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Originally published In Press as doi:10.1074/jbc.M403526200 on June 4, 2004

J. Biol. Chem., Vol. 279, Issue 32, 33114-33122, August 6, 2004
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Transcriptional Repressor DREAM Interacts with Thyroid Transcription Factor-1 and Regulates Thyroglobulin Gene Expression*

Marcos Rivas{ddagger}§, Britt Mellström{ddagger}, José R. Naranjo{ddagger}||, and Pilar Santisteban§

From the {ddagger}Dpto. Biología Molecular y Celular, Centro Nacional de Biotecnología, CSIC 28049 Madrid, Spain and the §Instituto de Investigaciones Biomédicas, CSIC 28029 Madrid, Spain

Received for publication, March 30, 2004 , and in revised form, May 18, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The thyroid-differentiated phenotype is characterized by a diversity of proteins whose expression is either unique to thyroid follicular cells, such as thyroglobulin (Tg)1 and thyroperoxidase, or restricted to a few cell types such as thyrotropin receptor and sodium iodide symporter (1-4). Three thyroid-specific transcription factors, thyroid transcription factor (TTF)-1, TTF-2, and Pax-8, are responsible for thyroid-specific gene expression (5). TTF-1 is a homeoprotein whose expression is restricted to thyroid, lung, and the developing brain (6). TTF-2 contains a forkhead domain and, apart from thyroid, is also expressed in Rathke's pouch (7). Pax-8 is a member of the paired box-containing proteins and is expressed in thyroid and in the brain and kidney during development (8). The transcriptional activity of these three thyroid-specific proteins is regulated by phosphorylation (9), by changes in the redox state (10), or by protein-protein interactions with other nucleoproteins (11-13). 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 (8, 14, 15). 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 (6). 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 (16).

The transcriptional repressor DREAM is a calcium-binding protein with homology to members of the recoverin family of neuronal calcium sensors (17). DREAM was identified through its binding to the downstream regulatory element (DRE) in the prodynorphin gene (17, 18). Binding of DREAM to DRE sequences is regulated by the level of nuclear Ca2+ (17), by the interaction with other nucleoproteins (19), and by the PI 3-kinase pathway (20). Unbinding of DREAM from the DRE sequence results in transcriptional derepression of target genes, e.g. prodynorphin (17). Consistent with this mechanism, DREAM-deficient mice show an up-regulation of prodynorphin expression in spinal cord, which results in a hypoalgesic phenotype (21). 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 (22). 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 (22).

In the periphery, expression of DREAM has been found in immune and reproductive organs as well as in the thyroid gland (17). 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 METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell 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 (23), 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 (24), which contains mutations in the TgDRE core as indicated in Fig. 3. pBLCAT2, pTKDRECAT, pDRETKCAT, pHD1Luc, and pHD1mDRELuc have been described elsewhere (17, 18). RSV-Luc and pRL-CMV were used to correct for transfection efficiency. Plasmids encoding wild-type TTF-1 (25), TTF-1{Delta}3 and TTF-{Delta}14 deletions, and Gal4-TTF-1 constructs have been previously described (26), as well as the plasmid pCMV-Pax-8 (27). For analysis of DREAM activity we used expression vectors containing wild-type DREAM (wtDREAM) (17) 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 (17). DREAM-L47,52V and DREAM-L155V encode LCDm-DREAM proteins with mutations in the first or the second LCD domain, respectively (19).



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  		FIG. 3.
The TgDRE site mediates Tg promoter repression by DREAM. A, schematic representation of the TACAT-3 reporter vector containing the minimal Tg promoter. Mutations in the TACAT-14 reporter are shown. B, FRTL-5 cells were transiently transfected with empty pcDNA3 (gray bars) or DREAM (black bars), together with the indicated reporter vector. Asterisks represent statistically significant differences between the means relative to corresponding controls. ***, p < 0.001, Student's t test. C, EMSA using purified recombinant proteins and oligonucleotide probes containing wild-type TgDRE or the mutations introduced in TACAT-14, as indicated.

 
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 (28). 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 (19). The Tg DRE sequence is: 5'-AAAGTGAGCCACTGCCCAGTCAAGTGTTCTTGA-3'. Oligonucleotides containing c-fosDRE or Sp1 have been previously described (17, 18). 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 (28) 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 {beta}-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 (22).

Northern Blot Analysis—Total RNA was isolated and blots prepared using 20 µg of total RNA as described (28). Hybridizations were performed with probes specific for Tg, TTF-1 (25), Pax-8 (27), DREAM (17), and {beta}-actin (29) 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'; {beta}-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 (22). 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 (30). Briefly, ~6 x 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 {beta}-actin forward 5'-GAAGCTGTGCTATGTGCCCTAGA-3' and reverse 5'-TGCCGATAGTGATGACCTGACCGT-3'. All ChIP results were confirmed in at least three separated experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DREAM Is Expressed in Follicular Thyroid Cells—To investigate the functional role of the high basal levels of DREAM mRNA in the thyroid gland (17) 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 (18). In keeping with previous reports (17, 18), 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. 46). 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.



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  		FIG. 1.
Detection 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.

 
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 (18) was reduced when compared with the basal activity of empty reporter vector pBLCAT2 (Fig. 1D). Moreover, in agreement with previous results in NB69 cells (18), 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 (18). 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 (27, 31). 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.



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  		FIG. 2.
The 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.

 
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 (24), 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 (24). 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 (32-34). Given that DREAM binds to a region overlapping the binding site for TTF-1 in the Tg promoter, we next investigated whether DREAM could interact with TTF-1. Coimmunoprecipitation experiments showed that an anti-DREAM antibody efficiently coimmunoprecipitated endogenous TTF-1 protein in FRTL-5 cells (Fig. 4A) whereas an anti-TTF-1 antibody efficiently immunoprecipitated DREAM (Fig. 4B). To investigate the domains in TTF-1 responsible for the interaction with DREAM we used pull-down assays using a GST-DREAM fusion protein and 35S-labeled in vitro-translated TTF-1 full-length protein or truncated forms (Fig. 5A). TTF-1{Delta}14 contains the N-terminal half including the homeodomain and the main transactivating domain, and TTF-1{Delta}3 harbors the central homeodomain and a second transactivation domain located at the C-terminal (Fig. 5A) (26, 35). Importantly, GST-DREAM was able to pull-down TTF-1 and TTF-1{Delta}14 but not {Delta}3, indicating that DREAM interacts with a region in the N-terminal of TTF-1 but not with the homeodomain, also present in TTF-1{Delta}3 (Fig. 5B). Taken together, pull-down and coimmunoprecipitation results suggest a direct protein-protein interaction between DREAM and the N-terminal region of TTF-1. Furthermore, since two leucine-charged residue-rich domains (LCDs) present in DREAM (Fig. 6A) have been previously shown to be involved in the interaction between DREAM and CREM or CREB proteins (19, 22), GST fusion proteins with the DREAM protein mutated in either LCD were also included in similar pull-down experiments. Interestingly, mutation of the LCDs did not affect the interaction with TTF-1 (Fig. 6B) indicating that the LCDs in DREAM do not mediate the interaction with TTF-1.



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  		FIG. 4.
DREAM directly interacts with TTF-1. Coimmunoprecipitation of TTF-1 (A) or DREAM (B) endogenously expressed in FRTL-5 cells using monoclonal antibody 1B1 against DREAM or an anti-TTF-1 antibody. No coimmunoprecipitation is observed using a non-related antibody. In both cases the input corresponds to 5% of the total.

 



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  		FIG. 5.
DREAM interacts with the N-terminal domains of TTF-1. A, schematic representation of TTF-1 proteins and truncated fragments. HD, homeodomain; A, activation domain; R, repression domain. B, pull-down analysis of the interaction between DREAM and TTF-1 In all cases the input corresponds to 10%.

 



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  		FIG. 6.
DREAM LCD mutants interact with TTF-1. A, schematic representation of the different domains in the DREAM protein. LCD, leucine-charged residue-rich domain; EF, EF-hand motif for Ca2+ binding. B, pull-down analysis of the interaction between DREAM LCD mutants and TTF-1. The input corresponds to 10%.

 
DREAM Interferes with TTF-1 Transactivating Function—The N-terminal region of TTF-1 contains the main transactivating domain in TTF-1. To investigate if binding of DREAM could affect TTF-1 transactivation, we cotransfected the pG5Luc reporter, containing five GAL4 binding sites, together with the DNA binding domain of GAL4 fused to the N-terminal activation domain of TTF-1 in construct {Delta}G-21 (Fig. 7A). The experiments were performed in HeLa cells, which do not express endogenous DREAM. As a control, the C-terminal activation domain of TTF-1 fused to Gal4 in construct {Delta}G-13 (Fig. 7A) was used in these experiments. The {Delta}G-21 fusion protein was more potent than {Delta}G-13 to transactivate the pG5Luc reporter as previously shown (26). Interestingly, while cotransfection with DREAM totally blocked {Delta}G-21 transactivation it did not affect the activity of {Delta}G-13 (Fig. 7B). Furthermore, the DREAM LCD double mutant that still is able to interact with TTF-1, repressed to the same extent the transactivation induced by {Delta}G-21 without affecting the activity of {Delta}G-13 (data not shown). To further support these results, the fusion protein {Delta}G-21 was coimmunoprecipitated with an anti-DREAM antibody when co-expressed with DREAM in HeLa cells (Fig. 7C). These results demonstrate that the interaction of DREAM with the N-terminal of TTF-1 blocks the activation domain in this region.



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  		FIG. 7.
DREAM blocks transactivation mediated by the N-terminal activation domain of TTF-1. A, schematic representation of the Gal4-{Delta}TTF-1 fusion proteins used. Box represents the Gal4 DNA binding domain fused to TTF-1 residues 51-123 ({Delta}G21) or 295-372 ({Delta}G13). B, basal activity and transactivation of the pG5 reporter by {Delta}G21 and {Delta}G13. Increasing amounts of DREAM expression vector in the transfection (1 or 2 µg) repress transactivation by {Delta}G21 but do not modify activation by {Delta}G13. C, coimmunoprecipitation of {Delta}G21-TTF-1 and DREAM after overexpression in HeLa cells.

 
DREAM Regulates Thyroglobulin Gene Expression in FRTL-5 Cells—To validate the functional significance of DREAM regulation of Tg gene expression, we prepared FRTL-5 clones stably transfected with either wtDREAM or EFm-DREAM. Four clones of each type showing expression of DREAM protein above the levels in mock-transfected cells were selected for the experiments. Importantly, overexpression of wtDREAM as well as EFmDREAM decreased Tg mRNA and Tg protein levels in FRTL-5 clones (Fig. 8 and data not shown). These results support the data from transient transfection experiments and confirm a role of DREAM in Tg gene expression in thyroid follicular cells. Stable overexpression of wtDREAM and the reduction in Tg gene expression were not associated with decreased cell viability as shown by the unchanged processing of poly(ADP-ribose) polymerase (PARP) (Fig. 8B), a marker of caspase 3- or 7-dependent apoptosis (13). Moreover, no difference was observed in the level of {beta}-actin (Fig. 8) between non-transfected FRTL-5 cells, mock-transfected clones, and clones overexpressing DREAM proteins.



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  		FIG. 8.
Characterization of stably transfected FRTL-5 clones expressing wtDREAM. A, Northern blot; B, Western blots from FRTL-5 transfectants expressing wtDREAM compared with control FRTL-5 mock-transfected cells.

 
TSH Regulates DREAM Repression of the Tg Promoter—Maintenance of the differentiated phenotype and Tg gene expression in thyroid follicular cells is largely controlled by TSH (36), a potent inducer of cAMP. Basal growth conditions for FRTL-5 cells include the presence of TSH in the culture medium. To further understand the regulation of the Tg promoter in follicular cells, we analyzed the effect of TSH on DREAM-mediated repression. For that we first checked binding to the TgDRE site in the presence and in the absence of TSH in the medium. Removal of TSH from the culture medium resulted in a substantial increase in the intensity of the TgDRE-retarded band and re-exposure to TSH or Ca2+ mobilization with ATP reduced the TgDRE band to control levels (Fig. 9A). This increase in the absence of TSH (compare lanes 1 and 2) was specific because it was not observed with a CRE probe and the same nuclear extracts (compare lanes 5 and 6 in Fig. 9A). Thus, similar to other cell systems, the increase in cAMP after TSH stimulation and the rise in calcium reduced binding of endogenous DREAM to the TgDRE probe. Consequently, unbinding from the TgDRE following TSH exposure induced Tg reporter transcription (Fig. 9B) and Tg mRNA levels (Fig. 9C). Importantly, TSH treatment has been shown to increase binding of TTF-1 to the Tg promoter (10), which would also contribute to increase Tg expression. Furthermore, treatment with 8-Br-cAMP or increase in cytosolic Ca2+ concentration following ATP, ionophores or caffeine administration to the culture medium, also resulted in transactivation of Tg gene expression (Fig. 9C). These data support a Ca2+- and cAMP-dependent derepression of the Tg gene in FRTL-5 cells, a mechanism first described in neuroblastoma cells (17, 19). To further substantiate a role for Ca2+ in the regulation of Tg expression, we performed transient transfections using a dominant negative DREAM mutant insensitive to calcium stimulation (EFm-DREAM). Cotransfection with EFmDREAM repressed Tg reporter activity, as previously shown for wtDREAM and partially blocked the effect of ATP- or caffeine-induced Ca2+ release on Tg transactivation (Fig. 9B and results not shown). Interestingly, EFmDREAM was without effect on TSH-induced Tg promoter activation (Fig. 9B).



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  		FIG. 9.
TSH regulates DREAM-dependent Tg gene expression. A, EMSA using nuclear extracts from FRTL-5 cells maintained in 6H medium (lanes 1 and 5) or 5H medium (lanes 2-4 and 6-8). TSH (lanes 3 and 7) or ATP (lanes 4-8) were added for 12 h. The TgDRE (lanes 1-4) or a CRE (lanes 5-8) were used as probes. B, FRTL-5 cells maintained in 5H medium were transfected with the Tg reporter TACAT-3 and the indicated expression vectors. Reporter activity was analyzed 12 h after treatment with TSH (black bars) or ATP (gray bars). RSV-Luc was used to correct for transfection efficiency. C, Northern blot analysis of Tg mRNA levels. FRTL-5 cells were maintained in 5H medium for 7 days and then incubated for 10 h with complete medium (6H) or the indicated treatments. Hybridization to the 18 S rRNA is shown.

 
To further strengthen the physiological relevance of DREAM in the regulation of the Tg promoter we performed chromatin immunoprecipitation experiments in FRTL-5 cells in the absence and in the presence of TSH in the culture medium. A fragment encompassing the TgDRE site in the Tg proximal promoter was specifically amplified after immunoprecipitation using an anti-DREAM antibody from chromatin of FRTL-5 cells maintained in the presence of TSH (Fig. 10, lane 6). Importantly, the amount of immunoprecipitated material and therefore the intensity of the amplified band was notably stronger, compare lanes 3 and 6, when the same protocol was applied to chromatin extracted from FRTL-5 cells grown in 5H media without TSH (Fig. 10, lane 3). As a control, differential amplification of the c-fos promoter in the presence or the absence of TSH treatment was observed in the same immunoprecipitated chromatin, whereas no amplification was detected with primers for the {beta}-actin proximal promoter (Fig. 10). The presence of a DRE site in the c-fos proximal promoter and the induction of c-fos following TSH stimulation have been previously reported (17, 37). Furthermore, no amplified band was observed with specific primers for human Tg and immunoprecipitated chromatin from HeLa cells (data not shown).



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  		FIG. 10.
Chromatin immunoprecipitation showing occupancy of the Tg promoter by endogenous DREAM in FRTL-5 cells, with or without TSH treatment. Input corresponds to 1% of the total amount of chromatin from FRTL-5 cells used in immunoprecipitation reactions. Control reactions lacking the anti-DREAM antibody are shown. Differential occupancy of the proximal c-fos promoter by DREAM is shown as a positive control. No amplification of the {beta}-actin promoter after chromatin immunoprecipitation with the DREAM antibody is shown as a negative control. PCR-amplified products were detected by autoradiography.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results suggest that in FRTL-5 thyroid follicular cells DREAM regulates Tg gene expression through a mechanism that involves direct binding to the Tg promoter and interaction with the N-terminal region of TTF-1. As a result, DREAM blocks TTF-1-mediated transactivation of Tg gene expression. The N-terminal region of TTF-1 contains the main activation domain (26, 38) and is responsible for the interaction between TTF-1 and Pax-8 that is essential for the activation of Tg gene expression (39). In addition, the N-terminal part of TTF-1 is required for the interaction between TTF-1 and co-activators like TBP, p300, and TAF (21, 38, 40). Thus, binding of DREAM to the N-terminal of TTF-1 may preclude the recruitment of other nucleoproteins to the mediator complex and in this way block TTF-1 transactivating function. A similar mechanism has been proposed after binding of DREAM to the KID domain in CREB, which blocks CBP recruitment and CRE-dependent transactivation (22). Furthermore, blockage of TTF-1-activated transcription has also been described for the co-repressor TDG, which binds to TTF-1 and blocks the second transactivating domain located at the C terminus (12).

Binding of DREAM or dominant negative DREAM mutants to DRE sites downstream from the TATA box in the prodynorphin and the c-fos genes repress their transcription in neurons and PC12 cells (17). In these conditions, the mechanism for the repression involves changes in the acetylation status of the chromatin.2 Early work using artificial reporters with DRE sites upstream from the TATA box did not show a DRE-mediated repressor effect in neuroblastoma cells (18). In thyroid follicular cells, however, DREAM represses Tg gene expression though the TgDRE is located upstream from the initiation site. Nevertheless, in these cells DREAM does not repress basal activity of a heterologous DRETKCAT reporter in which the DRE is upstream from the TATA box of the thymidine kinase promoter (data not shown). Accordingly, we propose a model in which the main mechanism is the blockage by DREAM of Tg transactivation by TTF-1. In addition, binding of DREAM to the TgDRE site might prevent the approach of other activators and/or favoring the recruitment of tissue-specific repressors. The latter mechanism could account for the lack of repressor activity of DREAM in thyroid follicular cells on the DRETKCAT reporter.

Thyroid follicular cell differentiation and thyroid function in the adulthood are maintained by the tonic TSH stimulation from the pituitary gland (36, 41, 42). TSH receptor activation leads to an increase in cAMP levels that ultimately induces Tg gene expression in vivo (36, 43). In addition, it has been proposed that at higher concentrations TSH can also trigger a simultaneous rise in cytosolic free Ca2+ (44). Here we show, that TSH stimulation reduces binding of DREAM to the Tg-DRE and derepresses Tg expression. This effect is not blocked by the Ca2+-insensitive mutant EFmDREAM indicating that, for the induction of Tg expression, TSH at physiological concentrations is not signaling through changes in Ca2+ concentration in FRTL-5 cells. Other proposed mechanisms by which TSH increases Tg expression are the increase in Pax-8 protein levels (38) and the repression of Hex, a homeodomain-containing transcriptional repressor, which like DREAM represses the Tg promoter by direct DNA binding to an area overlapping oligo C, the TTF-1 and Pax-8 binding region (16). Interestingly, recombinant DREAM does not bind in vitro to a probe containing exclusively the oligo C sequence (data not shown) indicating that DREAM does not bind exactly to the same region in the Tg promoter as the TTF-1/Pax-8 complex.

Binding of DREAM to DRE sequences is regulated by calcium, cAMP, and the PI 3-kinase pathway (17, 19, 20). In FRTL-5 cells grown in the absence of TSH, exposure to calcium ionophores or calcium release from cytosolic stores by ATP or caffeine resulted in a substantial increase in Tg promoter activity followed by an accumulation of Tg mRNA. The calcium-insensitive EFmDREAM mutant partially blocked the ATP-induced Tg expression indicating a calcium-dependent derepression mechanism for Tg transactivation in FRTL-5 cells similar to the one previously described in neurons (17). The physiological meaning of the Ca2+ signaling to regulate Tg gene expression in the thyroid gland remains to be further analyzed.

Early description of the DREAM repressor reported its presence both in the cytoplasm and in the nucleus of neuroblastoma cells and in HEK293 cells after transfection (17). Interestingly, work carried out in other laboratories has identified two proteins identical to DREAM, calsenilin, and KChIP-3, with specific functions in the cytoplasm (45, 46). In the first case, DREAM/calsenilin was reported to interact in neurons with presenilin 2 regulating its proteolytic fragmentation (45, 47) and the capacity of presenilins to release calcium from the endoplasmic reticulum (47). In the second case, DREAM/KChIP-3 was found to interact with voltage-dependent K+ channels of the Kv4 class modulating potassium currents in a calcium-dependent manner in the plasma membrane (46). Importantly, three proteins, KChIP-1, -2, and -4 with high homology to DREAM and similar activity on Kv4 channels and repressor properties (46, 48)3 have been so far described. While all four DREAM/KChIP genes are expressed in mouse brain, only DREAM and KChIP-2 mRNAs were detected in the thyroid gland. In FRTL-5 cells, we have observed the presence of DREAM in both cellular compartments (data not shown) indicating that in addition to the control of thyroid-dependent gene expression, DREAM may have other yet unknown functions in the cytoplasm that could be relevant for directing follicular cell differentiation and to regulate thyroid physiology.

TTF-1, also known as Nkx2.1 or T/ebp, has been found essential for the correct development of the ventral telencephalon and the lungs, as well as the thyroid and pituitary glands (6, 14, 49). Genetic ablation in TTF-1-/- mice resulted in early postnatal death (14). DREAM is expressed very early during embryonic life (50),4 and because DREAM regulates TTF-1-transactivating properties, it is tempting to speculate an important role for DREAM in thyroid and brain development and organogenesis during embryonic life. However, DREAM-/- mice are viable and do not show any gross morphological alteration in brain or in non-neural organs (21). Functional redundancy among the four members of the DREAM subfamily of calcium sensors (46, 48) may account for the lack of phenotype during embryonic development in DREAM knockout animals. Indeed, all four members of the DREAM family are expressed in the brain, while in the thyroid gland expression of KChIP-2 together with DREAM have been detected by RT-PCR. The functional significance of the redundant expression of different DREAM/KChIP genes coding for proteins with a similar function in several organs including the thyroid gland is presently unknown.


   FOOTNOTES
 
* This work was supported by grants from Direccion General Investigacion Cientifica y Técnica, Comunidad Autónoma de Madrid, and Fondo Investigaciones Sanitarias Seguridad Social, and the Human Frontiers Science Program RGP0156/2001B. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Both authors contributed equally to this work. Back

|| To whom correspondence should be addressed: Dpto. Biologéa Molecular y Celular, CNB-CSIC, Campus Cantoblanco, 28049 Madrid, Spain. Tel.: 34-91-5854682; Fax: 34-91-5854506; E-mail: naranjo{at}cnb.csic.es.

1 The 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. Back

2 F. Ledo, B. Mellström, and J. R. Naranjo, manuscript in preparation. Back

3 Link, W. A., Ledo, F., Torres, B., Palczewska, M., Madsen, T. M., Savignac, M., Albar, J. P., Mellström, B., and Naranjo, J. R. (2004) J. Neurosci. 24, 5346-5355. Back

4 J. R. Naranjo, W. A. Link, and B. Mellström, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Dr. L. D. Kohn (Edison Biotech Institute, Athens, OH) for FRTL-5 cells and R. Di Lauro (Stazione Zoologica, A. Dohrn, Naples, Italy) for expression vectors for TTF-1 and Gal4-TTF-1 as well as for the different Tg promoter constructs.



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