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INTRODUCTION |
Tissue-specific transcriptional regulation is often mediated by a
complex of cis-acting elements. The vast majority of the promoters of
genes expressed in a cell type-specific fashion contains a variety of
recognition sequences for tissue-specific and ubiquitous transcription
factors. It is well known that tissue-specific transcriptional regulation is mediated by a set of transcription factors whose combination is unique to the cell type. Thyroid follicular cells, the
most abundant cell population of the thyroid gland, represent a useful
model system to elucidate the mechanism operating in the establishment
and maintenance of cell type-specific expression. Thyrocytes are
responsible for thyroid hormone synthesis and are characterized by the
expression of a specific set of genes such as thyroglobulin
(Tg)1 and thyroperoxidase
(TPO), which are exclusively expressed in this cell type (1, 2), and by
the expression of genes expressed only in a few tissues other than the
thyroid, such as the thyrotropin-stimulating hormone receptor and the
sodium/iodide symporter.
The Tg and TPO promoters have been extensively studied, and multiple
factors have been shown to be required for their expression (3, 4). To
date, three transcription factors that specifically bind to and
regulate these promoters have been cloned (2). The three transcription
factors are as follows: thyroid transcription factor-1 (TTF-1), thyroid
transcription factor-2 (TTF-2), and Pax8. TTF-1 (also named NKx 2.1 and
T/EBP) is a homeodomain-containing protein expressed in embryonic
diencephalon, thyroid, and lung (5). TTF-2 is a forkhead
domain-containing protein expressed in pituitary and thyroid (6), and
Pax8 is a member of the murine Pax family of paired domain-containing
genes that is expressed in kidney, in the developing excretory system,
and in the thyroid (7). We have focused our studies on the molecular
mechanisms of action of TTF-1 and Pax8. These two transcription factors
are present together only in the thyroid, suggesting that this unique combination could be responsible for early commitment and
differentiation of thyrocytes. TTF-1 was originally identified as a
protein binding to a DNA sequence that is present three times on both
the Tg and the TPO promoters. Furthermore, TTF-1 was shown to be
involved in the activation of thyroid-specific gene expression (8). In
fact, TTF-1 is able to activate transcription from the Tg promoter and,
albeit at a much lower level, from the TPO promoter both in thyroid and
non-thyroid cells.
Parallel to its function in the thyroid, TTF-1 plays a critical role
also in lung morphogenesis and in respiratory epithelial cell gene
expression. It has been demonstrated that TTF-1 is required for the
transcriptional activation of surfactant proteins A, B, and C (SP-A,
SP-B, and SP-C) and Clara cell secretory protein (9-13).
ttf-1 gene inactivation in the mouse results in
severe thyroid and lung hypoplasia (14), and the homozygous mice are not viable. Recently, it has been published that TTF-1 and GATA-6 are
co-expressed in the respiratory epithelium in vivo and in respiratory epithelial cells in vitro. It has been also
demonstrated (15) that TTF-1 and GATA-6 directly interact and have a
synergistic effect on the transcriptional activation of the SP-C promoter.
The Pax gene family encodes for DNA-binding proteins that are involved
in the regulation of the development of a variety of tissues in
different species. Specifically, Pax8 has been demonstrate to be
required both for morphogenesis of the thyroid gland (16) and for
maintenance of thyroid-differentiated phenotype (17). The molecular
mechanisms involved in Pax8 control of differentiation have been
investigated in detail. Pax8 binds to a single site on the Tg and on
the TPO promoters, and in both cases, the Pax8-binding site overlaps
with one of the TTF-1-binding site (4, 18). In addition, Pax8 was
shown, in transient transfection assays, to activate transcription from
the TPO and the Tg promoters in non-thyroid cells (18). Recently,
direct evidence of the ability of Pax8 to activate transcription of
thyroid-specific genes at their chromosomal locus was obtained (17).
Interestingly, in Pax8 knockout mice the thyroid gland is barely
visible and lacks follicular cells (16). Recent studies
(19) have demonstrated that in rat differentiated thyroid cells in
culture (the PC Cl3 cell line), the thyrotropin-stimulating hormone
regulates the expression of both thyroglobulin and Pax8 genes by a
cAMP-mediated mechanism.
Taken together, all the data present in the literature to date strongly
suggest a fundamental role of Pax8 in the maintenance of functional
differentiation in thyroid cells.
Despite the critical role during development and in human diseases of
the Pax proteins, the biochemical basis of their function within the
cell nucleus is poorly understood.
The functional role of TTF-1- and Pax8-binding sites within the Tg and
TPO promoters has been studied in rat, bovine, and human Tg gene
promoters (18, 20-22). Some studies (17) have suggested that there
might be a functional cooperation between the two transcription factors
in the transcriptional activation of thyroid-specific promoters.
Recently, it has been proposed that TTF-1 and Pax8 cooperate in the
stimulation of the TPO (23) and of the human Tg (22) gene promoters.
However, the demonstration of a direct interaction has not yet been provided.
The aim of our study was to investigate a possible biochemical and
functional interaction between TTF-1 and Pax8. Indeed, we were able to
show a physical interaction between TTF-1 and Pax8, both in thyroid and
non-thyroid cells. Co-immunoprecipitation experiments performed using
thyroid cell extracts confirmed the existence of a complex between Pax8
and TTF-1 in vivo. Moreover, we observed an impressive
synergistic effect of the two transcription factors on the Tg promoter
activation. The synergism involves a specific region of the Tg promoter
(region C), that is the most proximal to the start of transcription
where TTF-1 and Pax8 binding sites overlap. In addition, by mutational
analysis we have identified the portions of TTF-1 and Pax8 proteins
responsible for the physical and functional interaction.
In conclusion, this paper describes a physical and functional
interaction between an homeodomain-containing factor and a paired domain-containing factor, both involved in the regulation of
tissue-specific transcription.
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EXPERIMENTAL PROCEDURES |
Plasmids--
The GST-Pax8 fusion protein was generated by PCR
amplification of Pax8 coding region and subsequent subcloning in the
EcoRI site of the pGEX-4T vector. The orientation of the
fragment and the correct frame of the fusion were verified by DNA sequencing.
3xFLAG-Pax8 was generated by PCR amplification of Pax8 coding region
and subsequent cloning in the EcoRI-XbaI sites of
the 3xFLAG CMV-10 vector (Sigma).
The plasmids used in transient transfection experiments have been
described previously and are as follows: Tg-CAT (3); Tg-CAT Acore (24);
TPO-LUC (4); CMV-TTF-1 (25);
1,
33,
2,
14,
3 (8); and
pCMV5-Pax8 (18). Expression vectors encoding Pax8 splicing variants are
described in Ref. 26.
The reporter construct Tg-CAT ABdel was generated by PCR amplification
of a small portion of the Tg promoter (region C) and subsequent
subcloning in SalI-HindIII sites of the same
reporter plasmid of Tg-CAT.
CMV-CAT and CMV-LUC plasmids were used as internal control in
transfection assays. The DNA of all plasmids was prepared by Qiagen
cartridges (Qiagen GmbH, Germany) and used for cell transfection.
Cell Culture and Transfection--
PC Cl3 and HeLa cell lines
have been described previously (27).
PC Cl3 cells were grown in Coon's modified F-12 medium (Seromed)
supplemented with 5% calf serum (Invitrogen) and six hormones and
growth factors as described by Ambesi-Impiombato and Coon (28).
HeLa cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum. For transactivation
experiments, cells were plated at a density of 3 × 105 cells/60-mm tissue culture dish, 5-8 h prior to
transfection. Transfections were carried out with the FuGENE 6 reagent
(Roche Diagnostics) as suggested by the manufacturer. The DNA/FuGENE ratio was 1:2 in all experiments.
Cells extracts were prepared after 48 h to determine either the
levels of CAT protein with a CAT enzyme-linked immunosorbent assay kit
(Roche Molecular Biochemicals) or LUC activities as described
previously (6).
Transfection experiments were done in duplicate and repeated at least
three times.
In Vitro Protein Interaction--
GST-Pax8 protein was purified
from BL21 (DE) LysS bacterial cells transformed with pGEX-Pax8. At
A600 = 0.6, isopropyl-1-thio-
-D-galactopyranoside (0.1 mM final) was added to the culture to induce the expression of the fusion protein, and cells were harvested 4 h later. Cells were resuspended in Lysis Buffer (1× PBS, 0.5 mM EDTA, 1 mg/ml lysozyme, 0.5 mM DTT, 1 mM
phenylmethylsulfonyl fluoride, protease inhibitors diluted 1:1000) and
sonicated. 1% Triton X-100 was added, and the cell extract was
centrifuged at 40,000 rpm for 40 min at 4 °C.
The supernatant was then subjected to affinity chromatography using
glutathione-agarose beads (Amersham Biosciences). After binding, beads
were washed three times with Washing Buffer (10 mM
Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM EDTA, 5 mM DTT).
GST-Pax8 was eluted with a buffer containing 10 mM
glutathione, 50 mM Tris-HCl, pH 8.0, 50 mM
NaCl, for 10 min at 4 °C. The eluted protein was store at
80 °C.
Protein concentration was judged from Coomassie Blue staining.
For the synthesis of bacterial TTF-1 protein,
Escherichia coli cells were transformed with pQE12-TTF-1, an
expression vector encoding for TTF-1 fused to a His6 tag at
the C terminus. The expression and the purification of TTF-1 protein
were performed as described previously (46).
Pull-down assays were performed by challenging 4 µg of GST or
GST-Pax8-purified proteins previously bound to glutathione-agarose beads with 3 mg of total protein extract prepared from PC Cl3 cells or
HeLa cells. The binding reactions were carried out for 90 min at
4 °C on a rotating wheel, and then the beads were washed several
times with a buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Triton X-100, 10% glycerol, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, and
proteases inhibitors (from Sigma). The bound proteins were eluted by
resuspending the beads directly in 2× SDS-PAGE sample buffer and
heating at 95 °C for 3-5 min. before loading on the gel.
The co-immunoprecipitation 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 then
centrifuged, and the agarose gel-bound proteins were washed several
times with a buffer containing 50 mM Tris-HCl, pH 7.5, 120 mM NaCl, 0.5% Nonidet P-40, 10% glycerol, and proteases inhibitors (from Sigma), resuspended in 2× SDS-PAGE sample buffer, and
heated at 95 °C for 3-5 min before loading on the gel.
Indirect Immunofluorescence--
Cells were grown directly on
glass coverslips for 72 h, fixed in 3.7% formaldehyde 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. The coverslips were subsequently incubated for 1 h with a
mixture of primary antibodies diluted in 0.5% bovine serum albumin in
PBS and, after PBS washing, incubated for 20 min with a mixture of
fluorescein-tagged goat anti-mouse and rhodamine-tagged goat
anti-rabbit secondary antibodies diluted 1:50 in 0.5% bovine serum
albumin in PBS. After final washings with PBS, the coverslips were
mounted on a microscope slide using a 70% solution of glycerol in
PBS.
Primary antibodies and sera were monoclonal anti-TTF-1 Ab-1 (clone
8G7G3/1) from Neo Markers (Lab Vision Corporation) and rabbit
polyclonal anti-Pax8 Ab187 (29).
Confocal Scanning Laser Microscopy--
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
63×/1.4 oil-immersion objective. Emitted fluorescence was detected
using a BP 505-530 bandpass filter for fluorescein isothiocyanate, and
a LP 560 long pass filter for TRITC. Pairs of images were collected
simultaneously in the green and red channels. High magnification images
were collected as 512 × 512 × 32 voxel images (sampling
distance 110 nm lateral and 350 nm axial).
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RESULTS |
Pax8 and TTF-1 Co-localize in the Nucleus of Thyroid Cells--
We
have examined the pattern of distribution of Pax8 and TTF-1 in PC cells
by immunolabeling and confocal microscopy. The nuclear localization of
both transcription factors in thyroid cells in culture has been already
shown by indirect immunofluorescence (19). The two antibodies that have
been used, Ab-1 (clone 8G7G3/1) against TTF-1 and Ab187 against Pax8,
are highly specific as indicated by immunoblot analysis (data not
shown). Confocal microscopy examination of dual-labeled samples
confirmed that the anti-TTF-1 (Fig.
1a) as well as the anti-Pax-8
(Fig. 1b) antibody stained only the nuclei, with the
exclusion of nucleoli, and did not give any staining outside the
nucleus. With either antibody the intensity of staining over individual
nuclei varied significantly suggesting a different degree of expression
of each transcription factor at the single cell level. At higher
magnification (Fig. 1, d and e), it clearly appeared that both factors were not diffused within the nucleus but
were instead localized in numerous interconnected nucleoplasmic domains, spread throughout the nucleus. The signals from the two immunostained transcription factors were acquired together, at high
resolution, by line-wise scanning. It has been thus possible to
determine that the patterns of distribution were quite similar and
frequently superimposed, even though not identical, as inferred from
the yellow output color (Fig. 1, c and
f).

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Fig. 1.
Co-localization by confocal microscopy
of Pax8 and TTF-1 in PC cells. PC cells were grown directly on
glass coverslips and stained for immunofluorescence with the Ab-1
(clone 8G7G3/1) anti-TTF-1 monoclonal antibody (a and
d), and the Ab187 anti-Pax8 polyclonal antibody
(b and e). Fluorescein isothiocyanate and TRITC
signals were acquired together at a confocal microscope, by line-wise
scanning. The overlay of the two signals is shown in c and
f. The nucleus within the square in
a-c is shown at higher magnification in d-f,
respectively. Bar, 5 µm
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Pax8 and TTF-1 Interact Both in Thyroid and Non-thyroid
Cells--
In order to determine whether Pax8 and TTF-1 could
physically associate, recombinant GST-Pax8 protein was purified from
bacteria and used in pull-down experiments with total protein extracts prepared from thyroid and non-thyroid cells. In particular, we used the
PC Cl3 cells as a source of thyroid-specific protein extracts and HeLa
cells transiently transfected with an expression vector encoding for
TTF-1 as a source of non-thyroid protein extracts containing exogenous
TTF-1. Results of the binding reactions show that TTF-1 protein is
specifically bound by GST-Pax8 but not by the unfused GST protein (Fig.
2A, compare lanes 2 and 4 with lanes 1 and 3). In
addition, the binding between Pax8 and TTF-1 occurs both in thyroid and
non-thyroid cells (Fig. 2A, lanes 2 and
4), suggesting that the molecular basis of this interaction
does not require a cell type-specific mechanism.

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Fig. 2.
Pax8 and TTF-1 interact both in
vivo and in vitro. A, GST
pull-down assay with GST-Pax8 immobilized on glutathione-Sepharose
beads and protein extracts prepared from PC Cl3 or HeLa cells
transiently transfected with the expression vector CMV-TTF-1. The
extract of either cell types was incubated with GST or GST-Pax8. Both
GST and GST-Pax8 beads were washed several times before being boiled,
run on 10% SDS-polyacrylamide gel, and analyzed by Western blot using
a TTF-1-specific antibody. Lanes 1 and 2, PC Cl3
protein extract. Lanes 3 and 4, protein extract
of HeLa cells transfected with CMV-TTF-1. B, full-length
purified bacterial TTF-1 was generated as described under
"Experimental Procedures" and incubated with GST-Pax8 and GST
control, separately. Both GST and GST-Pax8 beads were washed several
times before being boiled, run on 10% SDS-polyacrylamide gel, and
analyzed by Western blot using a TTF-1-specific antibody. Lane
1, purified bacterial TTF-1. Lane 2, TTF-1 protein
precipitated with GST for control. Lane 3, TTF-1 protein
precipitated with GST-Pax8. C, for the
co-immunoprecipitation 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 first a
TTF-1-specific polyclonal antibody and subsequently a monoclonal
anti-FLAG antibody (Sigma). Lane 1, protein extract of clone
3xFLAG-P8-7. Lane 2, protein extract of clone
3xFLAG-P8-8.
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Note that TTF-1 in thyroid cells (PC Cl3 cell line, Fig.
2A, lane 2) appears as two bands, although when
the cDNA is transiently transfected in non-thyroid cells (HeLa cell
line, Fig. 2A, lane 4), only the higher molecular
weight band is visible. The reason for this difference is still poorly understood.
To investigate the possibility that Pax8 and TTF-1 interact directly,
pull-down experiments were performed using the fusion protein GST-Pax8
and bacterial TTF-1 protein affinity-purified. The
glutathione-Sepharose beads loaded with GST-Pax8 were indeed able to
co-precipitate the bacterial TTF-1 protein (Fig. 2B,
lane 3), although the control reaction of beads loaded with the
GST protein did not co-precipitate any protein (Fig.
2B, lane 2). These results demonstrated that the
interaction between Pax8 and TTF-1, already observed in pull-down
assays with total protein extracts, is indeed a direct protein-protein interaction.
Pax8 Forms a Protein Complex with TTF-1 in Vivo--
To test
whether the interaction observed in vitro in GST pull-down
assays could be observed also in vivo, PC Cl3 thyroid cells were stably transfected with 3xFLAG-Pax8, an expression vector encoding
for Pax8 fused to the FLAG epitope. Several independent clones were
isolated, and the presence of 3xFLAG-Pax8 was determined by Western
blot analysis using a specific anti-FLAG monoclonal antibody (data not
shown). We identified 4 positive clones out of the 10 analyzed, and the
neomycin-positive clones that did not express 3xFLAG-Pax8 were used as
control of the subsequent experiments.
Anti-FLAG-agarose affinity gel was used to immunoprecipitate the
3xFLAG-Pax8 protein from total extracts prepared from clones 3xFLAG-P8-8 (positive clone) and 3xFLAG-P8-7 (negative clone). 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
co-immunoprecipitated protein only in the extract prepared from clone
3xFLAG-P8-8 (Fig. 2C).
Pax8 and TTF-1 Synergistically Stimulate Transcription from the Tg
Promoter--
It was reported previously that in transient
transfection assays in HeLa cells, TTF-1 is able to activate
transcription from a reporter construct in which the Tg minimal
promoter is subcloned upstream of the CAT gene (Tg-CAT (8)). In
contrast, in the same experimental model system Pax8 is a quite poor
activator of the Tg promoter (18). However, several lines of evidence indicate that Pax8 is a transcription factor strictly required for Tg
gene transcription in thyroid cells (17, 19, 20). To test whether Pax8
and TTF-1 could synergistically stimulate transcription from
thyroid-specific gene promoters, expression vectors encoding for Pax8
and TTF-1 were co-transfected in HeLa cells together with the reporter
construct Tg-CAT, separately or in combination. The two transcription
factors when transfected separately enhanced transcription from the Tg
promoter, albeit to a different extent as expected (Fig.
3). Interestingly, co-expression of TTF-1
and Pax8 led to a marked synergism in the activation of the Tg promoter
(Fig. 3). Moreover, a detailed analysis of the synergistic effect
demonstrated that for the functional interaction between Pax8 and TTF-1
on the Tg promoter the ratio of the two transcription factors is
crucial. It is worth mentioning that when a very small DNA
concentration of both transactivators are used, it is possible to
observe the synergism. In contrast, higher DNA concentrations still
have the ability of activating transcription from the promoter but do
not show the synergism any longer (data not shown). A clear
dose-dependent effect was observed as reported in other
studies (23).

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Fig. 3.
Synergistic activation of the thyroglobulin
promoter by Pax8 and TTF-1. HeLa cells were transiently
transfected with the reporter plasmid Tg-CAT containing the minimal
promoter region of the rat thyroglobulin gene, with or without the
expression vectors encoding for TTF-1 and Pax8. The cells were
subsequently harvested and assayed for CAT activity. Folds of
activation are considered as ratio between values obtained with and
without co-transfection of the expression vectors. CMV-LUC was added as
internal reference, and CAT values were normalized to the LUC activity.
Values are the mean of at least three independent experiments.
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TTF-1 N-terminal Domain Is Required for the Synergy with
Pax8--
Previous studies demonstrated that TTF-1 activates
transcription via a functional redundancy of the N-terminal and
C-terminal domains (8). To determine which of the two domains mediates the synergy with Pax8 in the transcriptional activation of the Tg
promoter, we have used deletion mutants described previously (8). The
mutants, named
14 and
3, were transfected in HeLa cells together
with the reporter construct Tg-CAT, in the absence or presence of Pax8.
As expected, both mutants were able to activate the Tg promoter as the
wild-type TTF-1 molecule (Fig.
4A). However, we demonstrated
that mutant
14 that contains the N-terminal activation domain and
the homeodomain is able to synergize with Pax8 on Tg-CAT-like wild-type
TTF-1 (Fig. 4A). On the contrary mutant
3, which contains the homeodomain and the C-terminal activation domain, lacks the ability
to exert the synergism with Pax8 (Fig. 4A). Therefore, we
conclude that even though in previous studies it was suggested that the
two activation domains of TTF-1 were functionally equivalent, only the
N-terminal domain of TTF-1 is able to synergize with Pax8. To map
further TTF-1 N-terminal sequences involved in transcriptional activation with Pax8, we have tested additional TTF-1 N-terminal deletion mutants described previously (8), such as
1,
2, and
33 (Fig. 4B). As before, the mutants were transfected in HeLa cells together with the reporter construct Tg-CAT, in the absence
or in the presence of Pax8. The results obtained for each of the
mutants tested indicate that the sequence downstream from amino acid 51 in TTF-1 N-terminal domain is required for the synergistic activation
with Pax8. In fact, deletion mutant
1 behaves as wild-type TTF-1
(Fig. 4B), suggesting that the region spanning between amino acids 1 and 51 is not essential for the synergy with Pax8. At the same
time, mutants
33 and
2 show a progressively reduced ability in
the cooperation with Pax8 (Fig. 4B), thus indicating that
the portion of the protein downstream from residue 51 is involved in
the synergism.

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Fig. 4.
The synergistic effect on the thyroglobulin
promoter requires TTF-1 N-terminal domain. A, schematic
representation of the different TTF-1 proteins used in transactivation
experiments. 3 contains the homeodomain and the C-terminal
activation domain. 14 contains the N-terminal activation domain and
the homeodomain. HeLa cells were transiently transfected with the
reporter plasmid Tg-CAT and the expression vectors encoding either
wild-type TTF-1 or the deletion mutants. Folds of activation are
considered as ratio between values obtained with and without
co-transfection of the expression vectors. CMV-LUC was added as
internal reference, and CAT values were normalized to the LUC activity.
Values are mean of at least three independent experiments.
B, schematic representation of TTF-1 N-terminal mutants.
HeLa cells were transfected as described before. Mutants 1, 33,
and 2 lack amino acids between 1 and 51, 1 and 92, and 1 and 102, respectively. CMV-LUC was added as internal reference, and CAT values
were normalized to the LUC activity. Values are mean of at least three
independent experiments.
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In order to test whether the different ability to synergize with Pax8
of mutants
14 and
3 was due to a different ability in
establishing a protein-protein interaction, we have performed pull-down
experiments with protein extracts prepared from HeLa cells transiently
transfected with the expression vectors encoding for
14 and
3
mutants. Results of the binding reactions show that
14 protein is
specifically bound by GST-Pax8, although
3 protein is not, thus
demonstrating that the biochemical interaction is required for the
synergism (Fig. 5).

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Fig. 5.
The interaction with Pax8 requires TTF-1
N-terminal domain. A, schematic representation of TTF-1
mutated proteins. 3 contains the homeodomain and the C-terminal
activation domain. 14 contains the N-terminal activation domain and
the homeodomain. B, GST pull-down assay with GST-Pax8
immobilized on glutathione-Sepharose beads and protein extracts
prepared from HeLa cells transiently transfected with the expression
vector encoding for 14 and 3 proteins. The extracts were
incubated with GST or GST-Pax8. Both GST and GST-Pax8 beads were washed
several times before being boiled, run on 10% SDS-polyacrylamide gel,
and analyzed by Western blot using a TTF-1-specific antibody (for
14) or an antibody (mAb 9E10 from Santa Cruz Biotechnology, Inc.)
recognizing the Myc tag epitope (for 3).
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Identification of Pax8 Domains Required for the Functional
Cooperation--
There are several reported isoforms of Pax8 protein
(26, 31). Among those, Pax8a is the full-length isoform that contains all 10 exons and encodes for the most abundant Pax8 protein species. Pax8b does not contain exon 8, although Pax8c lacks exons 7 and 8. In
this latter isoform, as a consequence of the splicing of two exons, the
shift in the reading frame generates a proline-rich C terminus and a
premature termination of translation (Fig.
6A).

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Fig. 6.
Pax8 C-terminal domain is involved in the
cooperation with TTF-1. A, schematic drawing of Pax8
isoforms. Pax8a is the full-length isoform. Pax8b lacks exon 8, although Pax8c lacks exon 7 and 8. The proline-rich domain arising due
to the alternative splicing results in the reading frameshift and
premature termination of translation. Mutant 287 lacks the
C-terminal domain downstream exon 6. PD, paired domain;
oct, octapeptide; HD/2 partial homeodomain.
B, HeLa cells were transiently transfected with the reporter
plasmid Tg-CAT, the expression vector encoding TTF-1, and the different
Pax8 isoforms or deletion mutant. Folds of activation are considered as
ratio between values obtained with and without co-transfection of the
expression vectors. CMV-LUC was added as internal reference, and CAT
values were normalized to the LUC activity. Values are mean of at least
three independent experiments. Pax8a and Pax8b synergistically activate
Tg promoter with TTF-1. Pax8c and 287 are not able to cooperate with
TTF-1.
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The C-terminal part of Pax8 full-length protein is rich in proline,
serine, and threonine (PST domain) and functions as a transcriptional
activation domain (32). In addition, data obtained studying various Pax
genes showed that the above regulatory module present in the C-terminal
region has been conserved among the members of the family (32).
The different Pax8 isoforms encode for proteins that differ in the C
terminus and therefore could differ, as suggested in previous studies,
in their transactivation properties (26). In all our experiments we
have used the full-length Pax8 cDNA, namely the Pax8a isoform. We
then asked whether the C terminus of the protein could be the portion
involved in the synergism between Pax8 and TTF-1 in the transcriptional
activation of the Tg promoter. To answer this question, we tested
whether Pax8a, Pax8b, and Pax8c behave differently with respect to the
synergism with TTF-1 in Tg activation in HeLa cells.
It was demonstrated previously (17) that the isoforms Pax8a, -b, and -c
are equally able to bind the same sequence derived from the Tg
promoter. Thus, we co-transfected expression vectors encoding for
Pax8a, Pax8b, and Pax8c, in the presence or absence of TTF-1, together
with the Tg promoter reporter construct in HeLa cells. The results
shown in Fig. 6B demonstrate that although Pax8a and Pax8b
are equally capable of functionally cooperating with TTF-1, the isoform
Pax8c is no longer able to do so. Accordingly, a deletion mutant,
287, lacking the entire C-terminal region, is also not able to show
any synergism when tested in the same experimental conditions (Fig.
6B). These results clearly demonstrate that the C terminus
of the protein is the portion of Pax8 involved in the synergism with
TTF-1 that we observed on the Tg promoter.
The TTF-1 and Pax8 Overlapping Binding Sites Are Involved in the
Synergism--
Pax8 binds to a single site on the Tg promoter, and the
binding site overlaps with one of the TTF-1-binding sites (18). To
elucidate the molecular mechanism taking place in the synergism between
the two transcription factors, we asked which of the three TTF-1-binding sites present on the Tg promoter was involved in the
observed effect. Thus, we investigated the effects of Pax8 and TTF-1 on
reporter constructs containing point mutations of the Tg promoter. We
first tested the Tg-CAT Acore mutant, which contains point mutations
that abolish the binding of TTF-1 at its distal site on the promoter
(site A (24)). Therefore, we co-transfected the Tg-CAT Acore promoter
mutant with both TTF-1 and Pax8 in the same experimental conditions
used for the wild-type promoter. Interestingly, we were able to observe
a strong synergism between TTF-1 and Pax8 in the transcriptional
activation of the promoter (Fig. 7). The
extent of the synergism on Tg-CAT Acore was comparable with that
observed on the wild-type Tg promoter. This result strongly suggest
that the molecular mechanisms at the basis of the synergistic effect
lie within the so-called region C of the Tg promoter (3, 18), which
contains the TTF-1-binding site proximal to the start of transcription
overlapping the Pax8-binding site. To test this hypothesis, we have
used the Tg-CAT Ccore mutant, which contains point mutations that
abolish the binding to region C of the promoter (Fig. 7). When we
tested this mutated promoter in co-transfection experiments in HeLa
cells, we did not observe any synergy. Thus, our results point out at a
molecular mechanism involving specifically site C of the Tg
promoter.

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Fig. 7.
Mapping of the cis-elements involved in
Pax8/TTF-1 synergy. A, schematic representation of the Tg
reporter constructs: wild-type (Tg-CAT), mutated in the
distal TTF-1-binding site (Tg-CAT Acore) and mutated in the
TATA box proximal Pax8 and TTF-1-binding sites (Tg-CAT
Ccore). Gray ovals represent TTF-1, and the white
rectangle represents Pax8. B, HeLa cells were
transiently transfected with the reporter plasmids containing the
wild-type Tg promoter (Tg-CAT), or with the mutated Tg-CAT
Acore and Tg-CAT Ccore promoters with or without the expression vectors
encoding for TTF-1 and Pax8. CMV-LUC was added as internal reference,
and CAT values were normalized to the LUC activity. Folds of activation
are considered as ratio between values obtained with and without
co-transfection of the expression vectors. Values are mean of at least
three independent experiments.
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Pax8 and TTF-1 Do Not Strongly Synergize on the TPO
Promoter--
Previous studies (4) have shown that in HeLa cells Pax8
strongly activates transcription from the TPO promoter. In order to
investigate whether the functional interaction between Pax8 and TTF-1
occurs on another thyroid-specific promoter structurally very similar
to the Tg one, HeLa cells were transfected with Pax8 and TTF-1 and the
reporter construct TPO-LUC (4). As expected Pax8 activates
transcription from the TPO promoter severalfold, whereas TTF-1 is less
efficient (Fig. 8). However, when
co-transfected together, Pax8 and TTF-1 are unable to synergize on the
TPO promoter (Fig. 8). These results indicate that although both
transcription factors bind to the Tg and TPO promoters in regions
displaying extensive sequence homology, their functional interaction is
promoter-specific.

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Fig. 8.
Pax8 and TTF-1 do not synergize on the TPO
minimal promoter. HeLa cells were transiently transfected with the
reporter plasmid TPO-LUC containing the minimal promoter region of the
rat thyroperoxidase gene, with or without the expression vectors
encoding for TTF-1 and Pax8. The cells were subsequently harvested and
assayed for LUC activity. Folds of activation are considered as ratio
between values obtained with and without co-transfection of the
expression vectors. CMV-CAT was added as internal reference, and LUC
values were normalized to the CAT activity. Values are mean of at least
three independent experiments.
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DISCUSSION |
In this study we show that the paired domain-containing
transcription factor Pax8 and the homeodomain-containing transcription factor TTF-1 co-localize in the nucleus of thyroid cells and
synergistically activate transcription from the promoter of
thyroglobulin, a thyroid-specific gene. A direct biochemical
interaction of the two factors was observed both in in vitro
and in vivo assays. This evidence, together with their well
established role during development/morphogenesis of the thyroid gland,
supports the hypothesis that a protein-protein interaction between
these two transcription factors may play a relevant role in the
expression of target genes in the thyroid. Recently, pax8
was shown to be a master gene for the maintenance of
thyroid-differentiated phenotype, being important for the
transcriptional activation of all the differentiation markers such as
thyroglobulin, thyroperoxidase, and sodium/iodide symporter genes (17).
Pax genes encode evolutionary conserved transcription factors that act
high up in the regulatory hierarchy controlling the development of
various organs. From the analysis of the transgenic or knockout mice,
it has become clear that pax genes are key regulators during crucial steps of the organogenesis of various tissues (33-36). In
particular, Pax8 knockout mice have a barely visible thyroid gland,
which is deprived of the follicular cells (16), and human patients
suffering from congenital hypothyroidism have been shown to carry
mutations in the Pax8 gene (37-39). TTF-1 is also an
important factor for thyroid morphogenesis. TTF-1 knockout mice lack
the thyroid and the pituitary gland and display severe defects in the
ventral area of the forebrain and in peripheral lung parenchyma (14).
In the present study, we show that Pax8 and TTF-1 are able to interact
directly in vitro in pull-down experiments, both in thyroid
and non thyroid cell extracts. In addition, by co-immunoprecipitation we also show that Pax8 and TTF-1 form a complex in vivo. Our
data provide the first strong evidence of the existence of a
biochemical interaction between these two transcriptional activators.
Moreover, we clearly show that the presence of both factors together
exclusively in thyroid cells has indeed a functional relevance. In
fact, their synergistic effect in the transcriptional activation of the
Tg promoter represents an example of how tissue-specific gene
expression may be achieved.
Previous results from our laboratory already suggested that TTF-1 and
Pax8 might cooperate in the transcriptional activation of Tg gene
expression in thyroid cells (17). Furthermore, it was recently claimed
that Pax8 and TTF-1 might cooperate in the regulation of
thyroid-specific gene expression (22, 23). However, none of
above-mentioned studies unravel a protein-protein interaction between
Pax8 and TTF-1. Interestingly, our previous data showed (18) that the
binding site of Pax8 on the Tg promoter overlaps one of the
TTF-1-binding sites and that, at least in vitro, the two
factors cannot bind together to the same DNA region. The results presented in this article provide the evidence of a direct interaction between Pax8 and TTF-1, which occurs via protein-protein interaction in
the absence of DNA. It is likely that the different experimental assays
used in the previous study are the reason of our failure to detect the interaction.
What could be the mechanism leading to the synergistic activation of
the Tg promoter by TTF-1 and Pax8 transcription factors? Interactions
among transcription factors that bind to separate promoter elements may
depend on various events, such as distortion of DNA structure. We have
now established that the molecular mechanisms leading to the
synergistic activity of Pax8 and TTF-1 implicate region C of the Tg
promoter (3, 18), which is where the binding sites of the two factors
overlap. It is, however, still unclear if the two proteins are both
binding to the DNA or if only one of them is binding and thus recruits
the other one to the promoter. Further investigation is required to
elucidate which protein is indeed bound to the DNA in vivo.
By mutational analysis we have determined the regions of Pax8 and TTF-1
proteins that are involved in the functional cooperation. TTF-1
contains two distinct activation domains, one at the N terminus and the
other at the C terminus, which have been shown to be functionally
equivalent (8). We demonstrated that only one domain, the N-terminal
domain, is able to mediate the synergism with Pax8. In addition, we
show that the synergy requires a biochemical interaction and that TTF-1 N-terminal domain is involved in such an interaction. At the same time
we have also established that the C-terminal portion of Pax8 protein is
necessary for the cooperative effect. In this part of the protein that
is very rich in proline, serine, and threonine lies the activation
domain of Pax8.
Moreover, it has become evident that, despite the high similarity of
the Tg and TPO promoter architecture, the molecular mechanisms leading
to transcriptional activation are different. In contrast with the
transactivation synergy observed on the Tg promoter, no such effect was
observed on the TPO promoter. Recently, Miccadei et al. (23)
observed a synergistic activity of TTF-1 and Pax8 that relies on the
TPO promoter/enhancer interplay. In their study they show that the
synergistic activity can be observed only in the presence of the
enhancer element. In our experiments we have used the minimal region of
the TPO promoter that is able to drive thyroid-specific gene expression
(4), and on this regulatory element we are not able to show any
synergism likely because of the lack of the upstream enhancer. It is
possible that Pax8 and TTF-1 interact in complex ways at cis-active
elements of target genes.
There is increasing evidence that TTF-1 functions cooperatively with a
number of the other transcription factors, forming complexes on
regulatory regions of target genes. TTF-1 interacts with retinoic acid
receptors and co-factors (40), AP-1 family members (41), BR22 (42),
CBP/p300 (43), TGD (T:G mismatch-specific thymine DNA glycosylase)
(44), and GATA-6 (15). Pax8, as it has already been observed for other
members of the pax gene family (30, 45), could play a role
in the recruitment to the Tg promoter of other factors, like TTF-1, and
one or more co-activators. Our current hypothesis is that, depending on
the promoter context within target genes, TTF-1 and Pax8 can act
separately or in cooperation with various factors or co-factors to
regulate gene expression. Thus, these different classes of
transcriptional regulatory molecules may form a network through
protein-protein and protein-DNA interactions in thyroid follicular
cells, allowing the fine-tuning of thyroid-specific gene expression.