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INTRODUCTION |
The mechanisms by which cells selectively activate the
transcription of a specific gene are essential. Tissue-specific
transcription factors that bind DNA sequences within the promoter are
the main mediators of tissue-specific gene expression (1). It has
become clear, however, that transcriptional activation of a given gene is defined not only by the activity of an individual factor or a single
DNA-binding site, but rather, depends on combinatorial interactions
between multiple proteins (2, 3). To understand the mechanism of
tissue-specific transcriptional activation, it is first necessary to
identify cis-regulatory elements and to characterize
tissue-specific transcription factors, and then to define
protein-protein interaction that determine their function.
The focus of our work has been to understand the regulatory mechanisms
underlying hormonal transcription of the thyroperoxidase (TPO)1 gene, a
tissue-specific enzyme expressed only in differentiated thyroid cells.
Its function involves the iodination and coupling of tyrosine residues
into the thyroglobulin molecule to generate thyroid hormones (4-6).
Both thyroglobulin and TPO are cell type-specific genes whose
respective promoters have been characterized (7, 8). With the use of
DNA binding assay, three thyroid-specific transcription factors have
been identified: TTF-1, TTF-2, and Pax-8 (9). Cloning of these three
proteins demonstrated that they are members of different transcription
factor families. TTF-1 and Pax-8 are homeo- and paired-containing
proteins, respectively (10, 11), and TTF-2 is a forkhead protein (12).
The three factors are expressed at the beginning of thyroid development (11-13) and are considered decisive in the maintenance of thyroid phenotype. All of them bind within the thyroglobulin and TPO promoter at similar positions (7, 8, 14) although the TPO promoter differs in
several aspects from that of thyroglobulin. It is thus approximately an
order of magnitude less active than the thyroglobulin promoter; the
Pax-8 protein overlapping the TTF-1-binding site has a different
position (14) and the ubiquitous transcription factors that bind to
both promoters occupy different sites in each one (7, 8).
Several ligands regulate the expression of the TPO gene through
alteration of the activity of the transcription factors that control
its expression. For example, we have recently demonstrated that the
transcription factor TTF-2 is under the hormonal control of the
thyrotropin (TSH) and the cAMP as well as to the insulin and
insulin-like growth factor I signaling pathways (15). TTF-2 binds to a
single site that acts as a hormone response element. This function
depends on multimerization and specific orientation of the
TTF-2-binding site (16). This suggests that TTF-2 is part of a complex
interaction network within the TPO promoter, whose final result is to
turn-on the specific expression of the TPO gene in response to external
hormonal stimuli.
As the binding site for TTF-2 functions in an orientation-specific
manner, we asked whether TTF-2 alone regulates the expression of this
gene or requires the action of neighboring sequences. Neighboring
regulatory elements of TTF-2 bind the thyroid-specific transcription
factor TTF-1 and the ubiquitous transcription factor UFB (8). Here we
show by transient transfection assays and site-directed mutagenesis
that the binding sites for TTF-2 and UFB are important for
hormone-induced expression at the TPO promoter. Furthermore, we show
that UFB is a binding site for members of the CTF/NF1 family of
transcription factors. These are a multiprotein family in which four
different genes have been cloned, NF1-A, NF1-B, NF1-C, and NF1-X
(17-19), as well as different isoforms generated by alternative
splicing (17, 20-22). Although their expression is fully ubiquitous,
differences can be detected in the distribution and abundance of their
different transcripts (23, 24). Strikingly, we have found that, as
occurs for TTF-2 (15), CTF/NF1 gene expression is up-regulated by TSH
and insulin in inducing the expression of the TPO gene. Furthermore, we
present evidence, from a GST pull-down assay, that these constitutive factors interact physically with TTF-2. This interaction appears to be
functional, since the TPO promoter activity and its hormonal response
are lost in transfection experiments in which the distance between the
CTF/NF1 and TTF-2-binding site has been altered. Thus the hormonal
control of TPO gene transcription, which takes place exclusively in
thyroid-differentiated cells, depends on the correct stereospecific
interaction of two hormonally expressed transcription factors: TTF-2
and CTF/NF1.
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EXPERIMENTAL PROCEDURES |
Materials--
Tissue culture medium, bovine TSH, and bovine
insulin were purchased from Sigma, and forskolin from Roche Molecular
Biochemicals (Mannheim, Germany). Donor, fetal calf serum, and
Dulbecco's modified Eagle's medium were from Life Technologies, Inc.
(Gaithersburg, MD); Nytran membranes were obtained from Schleicher & Schüll (Richmond, CA). TnT and luciferase assay kits were
purchased from Promega (Madison, WI). [
-32P]dCTP,
[
-32P]ATP, and [35S]methionine were from
ICN (Irvine, CA).
Cell Culture and Transfection--
FRTL-5 cells (ATTC CRL 8305;
American Type Culture Collection, Manassas, VA) were cultured as
described previously (25) in Coon's modified Ham's F-12 medium
supplemented with 5% donor calf serum and a six-hormone mixture
including 1 nM TSH and 10 µg/ml insulin (complete
medium). The effect of TSH and insulin were studied by starving
confluent or transfected cells for both hormones in the presence of
0.2% serum (basal medium) (26). After 4 days, each ligand was added to
the culture medium at the concentrations given. HeLa cells were
cultured in Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum. For transient expression assays, transfections were
performed by the calcium phosphate coprecipitation method, as described
for each cell line (16, 27). The plasmid RSV-CAT (28) was used to
correct for transfection efficiency. Luciferase and CAT activities of
cell extracts were determined as described (29, 30).
Promoter Constructs--
p420 TPO LUC containing the minimum TPO
promoter linked to the luciferase cistron (8) and the deletion p60 TPO
LUC (16) have been previously described. The deletions p120 and p92
TPOLUC, as well as insertions p120(+5) and p120(+10), were generated by the polymerase chain reaction on the p420 TPO LUC template, using as
flanking primers the 3' polymerase chain reaction primer LUC-1: 5'-GGATAGAATGGCGCCGGGCCTTTCTTTATG-3' and the following
oligonucleotides as 5' polymerase chain reaction primers: TPO-120,
5'-AAGAGCTCATACTAAACAAACAG-3'; TPO-92,
5'-AAGAGCTCGACACACAAGCACTTGGCAG-3'; TPO-120 (+5),
5'-AAGAGCTGACACACAAGCACTTGGCAGAAACGGATCAAATACTAAAC-3'; and TPO-120
(+10), 5'- AAGAGCTGACACACAAGCACTTGGCAGAAACGGATCCGACGAAATACTAAAC-3'. Amplified fragments were digested with SacI and
PstI and subcloned into pBS LUC-2 (8). The mutated
constructs, pBmm TPO LUC and pZm TPO LUC were previously described (8).
In the pBmm mutant the TTGG sequence is altered to GGTC and TTF-1 and
UFB binding activity is thus undetectable. pZm contains a 4-bp mutation
within the Z site, which interferes with TTF-2 binding. Constructs
containing tandem repeats of Z site (
93 to
73) in front of the
minimal promoter TATA LUC have been described elsewhere (16). The pBZ TATA LUC was made by insertion of a double strand synthetic
oligonucleotide containing the BZ (
118 to
73) region of the TPO
promoter 5'
3', into the SmaI site of the plasmid TATA
LUC (16).
Expression Vectors--
pSG-LexVP-16 has been previously
described (31). pBAT-hCTF-1 and pBAT-CTF/NF1-X constructs were obtained
by digesting the coding sequence of NF1/CTF1 and NF1-X from the
RSV-based expression vector (21) with XbaI/XhoI
and EcoRI/XbaI followed by a fill-in reaction and
ligation into the SmaI site of the plasmid pBAT (32). The
bacterial expression plasmid pGEX-4T3 was utilized to direct overexpression of the full-length GST-TTF-2 fusion protein. The full-length TTF-2 cDNA was digested with BamHI and
EcoRI and subcloned into the pGEX-4T3 vector. The mammalian
expression vector RSV-CTF/NF1-C has been previously described (21). The
expression vectors CMV-CTF/NF1-B and CMV-CTF/NF1-X (33) were kindly
provided by Dr. B. Gao (MCV-VCU, Richmond, VA).
RNA Analysis--
Total RNA was extracted by the guanidinium
isothiocyanate procedure (34). Polyadenylated RNA preparation was
performed with oligo(dT)-cellulose chromatography as described by Nebl
et al. (35). Thirty micrograms of total RNA or 5 µg of
poly(A)+ were denatured and fractionated on a 1% agarose
gel containing 3.7% formaldehyde. RNA was then blotted and fixed onto
Nytran membranes. The radioactive probes used included a 0.35-kb
HindIII/EcoRI fragment from the 3'-untranslated
region of rat TTF-2 (p3'UTRT) (12), a 0.6-kb EcoRI fragment
from rat TTF-1 (10), a 1.1-kb EcoRI fragment from the 5'-end
of human CTF-1 (17), a 0.5-kb PstI/EcoRI fragment
from the 5'-end of the hamster NF-1 X (18), a 1.5-kb EcoRI
from the 5'-end of hamster NF-1/Red (18), and a 1.0-kb PstI
fragment of p91
-actin (36). Hybridizations were carried out at
65 °C in 4 × SSC (1 × SSC is 0.15 M NaCl,
0.125 M sodium citrate), 10 mM EDTA, and 0.05%
SDS. After hybridization, the filters were washed at 65 °C for 30 min each in 3.3% phosphate buffer, pH 7.2, 0.1% SDS and successively
lower salt concentrations (2, 1, and 0.5 × SSC) before autoradiography.
DNase I Footprinting and Electrophoretic Mobility Shift
Assays--
For electrophoretic mobility shift assays (EMSA), the
nuclear protein fraction was extracted by the procedure described by Andrews and Faller (37). Protein concentration was measured according
to Bradford (38) with the Bio-Rad protein assay kit using bovine serum
albumin as standard. The recombinant purified NF-1 protein, used in
both EMSA and DNase footprinting, was kindly provided by Dr. M. Beato
(Institut fur Molecularbiologie und Tumorforshung, Marburg, Germany).
The footprinting probe, corresponding to the
257 to +30 region of the
TPO promoter was prepared by polymerase chain reaction using TPOF1
(
257 to
236): 5'-ATAAGAGAAACTCCCAGGAACC-3', and TPOF6 (+9 to +30):
5'-ACTTCAGAAATGTGAATCTCAA-3' labeled oligonucleotides as flanking
primers on the p420 template. DNase I footprinting reactions were
carried out in a 50-µl reaction volume as follows: recombinant NF-1
(5 or 25 ng) was preincubated with 5 × 104 cpm for 45 min on ice in 20 mM HEPES, pH 7.9, 50 mM KCl,
0.1 mM EDTA, 0.5 mM dithiothreitol, 10%
glycerol, and 0.4 mg/ml bovine serum albumin. DNase I digestion was
performed by the addition of 6 ng of DNase I in 10 mM
MgCl2, 2 mM CaCl2 and incubation
for 1 min at room temperature. Footprinting reactions were terminated by addition of 200 µl of a stop mixture (20 mM Tris-HCl,
pH 8.0, 1 M NaCl, 20 mM EDTA, 0.5% SDS, and
250 µg/ml proteinase K). The samples were incubated for 1 h at
45 °C, extracted with phenol-chloroform, ethanol precipitated, and
resuspended in formamide dye. Equal number of counts per sample were
loaded and resolved on an 8% sequencing gel, together with G and G + A
chemical sequencing reactions. Gels were fixed, dried, and visualized
by autoradiography. EMSA were carried out as described previously (39)
using 32P-labeled double-stranded oligonucleotide
UFB (5'-CAAGCACTTGGCAGAAACAAATAC-3') or consensus NF-1
(5'-CATATTGGCTTCAATCCAAA-3') derived from the MMTV promoter (40). For
supershift experiments, 1 µl of anti-NF-1/8199/2902 (kindly provided
by Dr. N. Tanese, New York University, New York) or preimmune serums
were added to the preincubation mixture.
GST-TTF-2 Fusion Protein and Pull-down Experiments--
An
overnight culture of the bacterial strand BL21 cells harboring plasmid
pGEX-4T3 or pGEX-TTF-2 was diluted 1:10 in a 2 × YT medium plus
ampicillin, pH 7.0, and cultured at 27 °C to optical density at 600 nm of 0.6-0.75. Protein expression was then induced by adding 0.1 mM isopropyl-
-D-thiogalactopyranoside and
cultures were incubated for an additional 2 h at the same
temperature. Cells were harvested and the fusion proteins purified as
described (41). The integrity of the GST fusion proteins bound to the beads was analyzed by resolution by 8% SDS-polyacrylamide gel electrophoresis (PAGE) and Coomassie Blue staining. Known amounts of
bovine serum albumin were included on the same gel for determination of
the yield. For in vitro translation in reticulocyte lysate, we used a coupled transcription/translation system (TnT, Promega) in
the presence of [35S]methionine (1000 mCi/mmol). The
protocol for the GST pull-down assay was essentially as described (42).
GST or GST-TTF-2 proteins (5 µg) immobilized on glutahione-Sepharose
4B beads were washed extensively with LBST-100 buffer (25 mM Hepes-KOH, pH 7.9, 100 mM NaCl, 6%
glycerol, 5 mM MgCl, 1 mM dithiothreitol,
0.05% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 5 mM EDTA) and the volume was raised to 180 µl with
LBST-100 buffer. Radioactively labeled protein (20 µl) was then added
and gently mixed at room temperature for 30 min, followed by a further
30-min incubation with gentle shaking at 4 °C. The beads were washed
four times with successively increasing NaCl concentrations (LBST-100,
LBST-300, and LBST-500), and the bound proteins were analyzed in 8%
SDS-PAGE followed by autoradiography.
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RESULTS |
Cooperativity of Regulatory Elements on the TPO
Promoter--
Previous results from our laboratory have demonstrated
that TPO promoter activity is hormonally regulated by TSH through the cAMP pathway, as well as by insulin and insulin-like growth factor I. This regulation is mediated mainly by the cis-regulatory
element (Z) to which the forkhead thyroid transcription factor-2
(TTF-2) binds (see Fig. 1A,
for cartoon). Further analysis showed that the TTF-2-binding site acts
as a hormone response element in a heterologous construct and that it
requires a specific orientation for activation of the TPO promoter
(16). This suggests that TTF-2 may require other bound factors for
activation of TPO expression gene.

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Fig. 1.
Identification of the B-site of the TPO
promoter as the element that cooperates with the TTF-2 binding
site. A-C, upper panels, are the schematic
diagram of the wild type TPO promoter (p420 TPO) and the different
constructs, described under "Experimental Procedures," linked to
the luciferase reporter gene (LUC). The deletions and
mutations generated on the wild type promoter are represented as a
double line (panels A and B) while the
tandem repeats generated on the TATA LUC are represented as a
single line (panel C). The protein-binding site
detected by footprinting assays (8) and the corresponding transcription
factors are indicated with different symbols. A-C,
lower panels, correspond to the TPO promoter activity
derived from 10 µg of each construct transiently transfected to
FRTL-5 thyroid cells. After transfection cells were maintained 72 h in the absence of serum (0.2%) without TSH and insulin (basal
medium). Then, the cells were treated with 1 nM TSH or 2 µM insulin for 24 h. Relative luciferase activity is
the value of light units normalizing the results to CAT activity
derived from 2 µg of RSV-CAT transfected to correct for transfection
efficiency. The TPO promoter activity is expressed as fold induction
over the basal levels (= 1) of hormone-depleted cells. The results are
the mean ± S.D. of four independent experiments.
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To identify the regulatory elements on the TPO promoter that cooperate
with the TTF-2-binding site, we transfected into FRTL-5 thyroid cells
different promoter constructs of this gene (Fig. 1, upper
panels) linked to the coding region of the firefly luciferase gene
(30). The transfected cells were cultured for 72 h in a minimal
medium depleted of TSH and insulin but supplemented with 0.2% serum to
ensure only a basal expression of the TPO (16, 43). TPO promoter
activity was enhanced by treatment of the cells with TSH or insulin for
24 h. The promoter activity was determined by luciferase activity
measurements while CAT activity derived from a co-transfected RSV-CAT
construct was used to correct for variability in transfection efficiency.
In this transfection assay, only the p420 TPO LUC and p120 TPO LUC
constructs showed hormone inducibility (Fig. 1A, lower panel). Neither p92 TPO-LUC nor p60 TPO-LUC, in which successive deletions were performed of the B and Z site, respectively, responded to TSH and insulin treatment. As both inducers enhanced the promoter activity of construct p120 but not p92 TPO LUC, the B element missing
in the p92 construct must be important for the hormone response.
The B element has been previously reported to bind the transcription
factors TTF-1 and UBF. Mutations introduced into both binding sites
(pBmm) that abolished binding by their respective factors (8)
drastically reduced hormone regulation of expression at the TPO
promoter (Fig. 1B). Mutations in the Z regulatory element destroying the binding of TTF-2 (pZm) also abrogated hormone
inducibility (Fig. 1B) indicating a concerted action of the
B and Z regulatory sites for hormone inducibility. The fact that the
multimerization of the Z element confers hormone inducibility to the
minimal promoter TATA LUC and a single Z element was unable (Fig.
1C) implies that the hormone regulatory activity of the Z
element comes from cooperative action with itself or possibly with
other transcription factor. To find out whether the B regulatory
element can confer hormone inducibility to a single Z element, we
cloned the B and Z regulatory units in front of the minimal promoter.
This construct in the transfection experiments produced an identical
result as p4xZ in its response to hormone treatment. We therefore
concluded that the hormonal response of the TPO promoter activity is
dependent on an active cooperation between TTF-2 and the factors
binding to the B site. It is important to mention that in the above
study the TSH effect was mimicked by forskolin (data not shown).
CTF/NF1 Proteins Bind to UFB-binding Site--
Analyses of the BZ
region (
120 to
73) of the TPO promoter by in vitro
footprinting with nuclear extracts from FRTL-5 thyroid and non-thyroid
Rat-1 cells had identified three different transcription factors that
bind to this sequence (8). Two of these are thyroid-specific and were
identified as TTF-1 and TTF-2, homeo and forkhead domains containing
proteins, while the third factor was characterized as a ubiquitous
factor (UFB) because it was found in both cell lines studied. Since UFB
has not yet been characterized, we analyzed the B promoter region for
potential recognition sequences for known ubiquitous transcription
factors. At position
100 bp from the transcription initiation site
within the B region is a TTGGCA motif that has been characterized as a
CTF/NF1-binding site in many eukaryotic promoters (18, 44-49).
Recombinant CTF/NF1 protein was therefore used in DNase I footprinting
experiments with the labeled TPO promoter fragment. The purified
CTF/NF1 protein protected the sequence 5'-AAGCACTTGGCAGAAACAA-3' from
position
112 to
95 (Fig.
2A), corresponding exactly to
the previously defined UFB-binding site (8). In EMSA in which we used
the UFB oligonucleotide (
112 to
95) and the recombinant CTF/NF1,
two protein complexes were formed possibly due to the intrinsic
property of the CTF/NF1 proteins to form homo- and heterodimers (20,
50) (Fig. 2B, lane 2). Both protein-DNA complexes were
specifically inhibited by addition of a 100-fold excess of unlabeled
oligonucleotide UFB (lane 3) as well as by a CTF/NF1
consensus sequence, derived from the mouse mammary tumor virus (MMTV)
promoter (51) (lane 4) and by the BZ oligonucleotide (
120
to
73 bp of the TPO promoter) (lane 5), but not by an
unrelated oligonucleotide (lane 6). If instead of the
recombinant CTF/NF1 protein, nuclear extracts of FRTL-5 cells were
used, a smear containing two distinct complexes was obtained (Fig.
2C, lane 2). These two complexes as well as the smear were
also competed by a 100-fold excess of unlabeled UFB (lane 3)
and the consensus CTF/NF1 oligonucleotide (lane 4) but not
by an unrelated oligonucleotide (lane 5). Preincubation of
the binding reaction with an anti-CTF/NF1 antiserum but not with a
preimmune serum, produced a supershifted band with a corresponding reduction in the CTF/NF1 complexes (Fig. 2C, compare
lanes 6 and 7). As a control of the supershift
assay the labeled oligonucleotide was preincubated with the CTF/NF1
antibody alone (lane 8) but this failed to produce the
supershifted band. A high molecular weight complex that barely entered
the gel was formed between the antibody and the labeled oligonucleotide
(Fig. 2C, lane 8). This comes from an interaction of the
antibody with the labeled DNA. Together, these data give sufficient
evidence to conclude that the UFB binding factor belongs to the CTF/NF1
family of transcription factors.

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Fig. 2.
Identification of UFB nuclear factor as a
member of the CTF/NF1 family of constitutive transcription
factors. A, DNase I footprinting analysis was performed
on the 257 to +30 TPO promoter fragment with 5 (lane 4) or
25 (lane 5) ng of recombinant CTF/NF1 protein, or with an
increased amount of bovine serum albumin (lanes 6 and
7). Lane 1 shows the DNase I digestion of the
probe in the absence of protein, and lane 2 is the G + A
reaction. The sequence of the protected region ( 112 to 95) is
represented, and the CTF/NF1 motif is marked with a box. The
A, B, Z, and C brackets correspond to
the protected regions previously identified with nuclear extracts of
FRTL-5 thyroid cells (8). B, EMSA of 5 ng of CTF/NF1
recombinant protein (lane 2) and the protected UFB sequence
identified and represented in panel A. For competition, a
100-fold excess of the same unlabeled oligonucleotide (lane
3), the consensus CTF/NF1 sequence of the MMTV promoter
(lane 4), the BZ oligonucleotide derived from the TPO
promoter (lane 5), or an unrelated oligonucleotide
(lane 6) were used. C, electrophoretic mobility
shift assay with 5 µg of nuclear extract from FRTL-5 cells and the
UFB oligonucleotide (lane 2). Competition was done with
100-fold excess of the unlabeled oligonucleotide UFB, with the CTF/NF1
consensus sequence or with an unrelated oligonucleotide (lanes
3-5, respectively). The supershift assay was performed with the
specific antibody (1/8199) -NF1 (lane 6). The preimmune
serum (lane 7) and the antibody alone without nuclear
extracts (lane 8) were used as control.
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CTF/NF1 Family Members Enhance TPO Promoter
Activity--
Mutation of the TGGCA motif, identified in the
present work as a CTF/NF1-binding site, is reported to decrease TPO
promoter activity (8). We have also shown that deletion or mutations in
the B regulatory element down-regulate hormone inducible expression of
TPO (Fig. 1, A and B). To determine the
functional activity of CTF/NF1 factors binding to the B regulatory
element of the TPO gene, transient transfection experiments were
carried out in HeLa cells. In these experiments, promoter constructs of
the TPO gene as well as the mutant version pBmm (Fig.
3, upper panel) were
co-transfected with expression vectors coding for various members of
CTF/NF1 family such as CTF/NF1-C, -B, or -X. A 5-fold increase in TPO
promoter activity was obtained in cells co-transfected with CTF/NF1-C
or -X whereas CTF/NF1-B showed no effect (Fig. 3, lower
panel). In the transfection, combinations of CTF/NF1-C and -X but
not -C and -B or -B and -X additively increased the TPO promoter
activity. This differential transactivation may be contributed by the
ability of the CTF/NF1 proteins to bind DNA and function as homodimers
and heterodimers (19, 20, 52). The pBmm TPO LUC construct, which
contains a mutated TGGCA motif (8), did not show an increased
transactivation after co-transfection of the different CTF/NF1 isoforms
(Fig. 3). These analyses demonstrate that binding of CTF/NF1 proteins
to the B regulatory region can functionally activate this promoter.

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Fig. 3.
Role of the CTF/NF1 transcription factors in
TPO promoter activity. Constructs containing the wild type TPO
promoter (p420 TPO LUC) and the mutation generated at the B site
(pBmm) (upper panel) were co-transfected into
HeLa cells with the expression vector pcDNA3.1 with no insert or
harboring the cDNA for CTF/NF1-C, CTF/NF1-B, or CTF/NF1-X, as
described under "Experimental Procedures." Luciferase activity
(lower panel) was determined as relative light units
normalizing to CAT activity derived from the RSV-CAT transfected to
adjustments of transfection efficiency. The TPO promoter activity is
expressed as fold induction over the wild type promoter (= 1). The
results are the mean ± S.D. of four independent
experiments.
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CTF/NF1 Isoforms Are Hormonally Regulated in FRTL-5 Cells--
The
contribution of the B regulatory element in the TPO promoter to induce
expression in the presence of TSH and insulin presupposes that either
one of the two or both factors binding to this region could be
hormonally regulated. We have already shown that TTF-2 expression is
controlled by TSH and insulin (15). To investigate whether CTF/NF1 is
also regulated by these hormones, we performed Northern blot studies
with poly(A)+ RNA from FRTL-5 cells maintained for 4 days
in a minimal medium and then treated for 24 h with 1 nM TSH, 2 µM insulin, or both hormones together.
As we have previously shown CTF/NF1-B and -X are expressed as 8.6- and
5.1-kb transcripts, respectively, whereas CTF/NF1-C is expressed as two
transcripts of 6.5 and 4.0 kb (35). The relationship between the 6.5- and 4.0-kb transcripts is not too clear but it is thought that the
6.5-kb is a primary transcript which is later processed to the 4.0-kb
transcript (35). TSH and insulin showed an interesting regulation of
expression of the CTF/NF1-C gene. These hormones slightly reduced the
level of the 6.5-kb transcript but drastically increased the level of the 4.0-kb transcript (Fig. 4,
lanes 1-3). With the other CTF/NF1 transcripts, TSH
slightly enhanced and insulin down-regulated the level of expression of
the CTF/NF1-B gene while no significant effect on the level of NF1-X
was observed. The same Northern blots were examined for the expression
of TTF-1 and -2 as well as the control
-actin gene (Fig. 4). The
expression of TTF-2 as in the case of the 4.0-kb transcript of
CTF/NF1-C was strongly enhanced by both TSH and insulin (Fig. 4,
lanes 5-7). A comparison of the expression of the CTF/NF1
and TTF-2 genes in the non-induced state and the fully induced state
show that the CTF/NF1-C and TTF-2 may contribute significantly to the
hormonal regulation of the TPO expression (Fig. 4, compare lane
1 with 4 and 5 with 8). The TTF-1
and
-actin mRNA levels were the same in the different
experimental approaches studied.

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Fig. 4.
Hormonal regulation of mRNA levels of the
transcription factors that bind to the cis-BZ
regulatory region of the TPO promoter. Poly(A)+ RNA
was extracted from FRTL-5 cells maintained 4 days in basal medium
(lanes 1 and 5) or treated with 1 nM
TSH (lanes 2 and 6), with 10 µg/ml insulin
(lanes 3 and 7) or with both hormones together
(lanes 4 and 8). The figure shows a
representative Northern blot hybridized subsequently with CTF/NF1-C,
CTF/NF1-B, CTF/NF1-X, TTF-2, TTF-1, and -actin probes. The size of
each transcript is indicated. The same results were obtained in three
independent experiments.
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As CTF/NF1 genes are known to be constitutively expressed, the
observation that they may be hormonally regulated deserves further
consideration. We therefore asked whether our findings at the mRNA
level reflect changes at the level of the CTF/NF1 proteins bound to the
UFB sequence of the TPO promoter. EMSA was performed with an
oligonucleotide derived from the UFB site of the TPO promoter and
nuclear extracts from non-treated and hormone-treated FRTL-5 cells.
With extracts derived from FRTL-5 cells in the absence of hormone, two
prominent protein-DNA complexes were formed which are indicated as 2 and 3 (Fig. 5A, lane 2).
Faster migrating complexes were also observed but these were not
consistently seen and may possibly have arisen as a result of minor
degradation of the CTF/NF1 protein. The DNA-binding domain of the
CTF/NF1 is known to be easily cleaved from the rest of the protein
(46). EMSA carried out with extracts of cells treated with TSH and
insulin showed quantitative and qualitative differences. In the
presence of TSH there was a shift in mobility of complexes 2 and 3 to
complexes 1 and 2 as well as an increase in the intensity of complex 2 (Fig. 5A, lane 3). Insulin treatment enhanced the intensity
of complex 3 without showing any qualitative changes in the complexes
compared with the pattern with extracts from non-stimulated cells (Fig. 5A, lane 4). A combined treatment with TSH and insulin
produced complexes qualitatively and quantitatively indistinguishable
from treatment with TSH alone (Fig. 5A compare lane
5 with lane 3). Thus TSH treatment appears to play a
dominant role in the changes that take place in the composition of the
CTF/NF1 in the regulation of the TPO promoter activity. The complexes
were specifically competed by addition of a 100-fold excess of
unlabeled UFB oligonucleotide (lane 7) but not by an
unrelated one (lane 6).

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Fig. 5.
The CTF/NF1 binding activity is regulated by
TSH. EMSA performed with the labeled UFB oligonucleotide
(panel A) or with the labeled CTF/NF1 consensus sequence
(panel B) and 5 µg of nuclear extract from FRTL-5 cells
maintained in absence of hormones (lanes 2), treated with
TSH (lanes 3), with insulin (lanes 4), or with
both hormones together (lanes 5). Competition experiments
were performed on cells treated with both hormones using an unrelated
(lanes 6) or a related oligonucleotide. The protein-DNA
complexes are indicated by arrows.
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To confirm the ability of TSH to regulate expression of CTF/NF1
proteins, we used the same extracts from the FRTL-5 cells in an EMSA
with a consensus CTF/NF1 oligonucleotide derived from the MMTV (Fig.
5B, lanes 1-6). These results were identical to those
obtained with the UFB oligonucleotide as TSH again caused a retardation
of the complexes whereas insulin did not have much of an effect. These
results together confirm that the composition of the CTF/NF1 proteins
was altered by TSH. In the Northern blot as well as in the EMSA the TSH
effect was mimicked by forskolin (data not shown).
To determine the TSH-induced changes at the level of the isoforms of
CTF/NF1, we performed the EMSA with the labeled consensus CTF/NF1
oligonucleotide and nuclear extracts from hormone-deprived cells or
treated 24 h with 1 nM TSH in the presence of two
anti-CTF/NF1 antibodies. These were
-CTF/NF1/8199 antibody that
recognize a N-terminal conserved sequence of all CTF/NF1 isoforms and
an
-CTF/NF1/2902 antibody that immunoreacted specifically with the C-terminal peptide (amino acids 419-435) of the CTF/NF1-C protein. In
the EMSA, the complexes formed with nuclear extracts from cells maintained in basal medium were recognized by the
-CTF/NF1/8199 antibody. This antibody reduced the intensity of the bands and at the
same time generated two supershifts, one of which could barely enter
the gel (Fig. 6, compare lanes
2 and 3). The slower migrating complexes obtained with
extracts from cells treated with TSH were also recognized by the
antibody forming complexes similar to that obtained with extracts from
the uninduced cells. The only difference being that the supershifted
complex that hardly entered the gel had a higher intensity (Fig. 6,
compare lanes 3 and 6). This indicates a increase
in the abundance of the CTF/NF1 proteins in this complex. To determine
the presence of an increased CTF/NF1-C as suggested by the Northern
blot experiments, we used the antibody
-CTF/NF1/2902 that
specifically recognized this protein. With extracts from uninduced
cells, only a weak supershifted band was observed indicating a low
level of the CTF/NF1-C in the complexes (Fig. 6, lane 4).
However, in extracts from TSH-treated cells, there was a shift to
higher mobility complexes as reported in Fig. 5 which in the presence
of
CTF/NF1/2902 generated a fairly strong supershifted band with a
drastic reduction of the complexes 1 and 2 (Fig. 6, compare lanes
5 and 7). These results together demonstrate that
TSH-treated FRTL-5 cells contain a lot more CTF/NF1-C protein compared
with the non-treated cells and confirm our results in the Northern blot
assay of TSH induced activation of CTF/NF1-C.

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Fig. 6.
Identification of the TSH-induced DNA
complex. EMSA from nuclear extracts (5 µg) of FRTL-5 cells
maintained in basal medium (lane 2) or treated 24 h
with 1 nM TSH (lane 5) and the labeled CTF/NF1
consensus oligonucleotide. Supershift assays were performed in both
basal and TSH-induced cells with the -CTF/NF1/8199 antibody
(lanes 3 and 6) or with -CTF/NF1/2902
(lanes 4 and 7). For specificity of the
supershift, antibodies were incubated with labeled oligonucleotides
without nuclear extracts (lanes 8 and 9). The
complexes found and the supershifts are indicated by
arrows.
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Physical Interaction between TTF-2 and Members of the CTF/NF1
Proteins--
The close proximity of the CTF/NF1 and TTF-2 binding
sites and inducibility of their expression suggested that they may
physically cooperate in the hormonal regulation of expression at the
TPO promoter. To investigate this idea, we examined the ability of in vitro-translated CTF/NF1 proteins to bind to a
bacterially expressed GST-TTF-2 fusion protein and the effect of
increasing the distance between these binding sites in transfection experiments.
In GST pull-down assay, radioactively labeled CTF/NF1-C, -X, or -B
bound to the immobilized GST-TTF-2 but not GST alone (Fig. 7, compare lanes 5, 9, and
12 with 4, 8, and 11). A radioactively labeled LexA-VP16 fusion protein in a parallel reaction failed to bind
to either GST or GST-TTF-2 (Fig. 7, lanes 6 and
7). This demonstrates the specificity of interaction of the
CTF/NF1 proteins with TTF-2.

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Fig. 7.
Physical interaction between transcription
factors TTF-2 and CTF/NF1. Pull-down assays were carried out with
full-length TTF-2-GST fusion protein and with different members of the
CTF/NF1 family. Lysate containing [35S]methionine-labeled
CTF/NF1-C, CTF/NF1-X, or CTF/NF1-B proteins was incubated with
TTF-2-GST fusion protein (lanes 5, 9, and 12, respectively) or GST (lanes 4, 8, and 11,
respectively) that had previously been coupled to glutathione-Sepharose
beads and processed as described under "Experimental Procedures."
The samples were resolved by SDS-PAGE analysis on a 10% polyacrylamide
denaturing gel. Labeled in vitro translated Lex-A VP-16
protein was incubated under the same conditions with GST (lane
6) or TTF-2-GST (lanes 7). Input (lanes 1-3
and 10) is reticulocyte lysate that had been programmed with
the indicated templates.
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To determine whether alteration of the distance between these two
factors can affect their function, we increased the spacing between
their binding sites by 5 and 10 nucleotides p120 (+5) and p120 (+10) to
generate half-helical and helical turns of the DNA helix (Fig.
8, upper panel). These
constructs, together with the p420 and p120 TPO LUC as control, were
transfected into the FRTL-5 cells and their activity determined in the
absence and presence of TSH and insulin. Insertion of 5 bp (p120
(+5)TPO LUC) led to a reduction of the basal level of expression but
this was restored by increasing the distance between the factors to 10 bp (Fig. 8A). Similar results were obtained after TSH and
insulin treatment except that the response of the p120 (+10) construct to TSH was even higher than in the wild-type situation (Fig.
8B). The significance of this is not clear but would suggest
that an increase in the distance of the two binding sites by 10 bp (one helical turn) may allow an even better interaction for transactivation than in the wild-type situation (Fig. 8A).

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Fig. 8.
Increasing the distance between TTF-2 and
NF-1 binding motifs impairs TPO promoter activity and the response to
TSH and insulin is lost. Upper panel, spatial model
representing the DNA double helix of the TPO promoter. The distance
between TTF-2 and CTF/NF-1-binding sites is maintained intact in the
p120 construct, as in the wild type promoter, or is separated by an
insertion of 5 (+5) or 10 (+10) bp, respectively, as described under
"Experimental Procedures." Lower panel, TPO
promoter activity derived from each construct transfected into FRTL-5
cells. A, luciferase activity was determined 48 h after
the transfection in the proteins extracts from confluent cells
maintained in complete medium. TPO promoter activity is expressed as
light units normalized by transfection efficiency and is calculated
relative to the activity of the wild type TPO promoter (p420 = 100%). The results are the mean ± S.D. of four different
experiments. B, after transfection, cells were maintained
72 h in the absence of serum (0.2%) without TSH and insulin
(basal medium). Then, the cells were treated with 1 nM TSH
and 2 µM insulin for 24 h. Relative luciferase
activity is the value of light units normalized by transfection
efficiency. The TPO promoter activity is expressed as fold induction
over the basal levels (= 1) of hormone-depleted cells. The results are
the mean ± S.D. of four independent experiments.
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DISCUSSION |
Thyroid-specific gene expression is achieved by a combination of
multiple regulatory elements within the promoter of specific genes. One
of the decisive genes that defines thyroid-specific function is the
enzyme TPO. We have previously shown that the promoter element of this
gene termed Z, where the thyroid-specific transcription factor TTF-2
binds, is the main mediator of the hormonal response of TPO
transcription (16). Since TTF-2 activity depends on multimerization and
specific orientation, we have characterized the B element at its 5'
adjacent region (see Fig. 1 for diagram) for possible cooperation with
TTF-2. In this work we have shown that the B element is essential for
the action of TTF-2 at the TPO promoter. We established this with the
use of transfection experiments involving deletion and site-directed
mutations of the B element in the TPO promoter. Our findings showed an
important role for the hormone-regulated expression of a sequence in
the B element which we identified as a binding site for CTF/NF1.
CTF/NF1 was originally identified as a host-encoded protein required
for efficient initiation of adenovirus replication in vitro
(53) and was later shown to function in the expression of several
cellular genes (23, 54-57). CTF/NF1 can stimulate transcription by
itself but binding sites for this factor are frequently found clustered
with binding sites for other transcription factors such as AP-1,
hepatocyte nuclear factor 3
, and steroid receptors (44, 58-62).
Perhaps one of the most extensively studied effects of CTF/NF1 is that
of modulating the action of steroid hormone receptors on expression at
the MMTV promoter. In this example steroid hormone receptors bind the
nucleosomally organized promoter of MMTV and makes it accessible for
CTF/NF1 to exert its transactivation effect (60, 63). The synthesis of
CTF/NF1 is itself not affected by the steroid hormone. In our study we have shown that CTF/NF1 is not only involved in action of steroid hormone but also of thyroid hormones. This time it was not the action
of a hormone receptor that was affected but rather the production of
the hormone. As TPO is one of the key enzymes in the production of
thyroid hormone, its regulation by CTF/NF1 is an example of a direct
influence of this group of transcription factors on hormone production
and action.
One important message of this paper is that the expression of CTF/NF1
is enhanced by TSH, via cAMP, and insulin. TSH increases the expression
of CTF/NF1-B and -C while insulin up-regulates CTF/NF1-C but
down-regulates CTF/NF1-B. This effect is specific as none of the
inducers has any effect on CTF/NF1-X. In the case of TSH-induced
expression of CTF/NF1-C we have further shown that this increase is
associated with an enhanced synthesis of CTF/NF1-C. So far CTF/NF1 have
been thought of as ubiquitously expressed genes. Recent studies have
shown that certain CTF/NF1 genes are up-regulated during the
metamorphic transition in Xenopus laevis. It is interesting
to note that the two CTF/NF1 genes whose expression was enhanced by
thyroid hormone and highly expressed during intestine remodeling turned
out to be CTF/NF1-B and -C (64). These are the same genes whose
expression we have identified as inducible by TSH and insulin. Analysis
of the promoter regions of these genes will reveal how they are
regulated by these hormones.
CTF/NF1 proteins bind DNA through a palindrome containing two TGGCA
sequences on opposite strands. Furthermore, several CTF/NF1 half-palindromes in many eukaryotic promoters are located close to
other DNA element. Gil et al. (18) proposed that the binding of CTF/NF1 to such a sequence is stabilized by protein-protein interactions. In the TPO promoter the TTF-2 and the CTF/NF1-binding sites are separated by one turn of the DNA helix, suggesting that proteins bound to each element would be located on the same side of the
DNA helix, in a position favorable for protein-protein contacts. Our
results that a separation of these binding sites by a further 5 bp
impaired TPO promoter activity but not by 10 bp, indicate that the
stereospecific positioning of these elements is an important and
necessary requirement for the transcriptional activity of the TPO promoter.
The synergistic interaction between CTF/NF1 and TTF-2 element may occur
at the level of DNA binding or at the level of protein-protein interaction. Pull-down assays demonstrate that TTF-2 is able to interact physically with CTF/NF1 proteins. It is notable that CTF/NF1-X
is not activated by TSH or insulin also interacted with TTF-2. Should
these interactions occur in vivo, it will mean that physical
association of the CTF/NF1 and TTF-2 is the main process of activating
the TPO promoter activity. The hormone induced expression of CTF/NF1-C
and -B therefore serves the main function of increasing the abundance
of the CTF/NF1 proteins for this interaction. This suggests that the
hormone enhanced expression of the CTF/NF1 transcription factors is to
ensure the availability of enough CTF/NF1 proteins for interaction with
TTF-2.
The interaction between CTF/NF1 and forkhead transcription factors
could be a general mechanism of action of both families of
transcription factors. A similar example has been reported in the
modulation of liver-specific albumin transcription by HNF3-
(55).
This idea is reinforced by the fact that another forkhead protein
HNF3
is able to bind to the TTF-2-binding site (65). This conserved
interaction between these families of transcription factors might be
explained by their specific properties. Thus, the CTF/NF1-binding site
is masked inside of the nucleosomal structure. The binding of the
forkhead proteins to their cognate site desestabilizes this structure
and makes the CTF/NF1 site accessible to exert its transactivation
effect. The ability of the forkhead domain to induce DNA bending (66)
would favor its contact with CTF/NF1 factors. This interaction could
plays an important role in cell type-specific transcription and would
be a widespread phenomenon that will be studied in the future.
Our data unequivocally demonstrate the importance of CTF/NFI
transcription factors for thyroid-specific gene expression and show
that some members of this family are hormonally inducible genes.
Furthermore, CTF/NF1 factors interact in vitro with TTF-2, this interaction is functional and constitutes a requirement for a
correct hormonal response of the TPO promoter.