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Molecular Endocrinology, doi:10.1210/me.2005-0463
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Molecular Endocrinology 20 (8): 1810-1824
Copyright © 2006 by The Endocrine Society

Functional Inactivation of the Transcription Factor Pax8 through Oligomerization Chain Reaction

Barbara D’Andrea1, Roberto Iacone1, Tina Di Palma, Roberto Nitsch, Maria Giuseppina Baratta, Lucio Nitsch, Roberto Di Lauro and Mariastella Zannini

Istituto di Endocrinologia ed Oncologia Sperimentale-Consiglio Nazionale delie Ricerche and Dipartimento di Biologia e Patologia Cellulare e Molecolare (B.D., R.I., T.D.P., R.N., L.N., R.D.L., M.Z.), Italy; S.E.M.M. European School of Molecular Medicine (M.G.B.), and CEINGE biotecnologie avanzate (M.G.B., R.D.L.) 80131 Naples, Italy

Address all correspondence and requests for reprints to: Mariastella Zannini, Istituto di Endocrinologia ed Oncologia Sperimentale-Consiglio Nazionale delie Ricerche, c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, Universita’ di Napoli Federico II, via Pansini, 5, 80131 Napoli, Italy. E-mail: stella{at}szn.it.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Among the approaches used to provide a functional inactivation of a target protein, we have chosen the recently described oligomerization chain reaction (OCR) strategy to functionally inactivate the transcription factor Pax8, a member of the Pax gene family expressed in thyroid cells. The OCR strategy is based on the fusion of the self-associating coiled-coil (CC) domain of the nuclear factor promyelocytic leukemia (PML) to target proteins that are able to self-associate naturally or that form heterocomplexes. In the thyroid tissue, the transcription factor Pax8 is involved in the morphogenesis of the gland and in the transcriptional regulation of thyroid-expressed genes. We have recently demonstrated that in thyroid cells Pax8 interacts biochemically and functionally with the transcription factor TTF-1 (thyroid transcription factor 1), and that such interaction leads to the synergistic activation of thyroglobulin (Tg) gene expression. Fusion of the CC domain to Pax8 leads to the formation of aberrant, nonfunctional high-molecular mass complexes to which TTF-1 is also recruited. The CC-Pax8 chimera inhibits the transcriptional activity of Pax8 and of TTF-1 on both synthetic and physiological promoters and prevents the synergistic activation of the Tg promoter mediated by these two transcription factors. Furthermore, the expression of the CC-Pax8 chimera in differentiated thyroid cells leads to the down-regulation of the endogenous expression of several differentiation markers such as Tg, sodium/iodide symporter, Foxe1, TTF-1, and thyroid oxidase 2. These results demonstrate that the OCR is a useful tool to functionally inactivate a transcription factor. Moreover, by this approach, we identified Foxe1, TTF-1, and thyroid oxidase 2 as new direct targets of Pax8 or TTF-1.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TO UNDERSTAND THE molecular mechanisms underlying complex biological phenomena such as cell differentiation, organogenesis, and cancer, a detailed knowledge of the regulation of gene expression is required.

Regulation of gene expression occurs mostly at the transcriptional level. A large variety of transcriptional mechanisms have been delineated, and most of them are based on the capacity of transcription factors to recognize specific DNA sequences and subsequently, by interfering with either the transcriptional machinery or chromatin structure, or interacting with other transcription factors, to modulate transcription of target genes. Therefore, the understanding of transcription factors function is an important goal for a better description of complex and important biological phenomena and diseases. Several genetic and functional approaches are available to study the function of a transcription factor. Sometimes, genetic ablation of a target gene is not the best approach to understand its role, and in such cases functional inactivation of the protein may be a better choice. Several methods have been widely and successfully used, such as antisense RNA, RNA interference technologies, or specifically engineered dominant-negative mutants. However, these methods cannot be considered universally applicable.

The oligomerization chain reaction (OCR) is a recently described strategy for the functional inactivation of a target protein: this technique is based on the fusion of the self-associating coiled-coil (CC) domain of the nuclear factor promyelocytic leukemia (PML) to a target protein. In acute promyelocytic leukemia, PML is fused to the retinoic acid receptor (RAR). In contrast with RAR, PML-RAR forms oligomeric complexes through the self-associating CC domain present in PML (1, 2, 3, 4). This property of the CC domain suggested that the presence of this region could be used to modify the function of a target protein. Indeed, two recent reports describe the OCR as an innovative strategy for the inactivation of target proteins that are able to self-associate (5) or that form heterocomplexes (6).

Thyroid cells are a good model system by which to study the mechanisms involved in the regulation of gene expression. In fact, they provide a coherent system to address several questions related to cell differentiation, organogenesis, and pathology. Differentiated thyroid cells are characterized by the expression of thyroid-specific genes such as thyroglobulin (Tg), thyroperoxidase (TPO), sodium/iodide symporter (NIS), TSH receptor, and two recently cloned genes encoding thyroid oxidases (ThOX1 and ThOX2) (7). Tg and TPO promoters have been extensively studied, and three transcription factors have been cloned that specifically bind to and activate these two promoters (8, 9). The three transcription factors are Pax8, thyroid transcription factor (TTF)-1, and Foxe1.

In recent years, we have focused our studies on the molecular mechanism of action of Pax8 and TTF-1. These two transcription factors are present together only in the thyroid tissue, suggesting that this unique combination could be responsible for early commitment and differentiation of the thyrocytes. The transcription factor TTF-1 is a homeodomain-containing protein and belongs to the Nkx-2 class of homeobox genes (10, 11). During mouse embryo development, TTF-1 is expressed in the thyroid anlage, in restricted areas of the developing brain, and in the lung bronchial epithelium (12). TTF-1 is required for proper development of these tissues, because null mice die at birth lacking lung parenchyma and the thyroid and have severe defects of the ventral area of the forebrain (13). In addition to its role in development, TTF-1 has been shown to regulate tissue-specific transcription in differentiated thyroid and lung cells (14, 15, 16, 17). Pax8 is a member of the murine Pax family of paired domain-containing genes and is expressed in embryonic and adult kidney and thyroid, and in neural tube (18, 19). The Pax gene family encodes for DNA-binding proteins that are involved in the regulation of development and differentiation of a variety of tissue in different species (20, 21, 22, 23). Interestingly, Pax8 knockout mice have a barely visible thyroid gland, which is deprived of follicles because thyroid follicular cells are missing in these mice (24).

Our laboratory has demonstrated that, in a thyroid cell line, Pax8 is required for the expression of all the differentiation markers (25). In addition, our data demonstrated that in the environment of the thyroid cell, Pax8 is able to transcriptionally activate the thyroglobulin gene in cooperation with TTF-1 (25). Recently, more evidence of a functional cooperation between these two transcription factors has been collected (26, 27). Specifically, studies from our laboratory have demonstrated that Pax8 and TTF-1 are able to interact directly in vitro and to form a functional complex in vivo responsible for a synergistic transcriptional activation of the Tg promoter (28).

To further assess the functional role of Pax8 and of the Pax8/TTF-1 heterocomplex, we have chosen a protein knockout approach. Hence, we investigated the possibility of using the OCR strategy to generate a dominant-negative Pax8 molecule.

Here we present data demonstrating that we have successfully inactivated the Pax8/TTF-1 transcription complex in thyroid cells in culture through OCR. To this end, we have generated a chimeric construct carrying Pax8 fused to the CC domain. The oligomerization reaction induced by the CC domain triggered the formation of aberrant, nonfunctional high-molecular mass complexes, leading to a functional inactivation of both Pax8 and TTF-1 transcription factors. In addition, this approach allowed us to successfully interfere with the expression of Pax8 and/or TTF-1 target genes in the context of a thyroid-differentiated cell line. In such a way we identified ThOX2, Foxe1, and TTF-1 as direct targets of Pax8 or TTF-1. Based on these results, we propose that the fusion of the CC domain to Pax8 generates an effective dominant-negative function of CC-Pax8 that alters the functional properties of wild-type Pax8 and TTF-1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of a Dominant-Negative Pax8 Protein by OCR
It has been suggested that the oligomerization reaction induced by the CC domain of PML could lead to the formation of aberrant, nonfunctional high-molecular mass complexes, both when this domain is fused to proteins that are able to self-associate (5), as well as when the target of the OCR is a heterooligomer (6). We investigated whether the Pax8/TTF-1 heterocomplex could be targeted by the OCR strategy.

To this end, we fused the CC domain of PML to the N terminus of full-length Pax8, thus generating the 3xFlag-CC-Pax8 construct. At first, we analyzed the cellular distribution of the CC-Pax8 chimera by Western blotting of fractionated cellular extracts prepared from HeLa cells transiently transfected with either 3xFlag-Pax8 or 3xFlag-CC-Pax8. Immunodetection with anti-FLAG-M2 revealed that the CC-Pax8 protein was, at least in part, delocalized in the cytosol compartment, whereas the wild-type Pax8 protein showed an exclusive nuclear localization, as expected (Fig. 1Go).


Figure 1
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  		Fig. 1. Subcellular Localization of the CC-Pax8 Chimera

Fractionated cellular extracts (N, nuclear fraction; C, cytoplasmic fraction), prepared from HeLa cells transiently transfected with 3xFlag-Pax8 and 3xFlag-CC-Pax8, were separated on SDS-PAGE and subjected to Western blot analysis with anti-Flag antibody. 3xFlag-Pax8 and 3xFlag-CC-Pax8 are indicated by arrowheads. The hybridization with {alpha}-tubulin and Sp1 antibodies assessed the validity of the experiment and the protein uniform loading and integrity. The graph shows the ratio between nuclear fraction and cytoplasmic fraction of each sample measured by densitometric analysis.

 
It was previously reported that, in transient transfection assays in HeLa cells, Pax8 is able to activate transcription from a reporter construct (CP5-CAT) in which its binding site is present five times in tandem upstream the chloramphenicol acetyltransferase (CAT) reporter gene (29). To investigate the functional activity of the CC-Pax8 chimera and the interference that this protein might generate on wild-type Pax8, expression vectors encoding for 3xFlag-Pax8 and 3xFlag-CC-Pax8 were cotransfected in HeLa cells together with the reporter construct CP5-CAT, separately or in combination. The transfection of 3xFlag-Pax8 alone led to an activation of the CP5 promoter, as expected (Fig. 2AGo). The expression of 3xFlag-CC-Pax8 had no effect on the CP5 promoter, although the chimera retains the transactivation domain (Fig. 2AGo). Interestingly, when cotransfected together, the expression of 3xFlag-CC-Pax8 prevents the activation of the CP5 promoter by 3xFlag-Pax8 (Fig. 2AGo). The expression vector encoding for 3xFlag-CC used as control had no effect on Pax8 wild-type transcriptional activity on CP5 promoter. These data demonstrate that the fusion of the CC domain to Pax8 results in the functional inactivation of the Pax8 protein and, at the same time, provide good evidence that the CC-Pax8 chimera behaves as a dominant-negative molecule on Pax8.


Figure 2
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  		Fig. 2. Effect of CC-Pax8 on Pax8 Transcriptional Activity

A, HeLa cells were transiently transfected with the reporter plasmid CP5-CAT and the expression vectors encoding 3xFlag-Pax8, 3xFlag-CC-Pax8, and 3xFlag-CC. Folds of activation are considered as ratio between values obtained with and without cotransfection of the expression vectors. CMV-LUC was added as internal reference, and CAT values were normalized to the LUC activity. Values are mean ± SD of at least three independent experiments. B, HeLa cells were transiently transfected with the reporter plasmid Tg-CAT and the expression vectors encoding 3xFlag-Pax8, 3xFlag-CC-Pax8, and 3xFlag-CC as described earlier. Values are mean ± SD of at least three independent experiments.

 
Subsequently, we repeated the same transactivation assays using a more physiological promoter. It was previously reported that, in transient transfection assays in HeLa cells, Pax8 is able to activate transcription from a reporter construct in which the Tg minimal promoter is subcloned upstream of the CAT gene [Tg-CAT (30)]. Therefore, we cotransfected the expression vectors encoding for 3xFlag-Pax8 and 3xFlag-CC-Pax8 in HeLa cells together with the reporter construct Tg-CAT, separately or in combination. Also, in these experiments, we obtained a dominant-negative effect of the CC-Pax8 chimera on Pax8 activity due to the specific fusion of the CC domain to the target protein and not to the CC domain by itself (Fig. 2BGo).

Transcriptional Inactivation of TTF-1 by Overexpression of CC-Pax8
Studies from our laboratory have demonstrated that Pax8 and TTF-1 are able to interact directly in vitro and to form a functional complex in vivo responsible for a synergistic transcriptional activation of the Tg promoter (28).

To investigate the possibility that the OCR strategy could be used to modify the function of the Pax8/TTF-1 transcription complex, a strict requirement for the chimeric Pax8 protein would be the ability to associate with TTF-1. To test whether CC-Pax8 could still interact with TTF-1 as wild-type Pax8, HeLa cells were transiently transfected with the expression vectors encoding for 3xFlag-CC-Pax8 and TTF-1. As positive control we have also transfected the cells with the expression vectors encoding for 3xFlag-Pax8 and TTF-1, whereas as negative control we have transfected the cells with the expression vectors encoding for 3xFlag-CC and TTF-1. Anti-Flag-agarose affinity gel was used to immunoprecipitate the 3xFlag-CC-Pax8, the 3xFlag-Pax8, and the 3xFlag-CC proteins from total extracts prepared from HeLa cells transiently transfected. Subsequently, the bound proteins were subjected to Western blot analysis after separation by SDS-PAGE. Western blot developed with a specific anti-TTF-1 polyclonal antibody showed the presence of TTF-1-coimmunoprecipitated protein in HeLa cells expressing the 3xFlag-CC-Pax8 and the 3xFlag-Pax8 proteins (Fig. 3AGo, lanes 1 and 2), whereas TTF-1 was not immunoprecipitated in HeLa cells expressing the 3xFlag-CC (Fig. 3AGo, lane 3). Therefore, we can conclude that the CC-Pax8 chimera, like the Pax8 protein, is able to interact with TTF-1 despite the presence of the CC oligomerization domain.


Figure 3
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  		Fig. 3. CC-Pax8 Interacts with TTF-1 and Interferes with Its Transcriptional Activity

A, For the coimmunoprecipitation experiment, 2 mg of total protein extract were incubated with anti-FLAG-agarose affinity gel. The bound proteins were separated on 10% SDS-PAGE and analyzed by Western blot using a TTF-1-specific polyclonal antibody. Lanes 1 and 4: protein extract of HeLa cells transiently cotransfected with the expression vectors encoding for 3xFlag-CC-Pax8 and TTF-1. Lanes 2 and 5: protein extract of HeLa cells transiently cotransfected with the expression vectors encoding for 3xFlag-Pax8 and TTF-1. Lanes 3 and 6: protein extract of HeLa cells transiently cotransfected with the expression vectors encoding for 3xFlag-CC and TTF-1. In particular, lanes 1–3 correspond to the coimmunoprecipitated samples, whereas lanes 4–6 correspond to the input samples. B, HeLa cells were transiently transfected with the reporter plasmid C5-CAT and the expression vectors encoding CMV-TTF-1, 3xFlag-CC-Pax8, and 3xFlag-CC. Folds of activation are considered as ratio between values obtained with and without cotransfection of the expression vectors. CMV-LUC was added as internal reference, and CAT values were normalized to the LUC activity. Values are mean ± SD of at least three independent experiments. C, HeLa cells were transiently transfected with the reporter plasmid Tg-CAT and the expression vectors encoding 3xFlag-Pax8, CMV-TTF-1, 3xFlag-CC-Pax8, and 3xFlag-CC as described earlier. Values are mean ± SD of at least three independent experiments. IP, Immunoprecipitation; WB, Western blot.

 
We showed that the CC-Pax8 chimera prevents the activation of the CP5 reporter by Pax8. To test whether the CC-Pax8 protein could interfere also with the transcriptional activity of TTF-1, expression vectors encoding for 3xFlag-Pax8 and TTF-1 were cotransfected in HeLa cells, separately or in combination, together with the reporter construct C5-CAT, a reporter construct in which the TTF-1-binding site is present five times in tandem upstream from the CAT reporter gene (31). The expression of TTF-1 alone led to an activation of the C5 promoter, as expected, whereas the coexpression of CC-Pax8 prevented the activation of the C5 promoter by TTF-1 (Fig. 3BGo). Also in this experiment, the expression of 3xFlag-CC had no effect on TTF-1 activation of its promoter, as already described for Pax8 on the CP5 promoter. These results suggest that the CC-Pax8 chimera is capable of interfering also with TTF-1 transcriptional activity.

It was previously reported that, in transient transfection assays in HeLa cells, TTF-1 is able to significantly activate transcription from the Tg promoter (31), and that Pax8 and TTF-1 synergistically stimulate transcription from this promoter (28). We asked whether the CC-Pax8 chimera could interfere with TTF-1 transcriptional activity not only on a synthetic promoter, such as the C5 one, but also in a more physiological context represented by the Tg promoter. Moreover, we were very much interested in understanding whether the CC-Pax8 chimera could also interfere with the Pax8/TTF-1 transcriptional synergy on the Tg promoter. Therefore, we cotransfected expression vectors encoding for TTF-1, 3xFlag-Pax8, and 3xFlag-CC-Pax8 in HeLa cells together with the reporter construct Tg-CAT, separately or in combination. As a control we used the expression vector encoding for 3xFlag-CC. When transfected alone, TTF-1 enhances transcription of the Tg promoter, as expected (Fig. 3CGo). Coexpression of TTF-1 and 3xFlag-CC-Pax8 led to a loss of transcriptional activation of the Tg promoter by TTF-1, as was seen for the C5 promoter (Fig. 3CGo). Interestingly, the chimera CC-Pax8 completely prevented the synergistic transcriptional cooperation of Pax8 and TTF-1 on the Tg promoter, whereas the expression of 3xFlag-CC had no effect on transcriptional activity of the Pax8/TTF-1 transcriptional complex (Fig. 3CGo).

These results, taken together, show that the CC-Pax8 chimera is able to interfere with the functional activity of Pax8 and TTF-1 transcription factors, either when these proteins act alone or when they act in cooperation, and that this interference is specifically due to the fusion of the CC domain to the target protein and not to the CC domain alone.

Subcellular Localization of CC-Pax8
The results described so far show that the CC-Pax8 chimera, like the Pax8 protein, retains the ability to associate with TTF-1, despite the presence of the PML-CC extraoligomerization domain. We have also demonstrated that the CC-Pax8 chimera acts as a dominant-negative molecule on wild-type Pax8 transcriptional activity and that the coexpression of TTF-1 and CC-Pax8 results in a functional inactivation of TTF-1. Moreover, we have shown that the fusion of CC domain to Pax8 prevents completely the synergistic transcriptional cooperation of Pax8 and TTF-1 on the Tg promoter.

It has been suggested that an oligomerization induced by the CC domain of PML would lead to the formation of aberrant, nonfunctional high-molecular mass complexes localized outside the nucleus (5, 6). To test whether the fusion of Pax8 to the CC domain of PML induced the formation of high-molecular mass complexes we analyzed cellular lysates from transiently transfected HeLa cells by size exclusion chromatography (SEC) followed by Western blot. 3xFlag-Pax8 and TTF-1 form a heterocomplex found after SEC peaked in fractions corresponding to an apparent molecular mass of about 330 kDa (Fig. 4AGo). In contrast, the 3xFlag-CC-Pax8 fusion protein and TTF-1 were found in fractions corresponding to an apparent molecular mass ranging from 500 to 1000 kDa (Fig. 4BGo). According to data reported in the literature (5, 6), the widespread distribution of the two proteins could be interpreted as a result of a heterogeneous population of differently sized heterooligomeric complexes.


Figure 4
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  		Fig. 4. CC-Pax8 Recruits TTF-1 into High-Molecular Weight Complexes

A, Total extracts from HeLa cells, transiently transfected with the expression vectors encoding 3xFlag-Pax8 and TTF-1, were subjected to SEC. SEC fractions were analyzed by Western blot using the appropriate antibodies as indicated. The fractions containing the Pax8/TTF-1 complex are indicated and correspond to a molecular mass of about 330 kDa. B, Total extracts from HeLa cells, transiently transfected with the expression vectors encoding 3xFlag-CC-Pax8 and TTF-1, were subjected to SEC. SEC fractions were analyzed by Western blot using the appropriate antibodies as indicated. The fractions containing the CC-Pax8/TTF-1 complex are indicated and correspond to a molecular mass ranging from 500–1000 kDa.

 
Next, we checked for the subcellular localization of the CC-Pax8 protein in the differentiated rat thyroid cell line PC-Cl3. Previous studies described the nuclear localization of TTF-1 and Pax8 in thyroid cells in culture by indirect immunofluorescence (32) as well as their colocalization in the nucleus of these cells (28). To study the pattern of distribution of the CC-Pax8 chimera in PC Cl3 cells, we transiently transfected the cells with an expression vector encoding for the CC-Pax8-enhanced yellow fluorescent protein (EYFP) fusion protein. Confocal microscopy examination showed that the CC-Pax8 chimera was localized outside the nucleus but not diffused in the cytoplasmic compartment (Fig. 5Go, A and D). In the same cells we investigated the localization of TTF-1 by immunolabeling with a specific polyclonal antibody followed by confocal microscopy analysis. Interestingly, the cells that expressed the CC-Pax8 chimera showed an anomalous cytoplasmic localization of TTF-1 (Fig. 5Go, B and E). The examination of dual-labeled samples (Fig. 5Go, panels merge C and F) demonstrated that the two proteins colocalized in the same perinuclear domains. In control experiments the Pax8-EYFP chimera colocalized with TTF-1 (Fig. 5Go, G, H, and I) in the nuclei of PC-Cl3 cells. These results are in agreement with the analysis that we performed on HeLa cell-fractionated protein extracts, which demonstrated a partial cytoplasmic localization of the CC-Pax8 chimera. Moreover, the above data suggested that the dominant-negative effect exerted by CC-Pax8 on wild-type Pax8 could be due to the formation of a delocalized heterooligomeric complex that also recruits the TTF-1 protein.


Figure 5
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  		Fig. 5. CC-Pax8 Delocalizes TTF-1

PC Cl3 cells were plated on glass coverslips and transiently transfected with either an expression vector encoding CC-Pax8-EYFP (A–F) or an expression vector encoding Pax8-EYFP (G–I). Cells were stained for immunofluorescence with the anti-TTF-1 antibody and examined by confocal microscopy. CC-Pax8-EYFP fluorescence is detected mainly in a perinuclear area (A and D) where it is concentrated in large clusters (white arrowheads). The same clusters are also stained by the anti-TTF-1 antibody (B and E). The merge of the two signals reveals the colocalization of CC-Pax8-EYFP and TTF-1 in this extranuclear location (C and F). Pax8-EYFP is instead localized in the nucleus (G) where it colocalizes with TTF-1 (H and I). N, Nucleus. Bar, 10 mm.

 
In Thyroid Cells CC-Pax8 Affects the Expression of Thyroid-Specific Differentiation Markers
To better evaluate the functional consequences of CC-Pax8 expression in the context of the thyroid cell, we generated stable clones of PC Cl3 cells expressing the CC-Pax8 chimera. Several independent clones were isolated, and CC-Pax8 expression was determined by Western blot analysis (data not shown). We obtained five positive clones that we named CCP8–6, CCP8–7, CCP8–10, CCP8–17, and CCP8–23.

It has been previously suggested that the transcription factor Pax8 may be involved in tumor cell growth (33, 34, 35). Therefore, we asked whether the dominant-negative CC-Pax8 chimera could allow us to characterize the role of Pax8 in the control of cell proliferation. By means of growth curve experiments, we compared the proliferation rate of wild-type PC Cl3 cells with that of the CC-Pax8-positive stable clones. The analysis shows that there is no significant difference in the proliferation rate of the CC-Pax8 clones analyzed compared with that of the wild-type PC Cl3 cells (Fig. 6AGo).


Figure 6
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  		Fig. 6. In Thyroid Cells CC-Pax8 Has No Effect on Cell Proliferation but Interferes with the Expression of Thyroid-Specific Genes

A, PC Cl3 cells and CCP8 clones (CCP8–6, CCP8–7, CCP8–10, CCP8–17, and CCP8–23) were grown in complete medium, and the proliferation rate was measured as number of cells at different time points. B, Protein extracts of PC Cl3 and CCP8 cells were separated on 7% SDS-PAGE and analyzed by Western blot using a Tg-specific polyclonal antibody. The graph shows the densitometric analysis of the bands corresponding to the Tg protein. The bottom panel of the figure shows hybridization with {alpha}-tubulin to ensure protein uniform loading and integrity. C, The differentiated phenotype of the CCP8–23 clone was analyzed by Q-PCR. The expression of the indicated thyroid-specific genes is reported. For each gene, values are the average ± SD of two independent experiments, normalized by the expression of {alpha}1-tubulin, and expressed as a percentage of the value measured in parental PC Cl3 cells. For each gene analyzed, an unpaired two-tailed Student’s t test has been performed to obtain the P value associated with the observed expression differences. The individual P values have been then corrected for the number of genes in analysis by applying the Bonferroni correction formula (**, P < 0.004; *, P = 0.01).

 
Subsequently, the CC-Pax8 stable clones were analyzed for the expression of endogenous Tg to evaluate the effect of CC-Pax8 on the expression of this thyroid-specific differentiation marker. The Western blot analysis, carried out with a polyclonal antibody that specifically recognizes the Tg protein, demonstrated a significant down-regulation of the endogenous Tg protein in all the five positive clones isolated, compared with that of wild-type PC Cl3 cells (Fig. 6BGo) and neomycin-resistant clones that did not express CC-Pax8 chimera, which were used as negative control (data not shown). These findings demonstrated that constitutive expression of CC-Pax8 in differentiated thyroid cells causes the down-regulation of endogenous Tg expression, due to the dominant-negative effect of the exogenous chimera on the Pax8/TTF-1 transcriptional complex.

This result was expected because it is well known that Pax8 and TTF-1 are key transcription factors for Tg expression (28). Hence we have chosen one stable clone expressing the CC-Pax8 chimera (clone CCP8–23) to further analyze the expression profile of other known thyroid differentiation markers. By quantitative real-time RT-PCR (Q-PCR), we analyzed the mRNA levels of several markers of thyroid differentiation. As control, the mRNA level of a housekeeping gene, {alpha}-1 tubulin, was also measured in each sample. After normalization of input cDNA for {alpha}-1 tubulin transcripts, mRNA levels for each gene were reported as the percentage of the level measured in parental PC Cl3 cells (Fig. 6CGo). Our results confirm the highly significant down-regulation of Tg gene expression described previously in the Western blot analysis. Moreover, they also show a significant down-regulation of NIS, Foxe1, ThOX2, and TTF-1 gene expression in this positive clone. Although statistically less significant, a small down-regulation of the TSH receptor gene is also observed.

Foxe1, ThOX2, and TTF-1 Are Novel Targets of Pax8 and TTF-1
The down-regulation of Tg and NIS genes observed in the CCP8–23 clone is in agreement with previous evidence that describe both these two genes as direct targets of Pax8 (25, 30, 36, 37, 38, 39). To understand whether Foxe1, ThOX2, and TTF-1 could be direct targets of Pax8, TTF-1, or both factors, we performed a computational analysis using the Transcription Factor Database (TRANSFAC pro 9.3; BIOBASE Co.). We searched for Pax8- and TTF-1-binding sites in a region of about 1000 bp in the 5'-flanking region of Foxe1, ThOX2, and TTF-1, and the analysis showed the presence of either Pax8 or TTF-1 consensus sequences in the genomic regions analyzed. To confirm the predictions of the TRANSFAC analysis, we performed EMSAs with oligonucleotides derived from the putative Pax8-binding sites and TTF-1 binding sites found in Foxe1, ThOX2, and TTF-1. The results show that oligonucleotides derived from the Pax8-binding site regions of Foxe1 and ThOX2, named Pax8-binding site-8 and Pax8-binding site-5, respectively, are able to form a protein-DNA complex when incubated with a nuclear extract prepared from PC Cl3 cells (Fig. 7AGo). To further demonstrate that Pax8 is indeed able to bind to the analyzed sites, we performed the EMSAs also with the paired domain of Pax8, expressed in bacteria and affinity purified. Both the Pax8-binding site-8 and Pax8-binding site-5 oligonucleotides are able to form a protein-DNA complex with Pax8 paired domain (Fig. 7AGo). In parallel, we performed EMSAs with the oligonucleotides derived from TTF-1 binding site regions of the analyzed genes. The results show that the oligonucleotide derived from the TTF-1 binding site regions of TTF-1 gene, named TBS-2, is able to form a protein-DNA complex when incubated with a nuclear extract prepared from PC Cl3 cells (Fig. 7BGo). To further demonstrate that TTF-1 is indeed able to bind to the analyzed site, we performed a supershift experiment with a polyclonal antibody that specifically recognizes TTF-1. The protein-DNA complex observed with the TTF-1 binding sites-2 oligonucleotide is clearly supershifted by the TTF-1 antibody (Fig. 7BGo). As control, in all our EMSAs, the nuclear extract or the Pax8 paired domain were incubated with an oligonucleotide derived from the Tg promoter (oligo C) containing a well-known binding site for both Pax8 and TTF-1 (30). Therefore, we can conclude that Pax8 is able to bind in vitro to the 5'-flanking region of Foxe1 and ThOX2 genes, whereas TTF-1 is able to bind in vitro to its own 5'-flanking region.


Figure 7
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  		Fig. 7. Pax8 and TTF-1 Bind Both in Vitro and in Vivo to the Identified Pax8- and TTF-1-Binding Sites

A, Nuclear extract of PC Cl3 cells or Pax8 paired domain produced in bacteria (indicated as PD) were incubated in a binding assay with oligonucleotides Pax8-binding site-5, Pax8-binding site-8, or oligo C derived from the thyroglobulin promoter (30 ). B, Nuclear extract of PC Cl3 cells was incubated, alone or together with the antibody against TTF-1, in a binding assay with either oligonucleotide TTF-1 binding sites-2 or oligo C. The arrowhead indicates the high-molecular weight DNA-proteins complex supershifted by the TTF-1 antibody. C, ChIP experiments performed on PC Cl3 cells showing the binding in vivo of Pax8 and TTF-1 to the Tg promoter (upper panel), of TTF-1 to its own promoter (middle panel) and of Pax8 to the Foxe1 promoter (lower panel). PBS, Pax8-binding site; TBS, TTF-1 binding site.

 
To demonstrate the ability of TTF-1 and Pax8 to interact with the previously described target genes also in vivo, we performed chromatin immunoprecipitation (ChIP) experiments on PC Cl3 cells. The cross-linked chromatin was immunoprecipitated using polyclonal antibodies against Pax8 and TTF-1. As control, to rule out unspecific background of the ChIP assay, we performed one reaction lacking the primary antibody and one using an unrelated antibody. The enrichment of the endogenous 5'-flanking region of Tg, TTF-1, and Foxe1 genes was monitored by PCR amplification using specific primers for the described sequences. We demonstrate that, in agreement with the in vitro data, both TTF-1 and Pax8 antibodies are able to immunoprecipitate the chromatin containing the Tg promoter (Fig. 7CGo). Moreover, the TTF-1 antibody immunoprecipitates the chromatin containing TTF-1 binding sites-2 localized in its own 5'-region, whereas the Pax8 antibody immunoprecipitates the chromatin containing the Pax8-binding site-8 localized in the Foxe1 5'-region (Fig. 7CGo). These results confirm the in vitro binding data presented in this paper, or already reported in the literature, and clearly demonstrate that Pax8 and TTF-1 are able to bind in vivo to the Tg, TTF-1, and Foxe1 5'-flanking regions.

To determine whether the identified Pax8-binding sites and TTF-1 binding sites in the 5'-flanking region of Foxe1, ThOX2, and TTF-1 genes could be effective transcriptional regulatory elements, we performed functional reporter gene assays. We subcloned the regions containing the Pax8- and TTF-1-binding sites investigated by EMSAs and ChIP experiments upstream from the LUC reporter gene in the pGL3-basic vector (Promega Corp., Madison, WI). The generated reporter plasmids were named 5'-Foxe1-LUC, 5'-ThOX2-LUC, and 5'-TTF-1-LUC and were transiently transfected in HeLa cells in the absence or in the presence of increasing concentrations of the expression vectors encoding Pax8 or TTF-1. As shown in Fig. 8Go, Pax8 activates transcription from both 5'-Foxe1-LUC and 5'-ThOX2-LUC, whereas TTF-1 activates transcription from the 5'-TTF-1-LUC reporter construct, in a concentration-dependent manner. These data indicate that we have identified functional Pax8- and TTF-1-binding sites in the Foxe1, ThOX2, and TTF-1 5'-flanking regions and clearly demonstrate that these genes are direct downstream targets of the two transcription factors.


Figure 8
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  		Fig. 8. Foxe1, ThOX2, and TTF-1 Are Direct Targets of Pax8 and TTF-1

HeLa cells were transfected with the reporter constructs 5'-Foxe1-LUC, 5'-ThOX2-LUC, and 5'-TTF-1-LUC in the absence or in the presence of increasing concentration of the expression vectors encoding Pax8 or TTF-1. Folds of activation are considered as ratio between values obtained with and without cotransfection of the expression vectors. CMV-CAT was added as internal reference, and LUC values were normalized to the CAT activity. Values are mean ± SD of at least three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Transcription factors play a pivotal role in the determination and maintenance of the cellular differentiated phenotype. Among the transcription factors that are involved in thyroid-specific gene expression, Pax8, a member of the Pax gene family, has been demonstrated to be required for the expression of all the thyroid-specific genes that are considered markers of the differentiated phenotype (25). Recently, it has been shown that Pax8 cooperates with the transcription factor TTF-1 (26, 27). Specifically, our laboratory has demonstrated that Pax8 and TTF-1 form a functional heterocomplex responsible for the synergistic transcriptional activation of the thyroglobulin promoter (28). To better understand the role of Pax8, we have chosen an approach based on the functional inactivation of the protein. In our study, we propose the successful application of the innovative OCR strategy for an effective functional inhibition of the transcription factor Pax8 and of the heterocomplex Pax8/TTF-1 in thyroid cells in culture.

The OCR is a recently described strategy for the functional inactivation of a target protein, based on the fusion of the self-associating coiled-coil (CC) domain of PML to target proteins that are able to self-associate naturally (5) or that form heterocomplexes (6).

Our results show that the fusion of the CC domain to Pax8 results in an OCR that alters the functional properties of Pax8 itself and of the Pax8/TTF-1 complex. In fact, we demonstrated that the CC-Pax8 chimera is able to interfere with the functional activity of the Pax8 protein behaving as a dominant-negative molecule on the wild-type Pax8. Moreover, the CC-Pax8 chimera retains the ability to associate with the TTF-1 protein and completely prevents its functional activity on target promoters. Accordingly, with the molecular models proposed in previous reports (5, 6), the fusion of the CC domain with Pax8 induces the formation of aberrant high-molecular weight complexes, to which the TTF-1 protein is also recruited. Therefore, the generation of this aberrant complex likely prevents the correct intracellular localization of Pax8 and TTF-1 and prevents the transcription factors from binding to the DNA. In fact, the expression of the CC-Pax8 chimera in PC Cl3 cells causes an anomalous localization of the endogenous TTF-1 protein that colocalizes with CC-Pax8 in perinuclear domains. Most importantly, the stable expression of the CC-Pax8 chimera in differentiated thyroid cells leads to a significant down-regulation of endogenous Tg, NIS, Foxe1, ThOX2, and TTF-1 expression. These data provide strong evidence of the ability of the CC-Pax8 molecule to interfere with the transcription of Pax8 and/or TTF-1 target genes in a physiological chromosomal context.

Pax genes belong to a family of nine developmental control genes that encode transcription factors, which act as essential players during the organogenesis and the differentiation of several organs (21, 22, 23, 40). In particular, the analysis of transgenic and knockout mice revealed that Pax8 has a key role in development and differentiation of the thyroid gland. In fact, Pax8 knockout mice have a very small thyroid gland, which is deprived of follicular cells (24), and patients suffering from congenital hypothyroidism have been shown to carry mutations in the Pax8 gene (41). However, until now, very little has been known about in vivo targets of Pax8. It has been demonstrated that Pax8 is required for the expression of thyroglobulin, TPO, and the sodium/iodide symporter, all genes considered markers of the differentiated phenotype (25). Nevertheless, the finding that Pax8 knockout mice exhibit a total absence of thyroid follicular cells (24), although indicating an essential role for this gene in thyroid organogenesis, could not contribute to a definitive assessment of the role of Pax8 in controlling the expression of the thyroid-differentiated phenotype, because the thyroid cell precursors disappeared before the onset of the Tg, TPO, and NIS gene expression. Moreover, the association between mutations in the Pax8 gene with human thyroid dysgenesis (41) underlines an important role for this protein in the organogenesis but could not contribute to a further understanding of its involvement on the expression of thyroid-specific genes.

In addition, the evidence that the transcription factors involved in the expression of the thyroid-differentiated phenotype have a central role in the study of loss of differentiation observed in tumors is of great relevance. The expression of activated forms of different oncogenes in thyroid cells in culture leads to loss of differentiation and abnormal proliferation (25, 42). The fundamental role of Pax8 in thyroid differentiation and the link observed between the loss of thyroid differentiation and tumors suggest that this transcription factor could also be involved in molecular mechanisms of thyroid tumors.

Hence, a full understanding of Pax8 function and the identification of its targets represent a very important goal for a better knowledge of the physiological and the pathological processes occurring in the thyroid gland.

In this paper, we show that the CC-Pax8 chimera behaves as a dominant-negative molecule for both Pax8 and TTF-1. In addition, we demonstrate that the functional inactivation of Pax8 and TTF-1, by means of the OCR strategy, causes a down-regulation of the expression of thyroid differentiation marker genes such as Tg, NIS, ThOX2, Foxe1 and TTF-1.

Tg and NIS are two known target genes of Pax8 and TTF-1 (25, 30, 37, 38); therefore, the down-regulation of the expression of these genes observed in our experiments is in agreement with previous evidence.

ThOX2 is a recently cloned thyroid-specific gene (7). It encodes a Ca2+-dependent flavoprotein presenting a reduced nicotinamide adenine dinucleotide phosphate-oxidase activity. The ThOX2 protein is colocalized with the TPO at the apical membrane of thyroid cells and represents the thyroid H2O2-generating system. The expression of this system is stimulated by the cAMP pathway through the TSH receptor, and its enzymatic activity is triggered by the Ca2+-phosphatidylinositol cascade (43). The results reported in this paper demonstrate that the expression of ThOX2 is under the control of the transcription factor Pax8. In fact, we have identified one Pax8-binding site in the 5'-flanking region of the ThOX2 gene, and we have demonstrated that Pax8 is indeed able to bind to this site and activate transcription from the DNA region containing it. Therefore, this is the first evidence that describes ThOX2 as a transcriptional target of Pax8.

Foxe1 is a member of the forkhead family of proteins. Recently, a report appeared in the literature in which the authors describe a dramatic decrease of Foxe1 protein levels in the thyroid of Pax8–/– mice (44). Our findings show that Pax8 is able to bind to one site in the Foxe1 5'-flanking region both in vitro and in vivo and activates transcription from this DNA region. Hence, our data support the previous report and provide direct evidence that Foxe1 expression is indeed regulated by Pax8.

At last, we also show that TTF-1 is able to bind to one site in its own 5'-flanking region both in vitro and in vivo and activates transcription from this DNA region when challenged in a functional reporter assay. Such a finding suggests that TTF-1 expression is controlled by a mechanism of autoregulation mediated by the binding of the transcription factor to its own 5'-flanking region. This observation is further supported by a previous report in which a possible autoregulation of TTF-1 is described (45).

Thus, we conclude that the CC-Pax8 dominant-negative molecule has been a successful tool to interfere with the functional activity of the transcription factor Pax8 and of its partner TTF-1. This interference allowed us to identify ThOX2, Foxe1, and TTF-1 as novel targets of Pax8 or TTF-1 action.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids
The 3xFLAG-cc-Pax8 construct was generated as follows. The cDNA corresponding to the CC domain (encoding amino acids from 200–339 of the PML protein) was amplified by PCR using the PML cDNA as template, and subcloned between the HindIII-BglII sites of the 3xFLAGCMV-10 (Sigma Chemical Co., St. Louis, MO) expression vector. The cDNA corresponding to Pax8 was generated by PCR amplification with a 5'-primer and a 3'-primer containing at their ends the BglII and XbaI sites, respectively. The fragment was subcloned into the corresponding sites of the expression vector 3xFLAGCMV-10 (Sigma) downstream the from the CC domain sequence. To generate pCC-Pax8-EYFP, the insert of 3xFLAG-CC-Pax8 (encoding the CC-Pax8 fusion) was amplified by PCR with oligonucleotides containing the HindIII restriction site at both ends and then inserted into the pEYFP-N1 vector (CLONTECH Laboratories, Inc., Palo Alto, CA). The DNA fragments containing the Pax8-binding site-8, Pax8-binding site-5, and TTF-1 binding sites-2 sequences were amplified by PCR using rat genomic DNA as template and a 5'-primer and a 3'-primer containing at their ends the KpnI and BglII sites, respectively. The fragments were subcloned into the corresponding sites of the plasmid pGL3-basic (Promega), and the generated constructs were named 5'-Foxe1-LUC, 5'-ThOX2-LUC, and 5'-TTF-1-LUC. All constructs were analyzed by DNA sequencing. The other plasmids used in transient transfection assays have been previously described and were as follows: pTACAT3 (15), cytomegalovirus (CMV)TTF1 (10), CP5-CAT (29), and C5-CAT (31).

CMV-CAT and CMV-LUC plasmids were used as internal controls in transfection assays. The DNA of all plasmids was prepared by QIAGEN cartridges (QIAGEN GmbH, Hilden, Germany) and used for cell transfections.

Cell Culture, Transfection Experiments, and Cell Proliferation Assays
PC Cl3 and HeLa cell lines have been previously described (46). PC Cl3 cells were grown in Coon’s modified F-12 medium (EuroClone, Leeds, UK) supplemented with 5% calf serum and a six-hormone mixture and growth factors as described by Ambesi-Impiombato and Coon (47).

HeLa cells were grown in DMEM supplemented with 10% fetal calf serum. For transient transfection experiments, 5–8 h before transfection HeLa cells were plated at a density of 3 x 105 cells/60-mm tissue culture dish, whereas PC Cl3 cells were plated at a density of 5 x 104 cells on 12-mm diameter glass coverslips 72 h before transfection. Transfections were carried out with the FuGENE 6 reagent (Roche Diagnostic, Indianapolis, IN) according to the manufacturer’s directions. The DNA/FuGENE ratio was 1:2 in all experiments. After 48 h PC Cl3 cells were analyzed by immunofluorescence, or HeLa extracts were prepared to determine either the levels of CAT protein with a CAT enzyme-linked immunosorbent assay kit (Roche Molecular Biochemicals, Indianapolis, IN) or LUC activities as described previously (48).

Transfection experiments were done in duplicate and repeated at least three times.

To generate stable clones, PC Cl3 cells were transfected with the 3xFLAG-CC-Pax8. Cells were selected with 400 µg/ml of G418 (Invitrogen, San Diego, CA). After 2 wk, G418-resistant clones were isolated, expanded, and examined by Western Blot.

To measure cell growth parameters, PC Cl3 and CC-P8 clones were plated at 104 cells per 60-mm plate. The cells were grown in F12 medium with 5% calf serum and six-hormone mixture and growth factors. The medium was changed every 24 h, after which cells were collected and counted.

Protein Extracts and Western Blot
For LUC/CAT assays, total extracts were prepared as previously described (49). Total extracts from HeLa cells transiently transfected were prepared using the EBC buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1% Triton X-100, 10% glycerol, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitors (Sigma). Protein extracts were then centrifuged (12,000 x g) for 30 min at 4 C.

Cell fractionation into cytoplasmic and nuclear fraction was prepared by a variation of the standard protocol of Dignam (50). Briefly, the cells were scraped in buffer A (10 mM HEPES, pH 7.8; 1.5 mM Mg Cl2;10 mM KCl; 0.5 mM DTT) and incubated at 4 C for 10 min; 10% Triton X-100 (1/30 of original volume) was added and extracts were centrifuged at 12,000 x g for 1 min. To the supernatants were added 0.11 volume of buffer B (0.3 M HEPES, pH 7.8; 1.4 M KCl; 30 mM MgCl2), incubated at 4 C for 30 min rotating, and then centrifuged at 12,000 x g for 20 min at 4 C. The recovered supernatant corresponded to cytoplasmic extract. The pellets of nuclei were resuspended in 1/5 of the original volume of buffer C (20 mM HEPES, pH 7.8; 25% glycerol; 0.42 M NaCl; 1.5 mM MgCl2; 0.2 mM EDTA; 0.5 mM DTT) and incubated at 4 C for 30 min, rotating. The protein extracts were centrifuged at 12,000 x g for 20 min. The supernatant removed corresponded to nuclear extract.

The protein concentration was determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). Proteins were separated by SDS-10% PAGE, and SDS-7% PAGE for Tg. Gels were blotted onto Immobilon P (Millipore Corp., Bedford, MA) for 16–18 h, and the membranes were blocked in 5% nonfat dry milk for 2 h at room temperature. Immunodetection was performed by using a TTF-1-specific polyclonal antibody (12), a Tg-specific polyclonal antibody (51), and a monoclonal anti-FLAG antibody (Sigma). Subsequently, the filters were developed using an enhanced chemiluminescence detection method (Pierce Chemical Co., Madison, WI) according to the manufacturer’s directions.

SEC and Coimmunoprecipitation
For SEC analysis 2 mg of total extract was loaded on a Superose 6 HR 10/30 gel filtration column equilibrated in sodium phosphate buffer, pH 7.2, containing 150 mM NaCl. For Western blot analysis, the indicated fractions were subjected to trichloroacetic acid precipitation before loading on SDS-PAGE.

The coimmunoprecipitation experiment was performed by incubating 2 mg of total protein extract with 20 µl of anti-FLAG-agarose affinity gel (Sigma) overnight at 4 C on a rotating wheel. The samples were centrifuged, and the agarose gel-bound proteins were washed several times with EBC buffer, resuspended in 2x SDS-PAGE sample buffer, and heated at 95 C for 3–5 min before loading on the gel.

Immunofluorescence and Confocal Laser Scanning Microscopy
Cells were transiently transfected on glass coverslips, fixed in 4% paraformaldehyde in PBS for 20 min at room temperature, permeabilized for 7 min in 0.1% Triton X-100 in PBS, and incubated for 10 min in 0.1 M glycine in PBS. Coverslips were subsequently incubated for 20 min with an anti-TTF-1 polyclonal antibody (12) diluted 1:100 in 0.5% BSA in PBS and, after PBS washing, incubated for 20 min with a rhodamine-tagged goat antirabbit secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1:50 in 0.5% BSA in PBS. After final washes with PBS, the coverslips were mounted on a microscope slide using a 50% solution of glycerol in PBS.

Images were collected with a Zeiss LSM 510 confocal laser scanning microscope, equipped with a 488-nm argon ion laser, a 543-nm HeNe laser, and a Plan-Apochromat 63x/1.4 oil-immersion objective. Emitted fluorescence was detected using a BP 505–530 band pass filter for EYFP and a LP 560 long pass filter for tetramethylrhodamine isothiocyanate. Pairs of images were collected simultaneously in the green and red channels.

RNA Extraction, cDNA Synthesis, and Q-PCR
Total RNA was prepared using TRIZOL Reagent (Invitrogen) according to the manufacturer’s directions. Total RNA (4 µg) was retrotranscribed using the Superscript First Strand Synthesis System for RT PCR (Invitrogen). Real-time PCR analysis was performed using an ABI Prism 7900HT sequence detection system and SYBR green chemistry (PE Applied Biosystems, Foster City, CA). Reactions were carried out in triplicate using, for each reaction, cDNA obtained from 130 ng of total RNA and 0.3 µM primers. The specific primers sets used for this analysis have been described previously (42).

ChIP
The cross-linking solution, containing 1% formaldehyde, was added directly to cell culture media. The fixation proceeded for 10 min and was stopped by the addition of glycine to a final concentration of 125 mM. PC Cl3 cells were rinsed twice with cold PBS plus 1 mM PMSF, and then scraped. Cells were collected by centrifugation at 800 x g for 5 min at 4 C. Cells were swelled in cold cell lysis buffer containing 5 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (pH 8.0), 85 mM KCl, 0.5% Nonidet P-40, 1 mM PMSF, and inhibitors cocktail (Sigma) and incubated on ice for 10 min. Nuclei were spun down by microcentrifugation at 2000 x g for 5 min at 4 C, resuspended in nuclear lysis buffer containing 50 mM Tris-HCl (pH 8), 10 mM EDTA, 0.8% sodium dodecyl sulfate (SDS), 1 mM PMSF and inhibitors cocktail (Sigma), and then incubated on ice for 10 min. Samples were broken by sonication into chromatin fragments of an average length of 500/1000 bp and then microcentrifuged at 16,000 x g. The sonicated cell supernatant was diluted 8-fold in ChIP Dilution Buffer containing 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.1), and 167 mM NaCl, and precleared by adding Salmon Sperm DNA/Protein A Agarose (Upstate Biotechnology, Inc., Lake Placid, NY) for 30 min at 4 C. Precleared chromatin from 1 x 106 cells was incubated with 1 µg of affinity-purified rabbit polyclonal antibody (anti-Pax8, anti TTF-1 and an unrelated one), or no antibody and rotated at 4 C for 16 h. Immunoprecipitates were washed five times with RIPA buffer containing 10 mM Tris-HCl (pH 8), 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS, 140 mM NaCl, and 1 mM PMSF; twice with LiCl buffer containing 0.25 M LiCl, 1% Nonidet P-40, 1% Na-deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.0), and then three times with TE (10 mM Tris-HCl, pH 8; 1 mM EDTA). Before the first wash, the supernatant from the reaction lacking primary antibody was saved as total input of chromatin and was processed with the eluted immunoprecipitates beginning at the cross-link reversal step. Immunoprecipitates were eluted by adding 1% SDS, 0.1 M NaHCO3 and reverse cross-linked by addition of NaCl to a final concentration of 200 mM and by heating at 65 C for 16 h. Recovered material was treated with proteinase K, extracted with phenol-chloroform-isoamyl alcohol (25:24:1) and precipitated. The pellets were resuspended in 30 µl of TE and analyzed by PCR using specific primers for the analyzed regions. The input sample was resuspended in 30 µl of TE and diluted 1:10 before PCR.

EMSAs
Double-strand oligonucleotides were labeled with {gamma}-32P ATP and T4 polynucleotide kinase and used as probes. The binding reactions were carried out in a buffer containing 20 mM Tris-HCl (pH 7.6), 75 mM KCl, 1 mM DTT, 10% glycerol, 1 mg/ml BSA, and 3 mg/ml polydeoxyinosinic deoxycytidylic acid. After 30 min of incubation at room temperature, free DNA and DNA-protein complexes were resolved on a 5% nondenaturating polyacrylamide gel and visualized with a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). The antibody used in the supershift experiments was incubated with the protein extract for 20 min before adding the probe.


   ACKNOWLEDGMENTS
 
We thank Saverio Minucci for providing the CC-domain plasmid and the Service of Molecular Biology of the Stazione Zoologica A. Dohrn of Naples for their technical assistance.


   FOOTNOTES
 
This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro and from the Regione Campania. T.D.P. was supported by a fellowship from the Federazione Italiana per la Ricerca sul Cancro.

Present address for R.I.: International Max Planck Research School, Technische Universität Dresden, BioInnovationZentrum, Am Tatzberg 47, 01307 Dresden, Germany.

Author Disclosure Summary: B.D., R.I., T.D.P., R.N., M.G.B., L.N., R.D.L., and M.Z. have nothing to declare.

First Published Online April 13, 2006

1 B.D. and R.I. equally contributed to the work. Back

Abbreviations: CAT, Chloramphenicol acetyltransferase; CC, coiled coil; ChIP, chromatin immunoprecipitation; CMV, cytomegalovirus; DTT, dithiothreitol; EYFP, enhanced yellow fluorescent protein; NIS, sodium/iodide symporter; OCR, oligomerization chain reaction; PML, promyelocytic leukemia; PMSF, phenylmethylsulfonylfluoride; Q-PCR, quantitative real-time PCR; RAR, retinoic acid receptor; SDS, sodium dodecyl sulfate; SEC, size exclusion chromatography; Tg, thyroglobulin; ThOX1, thyroid oxidase 1; TPO, thyroperoxidase; TTF-1, thyroid transcription factor 1.

Received for publication November 18, 2005. Accepted for publication April 3, 2006.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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