor TFIIH (49Bootsma D. Hoeijmakers J.H. Nature. 1993; 363: 114-115Crossref PubMed Scopus (199) Google Scholar, 50Drapkin R. Reardon J.T. Ansari A. Huang J.C. Zawel L. Ahn K. Sancar A. Reinberg D. Nature. 1994; 368: 769-772Crossref PubMed Scopus (408) Google Scholar). TDG has been previously suggested to be involved in transcription regulation because of its ability to interact in yeast with other transcription factors such as c-Jun, the retinoic acid receptor, and p73 (44Chevray P.M. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5789-5793Crossref PubMed Scopus (479) Google Scholar, 45Um S. Harbers M. Benecke A. Pierrat B. Losson R. Chambon P. J. Biol. Chem. 1998; 273: 20728-20736Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 51Minty A. Dumont X. Kaghad M. Caput D. J. Biol. Chem. 2000; 275: 36316-36323Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). TDG function in the regulation of Jun and p73 has not been explored, while TDG was reported to enhance weakly retinoic acid receptor activity (3–4-fold) (45Um S. Harbers M. Benecke A. Pierrat B. Losson R. Chambon P. J. Biol. Chem. 1998; 273: 20728-20736Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). However, association of these transcription factors with TDG in mammalian cells was not examined. In the present study, we report that TDG not only interacts with TTF-1 in yeast but also associates with TTF-1 in mammalian cells. TDG binds to a limited portion of the TTF-1 C terminus that corresponds to the C-terminal activation domain independently of the homeodomain. Binding to the C-terminal activation domain is necessary and sufficient for TDG binding, while TDG repression occurs only in the presence of the entire carboxyl-terminal region. A deletion of the region spanning from the end of the homeodomain to the beginning of the activation domain results in a mutant that can still bind to TDG but is not significantly inhibited. Interestingly, this region comprises the NK2-specific domain that has been proposed to control the transactivation properties of the NK2 family members (10Watada H. Mirmira R.G. Kalamaras J. German M.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9443-9448Crossref PubMed Scopus (87) Google Scholar, 11Harvey R.P. Dev. Biol. 1996; 178: 203-216Crossref PubMed Scopus (494) Google Scholar). The mechanisms through which TDG represses TTF-1 activity are unclear. Although TTF-1 binding to DNA in vitro is not inhibited by TDG, 2C. Missero and R. Di Lauro, unpublished data. in vivo TDG could sequester TTF-1 in a different nuclear compartment, preventing binding to target promoters. Alternatively, TDG could work as a repressor by recruiting a histone deacetylase. However, this possibility is unlikely, since TDG-mediated repression was not alleviated by the histone deacetylase trycostatin A, and exogenous expression of histone deacetylase 2 did not affect TTF-1 activity (data not shown). TDG strongly represses TTF-1-activated transcription in several cell lines; however, the functional significance of this repression in vivo is unclear. TTF-1 is expressed early in mouse development (embryonic day 8.5), although thyroid-specific gene expression is not turned on until later (embryonic day 15) (12Lazzaro D. Price M. de Felice M. Di Lauro R. Development. 1991; 113: 1093-1104Crossref PubMed Google Scholar). It has been postulated that during this time TTF-1 may be kept inactive by a repressor. TDG is highly expressed during early mouse embryogenesis (52Niederreither K. Harbers M. Chambon P. Dolle P. Oncogene. 1998; 17: 1577-1585Crossref PubMed Scopus (27) Google Scholar) and may prevent TTF-1-activated transcription. As a result of our search for TTF-1-interacting proteins, we have isolated also Ajuba and HIPK2. Ajuba was the only isolated TTF-1 interactor that was reported to be mainly cytoplasmic, although it contains a nuclear export signal, and it has been suggested to shuttle between the cytoplasm and the nucleus (41Goyal R.K. Lin P. Kanungo J. Payne A.S. Muslin A.J. Longmore G.D. Mol. Cell. Biol. 1999; 19: 4379-4389Crossref PubMed Google Scholar). Ajuba belongs to the Zyxin family of LIM proteins, and it has recently been shown to induce growth inhibition and spontaneous endodermal differentiation in embryonal cells (46Kanungo J. Pratt S.J. Marie H. Longmore G.D. Mol. Biol. Cell. 2000; 11: 3299-3313Crossref PubMed Scopus (114) Google Scholar). LIM domains mediate strong protein-protein interactions, and this feature may allow nonspecific association upon overexpression. TTF-1 was exclusively located in the nucleus even upon overexpression in COS-7 cells, while Ajuba was detected primarily in the cytoplasm, and no colocalization could be detected (data not shown). Thus, the physiological significance of the interaction between TTF-1 and Ajuba remains unclear. HIPK2 was the first member of a family of HIPKs identified for their ability to bind to NK2 and NK3 and to enhance their repressor activities (42Kim Y.H. Choi C.Y. Lee S.J. Conti M.A. Kim Y. J. Biol. Chem. 1998; 273: 25875-25879Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Subsequently, HIPKs were reported to interact also with other transcription factors, such as the androgen receptor, the glucocorticoid receptor, and p73 (51Minty A. Dumont X. Kaghad M. Caput D. J. Biol. Chem. 2000; 275: 36316-36323Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 53Moilanen A.M. Karvonen U. Poukka H. Janne O.A. Palvimo J.J. Mol. Biol. Cell. 1998; 9: 2527-2543Crossref PubMed Scopus (100) Google Scholar, 54Janne O.A. Moilanen A. Poukka H. Rouleau N. Karvonen U. Kotaja N. Hakli M. Palvimo J.J. Biochem. Soc. Trans. 2000; 28: 401-405Crossref PubMed Google Scholar). However, the ability of HIPKs to associate with these transcription factors in mammalian cells could not be shown. In the present study, we report for the first time the interaction between HIPK2 and a member of the family of the homeodomain-containing proteins. HIPK2 protein was difficult to detect even by exogenous expression in COS-7 cells. Given that its C terminus contains a PEST sequence, the protein may be very unstable. For these reasons, it is likely that association with other proteins in mammalian cells might have been difficult to obtain. Under our conditions, HIPK2 displays no effect on TTF-1 transcriptional activity in either thyroid or HeLa cells; thus, the role of this interaction remains to be established. The extent to which the molecules isolated in this study contribute to TTF-1 regulation in vivo remains to be elucidated. However, the identification and characterization of their interaction and, in particular, of TDG-mediated repression in culture cells sets the molecular basis to dissect their functional role in TTF-1 regulation and in thyroid gland and lung physiology. We deeply thank Dr. Primo Schar and Dr. Peter Byers for helpful discussion and critical reading of the manuscript. We are indebted to Dr. Marc Vidal for providing valuable advice, helpful discussion, and reagents for the yeast two-hybrid screening. We thank Dr. Paolo Dotto for generous support and encouragement during the first part of the project. We are grateful to Dr. J. Don Chen and Dr. Mark Pearson for providing the DNA constructs. We greatly appreciate Elio Biffali and Raimondo Pannone of the Molecular Biology Service at the Stazione Zoologica for excellent technical help and suggestions." @default.
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• W2031088051 title "The DNA Glycosylase T:G Mismatch-specific Thymine DNA Glycosylase Represses Thyroid Transcription Factor-1-activated Transcription" @default.
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