Persistent activation of the transcription factor Nuclear factor-κB (NF-κB) is central to the pathogenesis of many inflammatory disorders, including those of the lung such as cystic fibrosis (CF), asthma, and chronic obstructive pulmonary disease (COPD). Despite recent advances in treatment, management of the inflammatory component of these diseases still remains suboptimal. A20 is an endogenous negative regulator of NF-κB signaling, which has been widely described in several autoimmune and inflammatory disorders and more recently in terms of chronic lung disorders. However, the underlying mechanism for the apparent lack of A20 in CF, COPD, and asthma has not been investigated. Transcriptional regulation of A20 is complex and requires coordination of different transcription factors. In this review we examine the existing body of research evidence on the regulation of A20, concentrating on pulmonary inflammation. Special focus is given to the repressor downstream regulatory element antagonist modulator (DREAM) and its nuclear and cytosolic action to regulate inflammation. We provide evidence that would suggest the A20-DREAM axis to be an important player in (airway) inflammatory responses and point to DREAM as a potential future therapeutic target for the modification of phenotypic changes in airway inflammatory disorders. A schematic summary describing the role of DREAM in inflammation with a focus on chronic lung diseases as well as the possible consequences of altered DREAM expression on immune responses is provided.
Abstract Expression of the downstream regulatory element antagonist modulator (DREAM) protein in dorsal root ganglia and spinal cord is related to endogenous control mechanisms of acute and chronic pain. In primary sensory trigeminal neurons, high levels of endogenous DREAM protein are preferentially localized in the nucleus, suggesting a major transcriptional role. Here, we show that transgenic mice expressing a dominant active mutant of DREAM in trigeminal neurons show increased responses following orofacial sensory stimulation, which correlates with a decreased expression of prodynorphin and brain‐derived neurotrophic factor in trigeminal ganglia. Genome‐wide analysis of trigeminal neurons in daDREAM transgenic mice identified cathepsin L and the monoglyceride lipase as two new DREAM transcriptional targets related to pain. Our results suggest a role for DREAM in the regulation of trigeminal nociception. This article is part of the special article series “Pain” . image
Tissue-specific gene expression depends on the interaction between tissue-specific and general transcription factors. DREAM is a Ca2+-dependent transcriptional repressor widely expressed in the brain where it participates in nociception through its control of prodynorphin gene expression. In the periphery, DREAM is highly expressed in the thyroid gland, the immune system, and the reproductive organs. Here, we show that DREAM interacts with thyroid-specific transcription factor TTF-1 and regulates the expression of the thyroglobulin (Tg) gene. The mechanism also involves binding of DREAM to the thyroglobulin promoter and blockage of TTF-1-mediated transactivation. The TSH/cAMP pathway and Ca2+ signaling regulate DREAM-mediated transcriptional repression of the thyroglobulin gene. Furthermore, chromatin immunoprecipitation experiments in FRTL-5 cells confirmed that Tg is a bona fide target gene for DREAM transrepression in thyroid follicular cells. Tissue-specific gene expression depends on the interaction between tissue-specific and general transcription factors. DREAM is a Ca2+-dependent transcriptional repressor widely expressed in the brain where it participates in nociception through its control of prodynorphin gene expression. In the periphery, DREAM is highly expressed in the thyroid gland, the immune system, and the reproductive organs. Here, we show that DREAM interacts with thyroid-specific transcription factor TTF-1 and regulates the expression of the thyroglobulin (Tg) gene. The mechanism also involves binding of DREAM to the thyroglobulin promoter and blockage of TTF-1-mediated transactivation. The TSH/cAMP pathway and Ca2+ signaling regulate DREAM-mediated transcriptional repression of the thyroglobulin gene. Furthermore, chromatin immunoprecipitation experiments in FRTL-5 cells confirmed that Tg is a bona fide target gene for DREAM transrepression in thyroid follicular cells. The thyroid-differentiated phenotype is characterized by a diversity of proteins whose expression is either unique to thyroid follicular cells, such as thyroglobulin (Tg) 1The abbreviations used are: Tg, thyroglobulin; DRE, downstream regulatory element; CRE, cAMP response element; CREB, CRE-binding protein; EMSA, electrophoretic mobility shift assay; TTF, thyroid transcription factor; PI, phosphatidylinositol; TSH, thyroid-stimulating hormone; wt, wild type; CAT, chloramphenicol acetyltransferase; GST, glutathione S-transferase; RT, reverse transcriptase; ChIP, chromatin immunoprecipitation assay. 1The abbreviations used are: Tg, thyroglobulin; DRE, downstream regulatory element; CRE, cAMP response element; CREB, CRE-binding protein; EMSA, electrophoretic mobility shift assay; TTF, thyroid transcription factor; PI, phosphatidylinositol; TSH, thyroid-stimulating hormone; wt, wild type; CAT, chloramphenicol acetyltransferase; GST, glutathione S-transferase; RT, reverse transcriptase; ChIP, chromatin immunoprecipitation assay. and thyroperoxidase, or restricted to a few cell types such as thyrotropin receptor and sodium iodide symporter (1Civitareale D. Lonigro R. Sinclair A.J. Di Lauro R. EMBO J. 1989; 8: 2537-2542Crossref PubMed Scopus (321) Google Scholar, 2Francis-Lang H. Price M. Polycarpou-Schwarz M. Di Lauro R. Mol. Cell. Biol. 1992; 12: 576-588Crossref PubMed Scopus (208) Google Scholar, 3Spitzweg C. Joba W. Eisenmenger W. Heufelder A.E. J. Clin. Endocrinol. Metab. 1998; 83: 1746-1751Crossref PubMed Scopus (273) Google Scholar, 4Misrahi M. Loosfelt H. Atger M. Sar S. Guiochon-Mantel A. Milgrom E. Biochem. Biophys. Res. Commun. 1990; 166: 394-403Crossref PubMed Scopus (278) Google Scholar). Three thyroid-specific transcription factors, thyroid transcription factor (TTF)-1, TTF-2, and Pax-8, are responsible for thyroid-specific gene expression (5Damante G. Tell G. Di Lauro R. Prog. Nucleic Acids Res. Mol. Biol. 2001; 66: 307-356Crossref PubMed Google Scholar). TTF-1 is a homeoprotein whose expression is restricted to thyroid, lung, and the developing brain (6Lazzaro D. Price M. de Felice M. Di Lauro R. Development. 1991; 113: 1093-1104Crossref PubMed Google Scholar). TTF-2 contains a forkhead domain and, apart from thyroid, is also expressed in Rathke′s pouch (7Zannini 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 (217) Google Scholar). Pax-8 is a member of the paired box-containing proteins and is expressed in thyroid and in the brain and kidney during development (8Mansouri A. Chowdhury K. Gruss P. Nat. Genet. 1998; 19: 87-90Crossref PubMed Scopus (495) Google Scholar). The transcriptional activity of these three thyroid-specific proteins is regulated by phosphorylation (9Zannini 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 (68) Google Scholar), by changes in the redox state (10Kambe F. Nomura Y. Okamoto T. Seo H. Mol. Endocrinol. 1996; 10: 801-812PubMed Google Scholar), or by protein-protein interactions with other nucleoproteins (11Perrone L. Tell G. Di Lauro R. J. Biol. Chem. 1999; 274: 4640-4645Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 12Missero C. Pirro M.T. Simeone S. Pischetola M. Di Lauro R. J. Biol. Chem. 2001; 276: 33569-33575Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 13Decker P. Muller S. Curr. Pharm. Biotechnol. 2002; 3: 275-283Crossref PubMed Scopus (205) Google Scholar). Gene inactivation studies in mice have demonstrated that TTF-1, TTF-2, and Pax-8 are essential for the proper development and differentiation of the thyroid gland (8Mansouri A. Chowdhury K. Gruss P. Nat. Genet. 1998; 19: 87-90Crossref PubMed Scopus (495) Google Scholar, 14Kimura 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 (988) Google Scholar, 15De 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 (255) Google Scholar). Nevertheless, despite numerous studies in recent years, it is not yet possible to present an accurate model for their role in thyroid differentiation. One possibility is the involvement of additional, yet unknown, factor(s). In this regard, it has remained particularly elusive to understand the late expression of the Tg gene, from E14.5, when TTF-1, TTF-2, and Pax-8 are expressed from the beginning of thyroid development, at embryonic day E8.5 in the mouse (6Lazzaro D. Price M. de Felice M. Di Lauro R. Development. 1991; 113: 1093-1104Crossref PubMed Google Scholar). The action of a transcriptional repressor(s) in thyroid cells that may interact with TTF-1, TTF-2, or Pax-8 and block their activity at the promoter of thyroid-specific genes early during thyroid development has been proposed (16Pellizzari L. D'Elia A. Rustighi A. Manfioletti G. Tell G. Damante G. Nucleic Acids Res. 2000; 28: 2503-2511Crossref PubMed Scopus (59) Google Scholar).The transcriptional repressor DREAM is a calcium-binding protein with homology to members of the recoverin family of neuronal calcium sensors (17Carrion A.M. Link W.A. Ledo F. Mellstrom B. Naranjo J.R. Nature. 1999; 398: 80-84Crossref PubMed Scopus (488) Google Scholar). DREAM was identified through its binding to the downstream regulatory element (DRE) in the prodynorphin gene (17Carrion A.M. Link W.A. Ledo F. Mellstrom B. Naranjo J.R. Nature. 1999; 398: 80-84Crossref PubMed Scopus (488) Google Scholar, 18Carrion A.M. Mellstrom B. Naranjo J.R. Mol. Cell. Biol. 1998; 18: 6921-6929Crossref PubMed Scopus (88) Google Scholar). Binding of DREAM to DRE sequences is regulated by the level of nuclear Ca2+ (17Carrion A.M. Link W.A. Ledo F. Mellstrom B. Naranjo J.R. Nature. 1999; 398: 80-84Crossref PubMed Scopus (488) Google Scholar), by the interaction with other nucleoproteins (19Ledo F. Carrion A.M. Link W.A. Mellstrom B. Naranjo J.R. Mol. Cell. Biol. 2000; 20: 9120-9126Crossref PubMed Scopus (81) Google Scholar), and by the PI 3-kinase pathway (20Sanz C. Mellstrom B. Link W.A. Naranjo J.R. Fernandez-Luna J.L. EMBO J. 2001; 20: 2286-2292Crossref PubMed Scopus (86) Google Scholar). Unbinding of DREAM from the DRE sequence results in transcriptional derepression of target genes, e.g. prodynorphin (17Carrion A.M. Link W.A. Ledo F. Mellstrom B. Naranjo J.R. Nature. 1999; 398: 80-84Crossref PubMed Scopus (488) Google Scholar). Consistent with this mechanism, DREAM-deficient mice show an up-regulation of prodynorphin expression in spinal cord, which results in a hypoalgesic phenotype (21Cheng H.Y. Pitcher G.M. Laviolette S.R. Whishaw I.Q. Tong K.I. Kockeritz L.K. Wada T. Joza N.A. Crackower M. Goncalves J. Sarosi I. Woodgett J.R. Oliveira-dos-Santos A.J. Ikura M. van der Kooy D. Salter M.W. Penninger J.M. Cell. 2002; 108: 31-43Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Furthermore, through protein-protein interactions it has been recently shown that DREAM may regulate other transcriptional events not directly related to the presence of DRE sites (22Ledo F. Kremer L. Mellstrom B. Naranjo J.R. EMBO J. 2002; 21: 4583-4592Crossref PubMed Scopus (95) Google Scholar). Thus, the Ca2+-dependent DREAM/CREB interaction displaces CREB from CRE sites and prevents the recruitment of CBP to phospho-CREB, which results in a reduction of CRE-dependent transcription without DREAM binding to the CRE site (22Ledo F. Kremer L. Mellstrom B. Naranjo J.R. EMBO J. 2002; 21: 4583-4592Crossref PubMed Scopus (95) Google Scholar).In the periphery, expression of DREAM has been found in immune and reproductive organs as well as in the thyroid gland (17Carrion A.M. Link W.A. Ledo F. Mellstrom B. Naranjo J.R. Nature. 1999; 398: 80-84Crossref PubMed Scopus (488) Google Scholar). In this study, we have used the FRTL-5 thyroid follicular cell line to analyze the function of the transcriptional repressor DREAM in thyroid-specific gene expression. We show that DREAM interacts with the thyroid-specific transcription factor TTF-1 and regulates expression of the Tg gene. In addition, the mechanism involves binding of DREAM to the thyroglobulin promoter and blockage of transactivation mediated by TTF-1.MATERIALS AND METHODSCell Culture—Rat thyroid follicular FRTL-5 cells (ATCC CRL 8305; American Type Culture Collection) were kindly provided by Dr. L. D. Kohn (Edison Biotech Institute, Ohio University). The cells had the properties previously described (23Ambesi-Impiombato F.S. Parks L.A. Coon H.G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3455-3459Crossref PubMed Scopus (972) Google Scholar), were diploid, and their doubling time with TSH was 24-36 h. Cells were maintained in Coon's modified Ham's F-12 medium (Sigma) supplemented with 5% calf serum (Invitrogen) and six growth factor complement (6H) including TSH (0.5 milliunits/ml), insulin (10 μg/ml), somatostatin (10 ng/ml), hydrocortisone (10 nm), transferrin (5 μg/ml), and glycyl-histidyl-lysine (10 ng/ml). The culture medium referred to as 5H contains the same hormones as the 6H medium but without TSH and only 0.2% serum. Human HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum.Reporters and Expression Vectors—Two different Tg promoter constructs were used: TACAT-3 corresponds to the wild-type Tg promoter from -173 to +48 in the rat Tg gene and TACAT-14 (24Sinclair A.J. Lonigro R. Civitareale D. Ghibelli L. Di Lauro R. Eur. J. Biochem. 1990; 193: 311-318Crossref PubMed Scopus (95) Google Scholar), which contains mutations in the TgDRE core as indicated in Fig. 3. pBLCAT2, pTKDRECAT, pDRETKCAT, pHD1Luc, and pHD1mDRELuc have been described elsewhere (17Carrion A.M. Link W.A. Ledo F. Mellstrom B. Naranjo J.R. Nature. 1999; 398: 80-84Crossref PubMed Scopus (488) Google Scholar, 18Carrion A.M. Mellstrom B. Naranjo J.R. Mol. Cell. Biol. 1998; 18: 6921-6929Crossref PubMed Scopus (88) Google Scholar). RSV-Luc and pRL-CMV were used to correct for transfection efficiency. Plasmids encoding wild-type TTF-1 (25Guazzi S. Price M. De Felice M. Damante G. Mattei M.G. Di Lauro R. EMBO J. 1990; 9: 3631-3639Crossref PubMed Scopus (469) Google Scholar), TTF-1Δ3 and TTF-Δ14 deletions, and Gal4-TTF-1 constructs have been previously described (26De 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), as well as the plasmid pCMV-Pax-8 (27Zannini M. Francis-Lang H. Plachov D. Di Lauro R. Mol. Cell. Biol. 1992; 12: 4230-4241Crossref PubMed Scopus (271) Google Scholar). For analysis of DREAM activity we used expression vectors containing wild-type DREAM (wtDREAM) (17Carrion A.M. Link W.A. Ledo F. Mellstrom B. Naranjo J.R. Nature. 1999; 398: 80-84Crossref PubMed Scopus (488) Google Scholar) and different DREAM mutants; DREAM 2,3,4EF-hand mutant (EFmDREAM), contains mutations in three of the four EF-hand motifs resulting in a protein insensitive to calcium (17Carrion A.M. Link W.A. Ledo F. Mellstrom B. Naranjo J.R. Nature. 1999; 398: 80-84Crossref PubMed Scopus (488) Google Scholar). DREAM-L47,52V and DREAM-L155V encode LCDm-DREAM proteins with mutations in the first or the second LCD domain, respectively (19Ledo F. Carrion A.M. Link W.A. Mellstrom B. Naranjo J.R. Mol. Cell. Biol. 2000; 20: 9120-9126Crossref PubMed Scopus (81) Google Scholar).Transfections—Transient transfections in FRTL-5 and HeLa cells were carried out by the calcium phosphate DNA precipitation method. In experiments with TSH and ATP, the cells were grown in 5H medium and treated with 1 nm TSH or 0.5 mm ATP 12 h before harvest. Luciferase (Luc) and chloramphenicol acetyltransferase (CAT) activity were measured as described elsewhere (28Medina D.L. Rivas M. Cruz P. Barroso I. Regadera J. Santisteban P. Mol. Endocrinol. 2002; 16: 33-44Crossref PubMed Scopus (5) Google Scholar). In all cases, RSV-Luc (1 μg) or pRL-CMV (Renilla) (0.2 μg) were used to correct for transfection efficiency. Results are shown as relative activity compared with the controls in each experiment. The data are shown as mean ± S.D. of at least three independent experiments in triplicate. Statistical analysis of the results was performed using the Student's t test. For stable transfection, FRTL-5 cells received 10 μg of plasmid DNA expressing either wtDREAM, EFmDREAM, or empty pcDNA3 vector. Cells were selected after 3 weeks with 300 μg/ml G418 (Sigma).DNA Binding Assays—Band shift assays using 100 ng of recombinant DREAM or 10 μg of nuclear extracts from FRTL-5 cells were performed as previously described (19Ledo F. Carrion A.M. Link W.A. Mellstrom B. Naranjo J.R. Mol. Cell. Biol. 2000; 20: 9120-9126Crossref PubMed Scopus (81) Google Scholar). The Tg DRE sequence is: 5′-AAAGTGAGCCACTGCCCAGTCAAGTGTTCTTGA-3′. Oligonucleotides containing c-fosDRE or Sp1 have been previously described (17Carrion A.M. Link W.A. Ledo F. Mellstrom B. Naranjo J.R. Nature. 1999; 398: 80-84Crossref PubMed Scopus (488) Google Scholar, 18Carrion A.M. Mellstrom B. Naranjo J.R. Mol. Cell. Biol. 1998; 18: 6921-6929Crossref PubMed Scopus (88) Google Scholar). For supershift experiments the extracts were incubated for 1 h at room temperature in the presence of 1 μg of anti-Pax-8 (Santa Cruz Biotechnology), anti-TTF-1 (Biopat), or anti-DREAM (Ab 1013) antibodies and then incubated for 20 min with 80,000 cpm of probe in 20 μl of final volume.Western Blot Analysis—Nuclear or total cell extracts were prepared (28Medina D.L. Rivas M. Cruz P. Barroso I. Regadera J. Santisteban P. Mol. Endocrinol. 2002; 16: 33-44Crossref PubMed Scopus (5) Google Scholar) and protein concentration determined by the Bradford method (Bio-Rad). 30 μg of protein were resolved in SDS-PAGE and transferred to Protran membranes (Schleicher & Schuell). Anti-Tg antibody (0.5 μg/ml) was from Dako, anti-TTF-1 (0.5 μg/ml) from Biopat, anti-Pax-8 (0.5 μg/ml), anti β-actin (0.2 μg/ml), and anti-PARP (0.2 μg/ml) was from Santa Cruz Biotechnology. Affinity-purified anti-DREAM (0.5 μg/ml) (Ab 1013) was raised against recombinant DREAM (22Ledo F. Kremer L. Mellstrom B. Naranjo J.R. EMBO J. 2002; 21: 4583-4592Crossref PubMed Scopus (95) Google Scholar).Northern Blot Analysis—Total RNA was isolated and blots prepared using 20 μg of total RNA as described (28Medina D.L. Rivas M. Cruz P. Barroso I. Regadera J. Santisteban P. Mol. Endocrinol. 2002; 16: 33-44Crossref PubMed Scopus (5) Google Scholar). Hybridizations were performed with probes specific for Tg, TTF-1 (25Guazzi S. Price M. De Felice M. Damante G. Mattei M.G. Di Lauro R. EMBO J. 1990; 9: 3631-3639Crossref PubMed Scopus (469) Google Scholar), Pax-8 (27Zannini M. Francis-Lang H. Plachov D. Di Lauro R. Mol. Cell. Biol. 1992; 12: 4230-4241Crossref PubMed Scopus (271) Google Scholar), DREAM (17Carrion A.M. Link W.A. Ledo F. Mellstrom B. Naranjo J.R. Nature. 1999; 398: 80-84Crossref PubMed Scopus (488) Google Scholar), and β-actin (29Levi A. Eldridge J.D. Paterson B.M. Science. 1985; 229: 393-395Crossref PubMed Scopus (228) Google Scholar) labeled with [32P]dCTP by random priming.Qualitative RT-PCR—Qualitative RT-PCR was performed using total RNA from mouse thyroid gland or brain and specific primers, KChIP-1; forward 5′-GACACCACCCAGACAGGCTCT-3′ and reverse 5′-CAGAATGGCCAGTGTCCTCAGT-3′, KChIP-2; forward 5′-CAAGTTCACACGCAGAGAGT-3′ and reverse 5′-CCGAAGAATCACTGACAAAC-3′, DREAM/KChIP-3; forward 5′-AGCAAGAGGGAAGGCA-3′ and reverse 5′-GAAGAACTGGGAATAAATGA-3′, KChIP-4; forward 5′-CGTGAGAAGGGTGGAAAG-3′ and reverse 5′-GCAGGAGACGACGTTTTG-3′; β-actin, forward 5′-TGGAATCCTGTGGCATCCATGAAAC-3′ and reverse 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′. PCR amplification was carried out for 40 cycles: denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s.Protein-Protein Interaction Assays—For co-immunoprecipitation experiments, FRTL-5 cells were lysed in Nonidet P-40 lysis buffer containing 50 mm Tris, pH 8.0, 150 mm NaCl, 1% Nonidet P-40 and a protease inhibitor mixture (Calbiochem). Co-immunoprecipitation was performed overnight at 4 °C using monoclonal antibody 1B1 (22Ledo F. Kremer L. Mellstrom B. Naranjo J.R. EMBO J. 2002; 21: 4583-4592Crossref PubMed Scopus (95) Google Scholar). Immunocomplexes were captured with protein A/G-Sepharose for 1 h, and pellets were washed three times in Nonidet P-40 buffer. Protein complexes were eluted in SDS sample buffer and subjected to Western blotting.For pull-down studies, TTF-1 and its truncated forms were 35S-labeled in vitro using the transcription/translation T7-TnT system (Promega). Equimolar amounts of recombinant GST and GST-DREAM proteins (∼15-20 pmol) bound to glutathione-Sepharose (Amersham Biosciences) were incubated with the labeled proteins in interaction buffer (20 mm potassium Hepes, pH 7.5, 10% glycerol, 150 mm KCl, 2 mm MgCl2, 0.5 mm EGTA, 1 mm dithiothreitol, 0.1% Nonidet P-40, 0.5% Blotto (Bio-Rad), and protease inhibitor mixture (Calbiochem)). After five washes in the same buffer, bound proteins were eluted with SDS sample buffer, resolved in SDS-PAGE, and detected with fluorography.Chromatin Immunoprecipitation Assay—We performed chromatin immunoprecipitation using a previously published method (30Takahasi Y. Rayman J. Dynlacht B. Genes Dev. 2000; 14: 804-816PubMed Google Scholar). Briefly, ∼6 × 107 FRTL-5 cells cultured in 5H for 7 days and untreated or treated with 1 nm TSH were cross-linked, nuclei were collected, and chromatin were sonicated to a length of between 500 and 2000 bp. The sheared chromatin was directly precleared with blocked protein-A/G Sepharose and used for immunoprecipitation, with or without 10 μg affinity-purified polyclonal antibody 1013 for DREAM. Immunoprecipitated DNA was subjected to semiquantitative PCR with primers to amplify the promoter region of thyroglobulin forward 5′-CGGGAGCAGACTCAAGTAGAGG-3′ and reverse 5′-TTTATAGCACAGTGGCAAGCAGTG-3′; c-fos promoter forward 5′-CAGACTGAGACGGGGGTTGA-3′ and reverse 5′-GGCGAGGGGTCCAGGGGTAGAC-3′ or β-actin forward 5′-GAAGCTGTGCTATGTGCCCTAGA-3′ and reverse 5′-TGCCGATAGTGATGACCTGACCGT-3′. All ChIP results were confirmed in at least three separated experiments.RESULTSDREAM Is Expressed in Follicular Thyroid Cells—To investigate the functional role of the high basal levels of DREAM mRNA in the thyroid gland (17Carrion A.M. Link W.A. Ledo F. Mellstrom B. Naranjo J.R. Nature. 1999; 398: 80-84Crossref PubMed Scopus (488) Google Scholar) we used rat FRTL-5 thyroid follicular cells, a model that has been extensively used to study thyroid cell differentiation. Follicular cells represent the predominant phenotype in the thyroid gland and are responsible for the expression and secretion of thyroglobulin and thyroid hormones. Northern blot analysis detected the expression of DREAM mRNA in FRTL-5 cells (Fig. 1A). Western blot analysis using nuclear extracts from FRTL-5 cells (Fig. 1B) confirmed the Northern blot results and showed that the levels of DREAM protein in FRTL-5 cells are comparable to the levels in NB69 cells, the human neuroblastoma cell line where DREAM activity was first characterized (18Carrion A.M. Mellstrom B. Naranjo J.R. Mol. Cell. Biol. 1998; 18: 6921-6929Crossref PubMed Scopus (88) Google Scholar). In keeping with previous reports (17Carrion A.M. Link W.A. Ledo F. Mellstrom B. Naranjo J.R. Nature. 1999; 398: 80-84Crossref PubMed Scopus (488) Google Scholar, 18Carrion A.M. Mellstrom B. Naranjo J.R. Mol. Cell. Biol. 1998; 18: 6921-6929Crossref PubMed Scopus (88) Google Scholar), DREAM mRNA or protein was not detected in HeLa cells (Fig. 1, A and B). Post-translational modifications of the DREAM protein may account for the appearance of the double band in the Western blot. Alternatively, the multiple bands may correspond to other KChIP proteins closely related to DREAM (An et al., Ref. 46An W.F. Bowlby M.R. Betty M. Cao J. Ling H.P. Mendoza G. Hinson J.W. Mattsson K.I. Strassle B.W. Trimmer J.S. Rhodes K.J. Nature. 2000; 403: 553-556Crossref PubMed Scopus (834) Google Scholar). To assess this possibility we used total RNA isolated from mouse thyroid gland and specific primers for each one of the KChIP transcripts. RT-PCR-amplified bands corresponding exclusively to DREAM and KChIP-2 mRNAs are shown in Fig. 1C. As a positive control, amplification of all KChIPs in mouse brain total RNA is also shown. Direct PCR on rat genomic DNA did not amplify any of the DREAM/KChIP bands (data not shown). Taken together these data indicate that DREAM and KChIP-2 but not KChIP-1 or -4 are expressed in the thyroid gland.Fig. 1Detection of DREAM in thyroid-derived FRTL-5 cells.A, Northern blot analysis of total RNA from rat brain, HeLa, and FRTL-5 cells. B, Western blot analysis of nuclear extracts from FRTL-5 cells. For comparison, neuroblastoma NB69 and HeLa cells are shown. C, RT-PCR analysis of DREAM/KChIP isoforms expressed in the thyroid gland. For comparison, expression of all the DREAM/KChIP genes in mouse brain is shown. D, DRE-dependent repression of the pTKDRECAT; E, pHD1Luc reporters in FRTL-5 cells, compared with the absence of DRE in pBLCAT2 and to the DRE-mutated reporter pHD1mDRELuc, respectively. Plasmids RSV-Luc and pRL-CMV were used to correct for transfection efficiency. Asterisks represent statistically significant differences between the means relative to corresponding controls. ***, p < 0.001; **, p < 0.01, Student's t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further substantiate these results, we assessed DRE-dependent repression by the endogenously expressed DREAM protein in FRTL-5 cells. For that we performed transient transfections using reporter plasmids containing DRE binding sites. The activity of a TKDRECAT reporter containing the prodynorphin DRE (18Carrion A.M. Mellstrom B. Naranjo J.R. Mol. Cell. Biol. 1998; 18: 6921-6929Crossref PubMed Scopus (88) Google Scholar) was reduced when compared with the basal activity of empty reporter vector pBLCAT2 (Fig. 1D). Moreover, in agreement with previous results in NB69 cells (18Carrion A.M. Mellstrom B. Naranjo J.R. Mol. Cell. Biol. 1998; 18: 6921-6929Crossref PubMed Scopus (88) Google Scholar), the activity of a reporter containing 1.8 kb of the prodynorphin promoter with a single mutation in the DRE site pHD1mutDRELuc was significantly higher than the activity of a similar reporter pHD1DRELuc containing the wild-type prodynorphin promoter (Fig. 1E). No difference in activity of the different reporters was observed after transfection in HeLa cells (data not shown), as previously reported (18Carrion A.M. Mellstrom B. Naranjo J.R. Mol. Cell. Biol. 1998; 18: 6921-6929Crossref PubMed Scopus (88) Google Scholar). These data indicate that follicular thyroid cells express DREAM and that in FRTL-5 cells DREAM functions as a DRE-dependent transcriptional repressor.DREAM Binds To and Regulates the Thyroglobulin Promoter—Follicular cells represent the vast majority of the cell population in the thyroid gland and are responsible for the expression of the most representative gene in thyroid function, Tg, from which the thyroid hormones are derived. In a first attempt to understand the role of DREAM in thyroid physiology we focused on follicular cells and the potential role of DREAM in the mechanisms that control Tg gene expression. Sequence analysis of the Tg promoter showed the presence of a DRE core motif (AGTCAAG), hereafter referred to as TgDRE, 70 base pairs upstream from the TATA box (Fig. 2A). Interestingly, the TgDRE sequence overlaps with a key regulatory region known as the C element that includes the binding sites for TTF-1 and Pax-8, the two main regulators of Tg transcription and thyroid differentiation (27Zannini M. Francis-Lang H. Plachov D. Di Lauro R. Mol. Cell. Biol. 1992; 12: 4230-4241Crossref PubMed Scopus (271) Google Scholar, 31Santisteban P. Acebron A. Polycarpou-Schwarz M. Di Lauro R. Mol. Endocrinol. 1992; 6: 1310-1317Crossref PubMed Scopus (75) Google Scholar). Thus, we tested the possibility that DREAM could participate in Tg gene expression acting at the TgDRE site. Using recombinant DREAM in EMSA, a specific retarded TgDRE/DREAM complex was observed, which could be competed by cold TgDRE or c-fosDRE but was not affected by competition with cold unrelated oligonucleotides (Fig. 2B). Furthermore, EMSA using nuclear extracts from FRTL-5 cells resulted in a TgDRE retarded band competed by DRE-containing oligonucleotides (Fig. 2C and data not shown). Incubation with a DREAM antibody substantially reduced the TgDRE-retarded band indicating the involvement of endogenous DREAM in the retardation. Moreover, an antibody specific for TTF-1 supershifted the TgDRE retarded band while a Pax-8-specific antibody had only a modest effect (Fig. 2D). These results indicate that the TgDRE sequence is able to bind a nuclear complex containing at least DREAM and TTF-1.Fig. 2The Tg promoter contains a functional DRE.A, schematic representation of the Tg promoter occupancy by the DNA-binding proteins present in rat FRTL-5 cells. The DNA sequences of the TgDRE probe used in band shift assays are shown. The core DRE sequence in TgDRE is in bold and marked with an arrow. EMSA using the TgDRE as a probe together with recombinant DREAM protein (B) or nuclear extracts from FRTL-5 cells (C and D) is shown. Competitions with a 50-fold (B) or a 25- or 50-fold excess (C) of related (TgDRE and c-fosDRE) or non-related (Sp1) cold oligonucleotides are shown (B and C). In D, the TgDRE-retarded band is competed with an anti-DREAM antibody but only slightly affected with an anti-Pax-8 antibody. An anti-TTF-1 antibody supershifted the TgDRE band as indicated by the asterisk.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further analyze the functionality of the TgDRE, FRTL-5 cells were transiently transfected with a DREAM expression vector together with the TACAT-3 or TACAT-14 reporters (24Sinclair A.J. Lonigro R. Civitareale D. Ghibelli L. Di Lauro R. Eur. J. Biochem. 1990; 193: 311-318Crossref PubMed Scopus (95) Google Scholar), which contain the Tg proximal promoter wild type or with mutations that affect the TgDRE core, respectively (Fig. 3A). Overexpression of DREAM resulted in a 50% inhibition of the Tg reporter activity (Fig. 3B). By contrast, DREAM could not decrease the activity of the TACAT-14 reporter (Fig. 3B). Mutations in TACAT-14 impair binding of TTF-1, which accounts for its lower basal activity (24Sinclair A.J. Lonigro R. Civitareale D. Ghibelli L. Di Lauro R. Eur. J. Biochem. 1990; 193: 311-318Crossref PubMed Scopus (95) Google Scholar). Importantly, binding of DREAM to TACAT-14 was also blocked, while the mutations did not affect the binding of Pax-8 (Fig. 3C). Taken together, these data suggest that binding of DREAM to the TgDRE is important to repress the Tg promoter. However, because binding of TTF-1 to the TACAT-14 mutant was also abolished, an effect of DREAM on TTF-1 transcriptional activity on the Tg promoter cannot be discarded.DREAM Interacts with TTF-1—Increasing evidence suggests that TTF-1 functions cooperatively with a number of other transcription factors to form transcriptionally active complexes on regulatory regions of target genes (32Sever-Chroneos Z. Bachurski C.J. Yan C. Whitsett J.A. Am. J. Physiol. 1999; 277: L79-88PubMed Google Scholar, 33Yan C. Naltner A. Conkright J. Ghaffari M. J. Biol. Chem. 2001; 276: 21686-21691Abs
DREAM/calsenilin/KChIP3 is a calcium binding protein of the neuronal calcium sensor superfamily. DREAM interacts with DRE (downstream regulatory element) sites in the DNA to regulate transcription and with many proteins to exert specialized functions in different subcellular compartments. Work from different laboratories has identified a growing list of interacting proteins that constitutes the DREAM interactome. The knowledge of these interactions has greatly contributed to the understanding of the various physiological functions of DREAM.
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