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INTRODUCTION |
PTTG1is a
novel oncogene (1) that is overexpressed in multiple tumors including
carcinomas of lung, breast, colon, leukemia, and lymphoma as well as in
pituitary adenomas (2-5). The expression of PTTG in normal
tissues is restricted, with highest expression in the testis (1-3).
PTTG is expressed in a stage-specific manner in germ cells
during spermatogenic cycle, suggesting it may play a role in male germ
cell differentiation (6, 7). The level of PTTG expression is
increased in rapidly proliferating cells and is regulated in a cell
cycle-dependent manner, peaking in mitosis (8), suggesting
that PTTG may play a role in control of cell proliferation.
PTTG encodes a protein of 30 kDa without homology to other
known protein (1). It was recently shown that PTTG is a mammalian
securin that acts as a sister-chromatid separation inhibitor (9). PTTG
protein is predominantly localized to the cytoplasm with partial
nuclear localization (3, 10, 11). However, nuclear translocation of
PTTG can be facilitated either by interaction with PTTG binding factor
(10) or by activation of mitogen-activated protein (MAP) kinase
cascade (11).
A role for PTTG as a transcriptional activator has been proposed. The
C-terminal region of PTTG mediates transcriptional activation when
fused to a heterologous DNA binding domain (3). Transient cotransfection of PTTG and PTTG binding factor expression constructs leads to transcriptional activation the basic fibroblast growth factor
promoter (10). In a recent study, I demonstrated that activation of the
MAP kinase cascade by epidermal growth factor or an expression vector
for a constitutively active form of the MAP kinase kinase (MEK1) led to
stimulation of PTTG transactivation activity (11). PTTG is
phosphorylated in vitro on Ser162 by MAP kinase
and is linked to the MAP kinase cascade by direct interaction with MEK1
through a putative SH3-domain binding site located between amino acids
51 and 54 (11). Both the MAP kinase phosphorylation and the
MEK1-binding sites play an essential role in PTTG transactivation
function (11).
These previous studies indicate that PTTG plays a potentially important
role in regulation of transcription. However, target genes whose
expression is definitively regulated by PTTG remain unknown. To address
the functional properties of PTTG and to examine its effect on
endogenous target genes, I developed cell lines in which
PTTG expression is tightly regulated by an inducible promoter (12, 13). DNA array-based gene expression profiling revealed a
small number of genes exhibiting altered expression after
PTTG induction. Among these candidate targets, the most dramatic induction was the c-myc oncogene. The
c-myc gene has a critical role in the control of cellular
proliferation. Deregulated c-myc is associated with a
variety of tumors. Overexpression of c-Myc protein stimulates cell
cycle progression, causes transformation, blocks differentiation, and
induces apoptosis in low serum (14). I observed that induction of
c-myc expression by overexpression of PTTG resulted in
increased cell proliferation and colony formation, supporting the role
of PTTG in regulation of cell growth.
It has been established that the C-terminal portion of PTTG contains a
transcriptional activation domain (3, 10, 11). However, it is not known
whether PTTG directly binds to DNA, and the DNA-binding site has not
been identified. I have therefore used c-myc promoter to
characterize PTTG interaction with DNA. Here I show that PTTG binds to
c-myc promoter near the transcription start site and forms a
complex with the ubiquitous transcription activator USF1. I have mapped
PTTG DNA binding domain to a region between amino acids 60 and 118. In
addition I demonstrate that transcriptional activation of
c-myc gene by PTTG requires its DNA binding activity.
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MATERIALS AND METHODS |
Antibodies--
Antibodies for USF-1 and YY1 were obtained from
Santa Cruz Biotechnology. C-Myc and His antibodies were purchased from
Berkeley Antibody Co. and CLONTECH,
respectively. Anti-PTTG anti-serum was described previously (1).
Generation of Cell Lines with Inducible PTTG Expression--
The
PTTG cDNA was cloned into the pcDNA3.1/His vector (Invitrogen).
The coding region of PTTG and the histidine tag was then amplified by
PCR with SstII restriction site attached to both ends. The
PCR product was cloned into pTRE vector (CLONTECH), resulting in pTRE-PTTG. Site-directed mutagenesis was performed to
change Ser162 to alanine in pTRE-PTTG, using the QuikChange
mutagenesis kit (Stratagene) following the manufacturer's
instructions. The resulting plasmid was termed pTRE-mPTTG. PTRE-PTTG or
pTRE-mPTTG was cotransfected into HeLaS3 Tet-off cell line with pTK-Hyg
vector (CLONTECH). The cells were selected in
medium containing 200 µg/ml hygromycin. Two weeks after transfection,
hygromycin-resistant clones were isolated and screened for integration
of PTTG by PCR. The clones that express PTTG were expanded into cell
lines. The cell lines were maintained in growth medium containing 2 µg/ml doxycycline (Dox). The expression of PTTG was induced by
doxycycline withdrawal. The level of PTTG induction was determined by
Northern and Western blot analysis using standard protocols. The cell
lines that have the highest level of induced PTTG expression after Dox
withdrawal were used for latter analyses.
Expression Profiling--
Total RNA was isolated from
HeLaS3-PTTG cells at 0 and 24 h after PTTG induction. The RNA was
labeled with [32P]dATP by reverse transcription, and the
labeled cDNA was purified by column chromatography. The probe was
hybridized to Atlas Human Array (CLONTECH)
following the manufacturer's instructions. After quantitative
analysis, candidate PTTG targets were assembled by identifying genes
that displayed an expression change of at least 2-fold. Some potential
targets were verified by Northern analysis. Probes for c-myc
and MEK1 were generated by PCR according to published sequences. The probe for HSP70 was kindly provided by Dr. R. Morimoto (Northwestern University).
Proliferation and Colony Formation Assays--
Cell
proliferation was assayed using the Celltiter 96 nonradioactive cell
proliferation assay kit (Promega) according to the manufacturer's
instructions. Five thousand cells were seeded in 96-well plates (6 wells for each clone in each assay) and incubated at 37 °C for 24 to
72 h in the presence or absence of Dox. At each time point, 15 µl of the dye solution were added to each well and incubated at
37 °C for 4 h. One hundred microliters of the
solubilization/stop solution were then added. After 1 h of incubation, the contents of each well were mixed, and the absorbency at
595 nm was recorded using an enzyme-linked immunosorbent assay reader.
Absorbency at 595 correlates directly with the number of cells in each
well. For the colony formation assay, 104 cells were plated
on 35-mm plates and grown with or without Dox. After 14 days in
culture, the colonies were fixed and stained with 0.1% crystal violet
and counted.
Transient Transfection Assays--
The c-myc
promoter-luciferase reporter constructs HBM-Luc and XNM-Luc were kindly
provided by Dr. Linda Penn (University of Toronto) and were described
previously (15). Mutagenesis of XNM-Luc was performed using QuikChange
site-directed mutagenesis kit according to manufacturer's protocol
(Stratagene). The construction of wild type and mutant PTTG expression
plasmids was reported previously (11). NIH 3T3 cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% calf serum
100 units/ml penicillin and 100 µg/ml streptomycin. Transfections
were performed using calcium phosphate precipitation as described
previously (7). All transfections were performed in triplicates, and
each DNA construct was tested in at least three independent
experiments. Forty-eight hours post-transfection cells were lysed in
0.25 M Tris, pH 7.8, with three freeze and thaw cycles.
Cell lysates (50 µg/assay) were assayed for luciferase activity as
previously described (7).
DNaseI Footprint and Gel Mobility Shift Assays--
Nuclear
extracts were prepared from HeLaS3-PTTG cells using the method
described by Dignam et al. (16). The probe used in DNaseI
footprinting assays is the 182-bp fragment containing +34 to
147 bp
of the c-myc sequence. To generate the probe, either the
upstream primer 5'-cgtccctggctcccctcctgcct-3' or the downstream primer
5'-agatctcgagctagcacgcgtaag-3' was end-labeled using
[
-32]ATP and T4 polymerase kinase (Life Technologies,
Inc.) and was then used with the unlabeled downstream or upstream
primer, respectively, in PCR reactions using HBM-Luc as template.
104 cpm of the probe was used in each reaction. The binding
reaction was performed in a 25-µl reaction containing 20 mM HEPES, pH 7.9, 20% glycerol, 1 mM EDTA, 2 mM MgCl2, 50 mM NaCl, and 0.5 mM dithiothreitol with either 50 ng of purified Sp1 protein
(Promega) or 5-50 µg of HeLa cell nuclear extracts. Poly(dI-dC) was
included as nonspecific competitor. Binding was at room temperature for
30 min. DNaseI digestion was carried out at room temperature for 60-90
s using 1-5 units of DNaseI (Ambion) depending upon protein
concentration. The reaction was terminated by adding 30 µl of 2×
stop buffer containing 15 mM EDTA, 0.2% SDS, and 40 µg/ml salmon sperm DNA. Nuclear acids were extracted with phenol,
ethanol-precipitated, and separated on 6% sequencing gels alongside a
G+A ladder.
Primers (only the upper strand sequences are shown) used in gel
mobility shift assay include the following: 1) Myc wild type: 5'-ttatctaactcgctgtagtaattccagtgagaggcagagggagcgagc-3'; 2) Myc M1:
5'-ttatctaactcgctgtagagataggagtgagaggcagagggagcgagc-3';
3) Myc M2:
5'-ttatcagacagactgtagtaattccagtgagaggcagagggagcgagc-3'; 4) Myc GH1: 5'-ttatctaactcgctgtagtaattc-3'; 5) Myc GH2:
5'-cagtgagaggcagagggagcgagc-3'; 6) MycGH1 m1:
5'-ttatctaactcgacttagtaattc-3'; 7) MycGH1 m2:
5'-ttatctaactcgctggcataattc-3'; 8) Myc CH1 m3:
5'-gccgataactcgctgtagtaatcc-3'. The nucleotide substitutions are underlined.
Constructions and expression of various regions of PTTG as
glutathione S-transferase fusion proteins were described
previously (11). For gel shift assays, the glutathione
S-transferase portion of the fusion proteins was removed by
thrombin protease digestion. Binding conditions were the same as
described above for DNaseI footprinting assays. The reactions were
resolved on 4.5% native polyacrylamide gels in 0.5× Tris-buffered
EDTA running buffer.
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RESULTS |
Inducible Expression of PTTG--
To study the functional
properties of PTTG, I established inducible, tetracycline-regulated
expression of PTTG in HeLa S3 cells. I cloned the His-tagged
PTTG into pTRE expression vector (pTRE-PTTG). This plasmid
together with pTK-Hyg was cotransfected into Tet-off HeLa S3 cell line.
Hygromycin-resistant clones were selected and screened for induced
expression of PTTG in the absence of doxycycline. Low levels
of endogenous PTTG mRNA were detectable when cells were
grown in the presence of doxycycline (band not visible in the photo
shown in Fig. 1), and drug withdrawal led to induction of the expected 900-bp transfected PTTG
transcript (Fig. 1A). The inducible protein expression was
analyzed by immunoblotting using anti-His antibody against the
N-terminal epitope tag. As shown in Fig. 1B, anti-His
antibody detected a protein of about 30 kDa only in induced cells
(i.e. doxycycline). These results indicate that
PTTG expression is tightly regulated by tetracycline in the
established the cell line (designated HeLaS3-PTTG). I also generated
cell lines with inducible expression of a transactivation-defective mutant PTTG (11) containing Ala substitution of Ser162.
These cells (designated HeLaS3-mPTTG) also demonstrated tightly regulated mutant transcript (Fig. 1A) and protein (Fig.
1B).

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Fig. 1.
Expression of wild type (wt)
and mutant (mut) PTTG in Tet-off HeLaS3 cells.
Total RNA (20 µg) and protein (50 µg) from HeLaS3-PTTG and
HeLaS3-mPTTG cells grown in the presence or absence (24 h) of
doxycycline was analyzed by representative Northern (A) and
Western blot assays (B). The bottom panel of
A shows ethidium bromide staining of the 28 S rRNA.
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Gene Expression Profiling to Identify PTTG Targets--
A
potential role for PTTG in transcriptional regulation has been
suggested by its ability to enhance transactivation of transiently transfected reporter constructs (3, 10, 11). However, target genes
whose expression is definitively regulated by PTTG remain unknown.
Therefore, I searched for endogenous genes whose expression level might
be altered after inducible expression of PTTG. Total RNA was
isolated from HeLaS3-PTTG at 0 and 24 h after PTTG
induction, labeled with 32P, and hybridized to human
cDNA expression trial array filters representing 84 known
transcripts (CLONTECH). A gene was considered a
candidate PTTG target if it exhibited an expression change
of at least 2-fold. To distinguish reproducible expression changes from
those due to random biological variation, probes complimentary to the
candidate PTTG targets were used to screen Northern blots containing total RNA from HeLaS3-PTTG cells 24 h after
PTTG induction. Of the 84 genes investigated by array
hybridization, five genes showed increased expression after
PTTG induction. These include c-myc oncogene,
MEK1, MEK3, protein kinase C
-1
(PKC
-1), and the heat shock protein
HSP70. The increased expression of c-myc, MEK1, and HSP70 was confirmed by Northern blot
analysis (Fig. 2).

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Fig. 2.
Representative Northern blot of candidate
PTTG targets. Total RNA (20 µg) from HeLaS3-PTTG cells grown in
the presence or absence (24 h) of doxycycline was analyzed. The
bottom panel of A shows ethidium bromide staining
of the 28 S rRNA.
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Induction of c-Myc by PTTG--
Myc is an important regulator of
many cellular processes, including growth promotion, differentiation,
and apoptosis (17-19). Identification of c-myc as a
potential target for PTTG is of particular interest; I therefore
investigated further the relationship between these two oncogenes. I
performed a time course study on the expression of c-myc
after induction of PTTG by tetracycline withdrawal. As shown
in Fig. 3, both c-myc mRNA
(Fig. 3A) and protein (Fig. 3B) were induced
within 6 h of induction of wild type PTTG expression. However, there was no change in either c-myc mRNA (Fig.
3C) or protein (Fig. 3D) expression after induction of the
transactivation-defective mutant PTTG expression. These
results indicate that transcriptional activation function of PTTG is
required to induce c-myc expression.

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Fig. 3.
The time course of induction of PTTG and
c-Myc in HeLaS3-PTTG (panels A and B)
and HeLaS3-mPTTG (panels C and D)
cells after withdrawal of doxycycline. Total RNA and protein
extract were made at 0, 6, and 24 h after doxycycline withdrawal
and analyzed by Northern (panels A and C) or
Western (panels B and D) blot assays.
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Many studies have shown that the c-myc gene has a critical
role in the control of cellular proliferation. Overexpression of c-Myc
protein stimulates cell cycle progression, causes transformation, blocks differentiation, and induces apoptosis in low serum (14). I
sought to test whether induction of PTTG expression in
HeLaS3-PTTG cells has any effect on growth property of these cells. As
shown in Fig. 4A, when cells
were grown in the presence of tetracycline, no significant differences
in growth rate were observed between HeLaS3-PTTG and HeLaS3-pCMV (cell
line that expresses the vector). When induced with tetracycline
withdrawal, the growth rate of cells expressing PTTG was
increased more than 2-fold. In contrast, in HeLaS3-mPTTG cells,
induction of mutant PTTG after tetracycline withdrawal led to slightly
retarded cell growth (Fig. 4A). I then measured the ability
of these cells to form colonies in the presence or absence of
tetracycline. A 2-fold increase in colony formation was observed
after the induction of PTTG (Fig. 4B). In
contrast, withdrawal of tetracycline had no effect on colony formation
in HeLaS3-pCMV or HeLaS3-mPTTG. These results suggest that
PTTG may exert its effects on cell proliferation through
activation of the c-myc oncogene.

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Fig. 4.
Increase in cell proliferation and colony
formation after induction of PTTG. A, cell
proliferation assay. Five thousand cells were seeded in 96-well plates.
The growth rate of cells was assayed at 0, 24, 48, and 72 h after
doxycycline withdrawal by cellular conversion of tetrazolium into
formazan. Absorbency at 595 nm correlates directly with the number of
cells in each well. Open circles, HeLaS3-pCMV
(Dox+); filled circles, HeLaS3-pCMV
(Dox ); open squares, HeLaS3-PTTG
(Dox+); filled squares, HeLaS3-PTTG
(Dox ); open triangles, HeLaS3-mPTTG
(Dox+); filled triangles, HeLaS3-mPTTG
(Dox ). Results represent the average of three independent
experiments, n = 12. B, colony formation
assay. Cells were plated at 104 cells/35-mm dish with or
without doxycycline. Plates were stained with crystal violet after 14 days in culture, and colonies were counted. Open bars,
Dox+; filled bars, Dox . Results
represent the average of three independent experiments,
n = 6. wt, wild type; mut,
mutant.
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PTTG Activates c-Myc Transcription in Transfected
Cells--
To test whether PTTG induces c-myc expression by
activating its transcription, I transfected NIH3T3 cells with fusion
constructs containing either 2.5 kilobase pairs (HBM-Luc) or 142 bp (XNM-Luc) of c-myc 5'-flanking region (15) fused to the
reporter gene luciferase. As shown in Fig.
5, the luciferase activity of both HBM-Luc and XNM-Luc was increased 2.5-fold when cotransfected with a
wild type PTTG expression plasmid, whereas there was no change in
reporter gene activity when the cells were cotransfected with either
the pCMV vector or transactivation-defective mutant Ser162
Ala (11). These results suggest that PTTG is able to
transactivate c-myc transcription and that the DNA sequences
required for PTTG transactivation are located within 142 bp of the
c-myc 5'-flanking region.

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Fig. 5.
PTTG activates c-myc
transcription. NIH 3T3 cells were transiently transfected
with the indicated plasmids and assayed for luciferase reporter gene
activity (represented as fold of induction over promoter-less pGL2).
Values represent the mean ± S.E. (n = 9, average
of three independent experiments). wt, wild type.
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PTTG Binds to c-Myc Promoter in a Complex with USF1--
To
determine whether PTTG transactivates c-myc transcription by
directly interacting with its promoter, I performed DNaseI footprinting
experiments using nuclear extract prepared from HeLaS3-PTTG cells.
Purified Sp1 protein was used as a control for experimental conditions.
As shown in Fig. 6, Sp1 protected two
regions on c-myc promoter corresponding to its consensus
binding sites. In the presence of the nuclear extract, a region between
20 and +28 in the c-myc 5'-flanking region was protected
from DNaseI digestion (Fig. 6, plus strand DNA was labeled), suggesting
that nuclear proteins in HeLaS3-PTTG cells specifically interact with
this DNA sequence. Similar results were obtained when minus-strand DNA
was labeled (data not shown).

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Fig. 6.
Interaction of HeLaS3-PTTG cell nuclear
proteins with the c-myc promoter. DNaseI
footprint assay. Lane 1, G+A ladder; Lanes 2 and
7, DNA probe only; lane 3, with 50 ng of purified
Sp1 protein; lanes 4-6: 5, 25, and 50 µg of HeLaS3-PTTG
nuclear extract. The regions protected by Sp1 and HeLaS3-PTTG nuclear
extract are indicated.
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To determine whether the nuclear proteins that interact with this
specific DNA sequence in the c-myc gene include PTTG, I performed gel mobility shift assays using the sequence between
20 and
+28 as the probe. As shown in Fig.
7A, incubation of HeLaS3-PTTG nuclear extract with the probe resulted in two mobility-shifted bands
(lane 2) that were competed with unlabeled, homologous
oligonucleotide (lane 3) but not by unrelated
oligonucleotide (lane 4). The addition of anti-PTTG antibody
to the reaction led to super shift of the upper band (lane
6), whereas pre-immune serum had no effect (lane 5).
These results suggest that PTTG is present in the protein complex that
interacts with the c-myc 5'-flanking sequence.

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Fig. 7.
A, interaction of PTTG and USF1
with the 48-bp DNaseI footprint sequence. Lane 1, free
probe. Lanes 2-9, +20 µg of HeLaS3-PTTG nuclear extract.
Lane 3, +100× unlabeled homologous oligonucleotide.
Lane 4, +100× unlabeled unrelated oligonucleotide.
Lane 5, +pre-immune serum. Lane 6, + anti-PTTC
antibody. Lane 7, + anti-USF-1 antibody. Lane 8, + anti-PTTC and anti-USF-1 antibodies. Lane 9, + anti-YY1
antibody. B, interaction of recombinant PTTG with the 48-bp
DNaseI footprint sequence. Lane 1, free probe. Lanes
2-5, +500 ng of PTTG expressed in and purified from E. coli. Lane 3, +100× unlabeled homologous
oligonucleotide. Lane 4, +100× unlabeled unrelated
oligonucleotide. Lane 5, + anti-PTTC antibody.
Mobility-shifted and the super-shifted bands are indicated by
arrows and an arrowhead, respectively.
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The sequence between
20 and +28 of the c-myc gene includes
two adjacent initiator elements (consensus: YYANTYY) (20), Inr-A (
4
to +7) and Inr-B (
7 to
17), that are potential binding sites for
transcription factors USF-1 (21), YY1 (22), and TFII-I (21). I
therefore tested whether any of these factors are present in the
mobility-shifted bands. Fig. 7A shows that inclusion of anti-USF1 antibody resulted in the disappearance of the upper band of
the two mobility-shifted bands (lane 7), suggesting that USF1 is part of the protein complex that interacts with the 48-bp c-myc sequence. The addition of both anti-PTTG and USF1
antibodies resulted in the disappearance of the upper band as well as a
super-shifted band (lane 8), suggesting that USF1 was in the
same protein complex with PTTG. The addition of anti-YY1 antibody did
not cause any change in the mobility-shifted bands (lane
9).
To test whether PTTG by itself could also bind to this DNA sequence in
the c-myc promoter, PTTG was expressed in and purified from
Escherichia coli as a glutathione S-transferase
fusion protein. After thrombin protease digestion to remove glutathione
S-transferase from the fusion protein, purified PTTG was
used in gel mobility shift assays. As shown in Fig. 7B,
incubation of PTTG with the probe resulted in a mobility-shifted band
(lane 2) that was competed with unlabeled, homologous
oligonucleotide (lane 3) but not by unrelated
oligonucleotide (lane 4). The addition of anti-PTTG antibody
to the reaction led to the appearance of a super-shifted band
(lane 5). These results suggest that PTTG is capable of DNA binding by itself.
Identification of the DNA-binding Site for PTTG--
To further
define the DNA sequence required for PTTG DNA binding, I generated
oligonucleotides that contain various mutations within the 48-bp
c-myc gene (see "Materials and Methods") sequence and
performed gel shift assays to test PTTG interaction with these sequences.
Initially, I tested whether mutations within the initiator elements
(Inr) affected PTTG DNA binding. In the adenovirus-2 major late
promoter (AdMLP) Inr, which shares substantial homology with c-myc Inrs, mutation of residues
1 and
2 or +2, +3, and
+4 relative to the transcription start site reduced USF1 binding to Inr
element without significantly affecting basal transcription levels
(23). Based on these observations, I made analogous mutations (Fig. 8A) within the
c-myc Inrs to determine whether these elements play a role
in PTTG DNA binding. As shown in Fig. 8B, incubation of
oligonucleotide containing point mutations within Inr-A (Myc M1,
panel A) and recombinant PTTG resulted in a mobility-shifted band (lane 2) that was competed by unlabeled homologous
(lane 3) but not by unrelated (lane 4)
oligonucleotide. The addition of anti-PTTG antibody resulted in a
super-shifted band (lane 5). When HeLaS3-PTTG nuclear
extracts were included, two mobility-shifted bands were present
(lane 6). They were competed by unlabeled Myc M1 (lane
7) but not by unrelated (lane 8) oligonucleotide. The addition of anti-USF1 antibody, however, did not lead to the
disappearance of the upper band (lane 9) as observed when
wild type oligonucleotide was used (Fig. 7A, lane
7). A super-shifted band was observed in the presence of anti-PTTG
antibody (lane 10). These results suggest that mutations
within Inr-A do not affect PTTG DNA binding, whereas they abolish USF1
DNA binding. I have also tested the effects of mutations within Inr-B
(Fig. 8A, Myc M2) on PTTG and USF1 DNA binding, and the
results indicated that mutations within this initiator element did not
affect DNA binding of either PTTG or USF1 (data not shown).

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Fig. 8.
Effect of mutations within the initiator
elements on PTTG and USF1 DNA binding. A, wild type
(wt) and mutant oligonucleotides (upper strand sequences).
Nucleotide substitutions are underlined. B, Gel
shift assay using Myc M1 as the probe. Lane 1, free probe.
Lanes 2-5, +500 ng of recombinant PTTG. Lanes
6-10, + 20 µg of HeLaS3-PTTG nuclear extract. Lanes
3-7, +100× unlabeled Myc M1 oligonucleotide. Lanes 4 and 8, +100× unlabeled unrelated oligonucleotide.
Lanes 5 and 10, + anti-PTTG antibody. Lane
9, + anti-USF-1 antibody. Mobility-shifted and the super-shifted
bands are indicated by arrows and an arrowhead,
respectively.
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I then divided the 48-bp c-myc sequence into two parts;
probe GH1 contains sequence
20 to +4, and GH2 contains +5 to +28. Fig. 9A shows that incubation
of GH1 probe with either recombinant PTTG (lane 2) or
HeLaS3-PTTG nuclear extracts (lane 6) resulted in a
mobility-shifted band. This band was competed by unlabeled GH1
(lanes 3 and 7) but not by GH2 (lanes
4 and 8). The addition of anti-PTTG antibody resulted
in a super-shifted band (lanes 5 and 9). One the
other hand, incubation of GH2 probe with either recombinant PTTG
(lanes 11-13) or HeLaS3-PTTG nuclear extracts (lane
14-16) resulted in no specific mobility-shifted band. These results indicate that the DNA-binding site for PTTG resides between position
20 to +4.

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Fig. 9.
Identification of PTTG DNA-binding site.
A, PTTG-binding site is located between 20 and +4 in c-Myc
gene. Gel mobility shift assays were performed using either recombinant
PTTG or HeLaS3-PTTG nuclear extracts. Probe GH1 and GH2 contain
sequences 20 to +4 and +5 to +28 of the c-myc gene,
respectively. Lanes 1 and 10, free probes.
Lanes 2-5 and 11-13, + 500 ng of recombinant
PTTG (rPTTG). Lanes 6-9 and 14-16, + HeLaS3-PTTG nuclear extracts. Lanes 3 and 7,
+100× unlabeled GH1, Lanes 4, 8, 12,
15, +100× unlabeled GH2. Lanes 5, 9,
13, and 16, + anti-PTTC antibody. B,
oligonucleotides (upper strand sequences) used in gel shift assays in
panel C. Nucleotide substitutions are underlined.
wt, wild type. C, effects of mutations within GH1
on PTTG DNA binding. Lanes 1, 7, 13,
and 17, free probes. Lanes 2-6,
8-12, 14-16, and 18-20, + nuclear
extracts. Lanes 3 and 9, +100× unlabeled GH1
wild type. Lanes 4 and 10, +100× unlabeled GH1
m1. Lanes 5, 11, and 15, +100×
unlabeled GH1 m2. Lane 19, + 100× unlabeled GH1 m3.
Lanes 6, 12, 16, and 20, + anti-PTTC antibody. The probes used are indicated on the top of the
panel. Mobility-shifted and the super-shifted bands are
indicated by arrows and an arrowhead,
respectively.
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I then made point mutations within this 24-base pair sequence (Fig.
9B). As shown in Fig. 9C, incubation of wild type
oligonucleotide with HeLaS3-PTTG nuclear extracts resulted in a
mobility-shifted band (lane 2) that was competed by 100×
unlabeled homologous sequence (lane 3) and was super-shifted
by anti-PTTG antibody (lane 6). The addition of 100× excess
of cold GH1 m1 resulted in very little competition (lane 4),
and addition of the same molar excess of GH1 m2 had no effect
(lane 5). When GH1 m1 was used as a probe, it generated a
mobility-shifted band in the presence of nuclear extracts (lane
8). The band was competed by both unlabeled wild type and m1
(lanes 9 and 10) but not by m2 oligonucleotide
(lane 11). The affinity of nuclear protein binding to m1
probe was lower than that to the wild type probe (compares lanes
2-6 to 8-12). When GH1 m2 and m3 were used as the
probes, no specific mobility-shifted band was observed (Fig.
9C, lanes 13-20). These results suggest that the
sequences between
3 and
5 (mutated in m2) and between
15 and
20
(mutated in m3) are required for PTTG DNA binding.
Mapping PTTG DNA Binding Domain--
PTTG does not contain any
typical DNA binding motif, and the amino acids required for PTTG DNA
binding have yet to be identified. I therefore sought to determine the
DNA binding domain for PTTG. Various parts of PTTG were expressed in
and purified from E. coli. After thrombin protease
digestion, the purified proteins were used in gel mobility shift
assays. The integrity of the proteins were verified by
SDS-polyacrylamide gel electrophoresis followed by Coomassie Blue
staining. As shown in Fig. 10,
truncated proteins containing either the N-terminal 118 amino acids or
amino acids between 61 and 199 generated a mobility-shifted band
(lanes 2 and 6) that was competed by unlabeled
homologous oligonucleotide (lanes 3 and 7) but
not by unrelated oligonucleotide (lanes 4 and 8).
The addition of anti-PTTG antibody resulted in a super-shifted band for
PTTG (1) (lane 5). Antibody was not included in
reactions with PTTG (61) because the epitope is located at the
N-terminal 17 amino acids. PTTG deletion mutant containing amino acids
119-199 did not a generate mobility-shifted band (lanes
9-11). These results indicate that the PTTG DNA binding domain
resides between amino acids 61 and 118.

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Fig. 10.
Mapping PTTG DNA binding domain.
Various parts of PTTG were expressed in and purified from E. coli and used in gel mobility shift assays with Myc wild type
probe. Lane 1, free probe. Lanes 2-11, + 500 ng
of recombinant protein as indicated on the top of the panel.
Lanes 3, 7, and 10, +100× unlabeled
homologous oligonucleotide. Lanes 4, 8, and
11, +100× unlabeled unrelated oligonucleotide. Lane
5, + anti-PTTC antibody. Mobility-shifted and the super-shifted
bands are indicated by arrows and an arrowhead,
respectively.
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PTTG DNA Binding Is Required for Its Transactivation of c-Myc
Gene--
To test whether binding of PTTG to DNA is required for its
transactivation function, I first determined the effect of mutations within PTTG-binding site on PTTG transactivation of c-myc
gene. The same nucleotide substitutions between position
3 and
5
(m2) and
15 to
20 (m3) were made in XNM-Luc by site-directed
mutagenesis. Fig. 11A shows
that cotransfection of PTTG expression vector resulted in a 3-fold
increase in wild type c-myc promoter activity (XNM-Luc); mutations between
15 and
20 (m3XNM-Luc) abolished this activation without affecting the basal promoter activity. Mutations between
3
and
5 (m2XNM-Luc) not only abolished PTTG-activated transcription but
also reduced basal promoter activity. The decrease in c-myc basal promoter activity in m2XNM-Luc may be a result of mutations near
the transcriptional initiation site. I then tested whether DNA
binding-defective mutant PTTG (119) could still activate
c-myc transcription. As shown in Fig. 11B, PTTG
(119) by itself had little effect on c-Myc promoter activity. When
cotransfected with wild type PTTG, it inhibited activation of
c-myc promoter by wild type PTTG. These results indicate
that PTTG DNA binding is required for its transactivation function.

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Fig. 11.
PTTG DNA binding is required for its
transactivation. NIH 3T3 cells were transiently transfected with
the indicated plasmids and assayed for luciferase reporter gene
activity (represented as fold of induction over promoter-less pGL2).
Values represent mean ± S.E. (n = 9, average of
three independent experiments). A, effect of mutations
within PTTG DNA-binding site on PTTG transactivation of
c-myc promoter. B, deletion of PTTG DNA binding
domain (DBD) abolishes its transactivation.
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DISCUSSION |
PTTG has been implicated in many types of human
neoplasm. However, the mechanisms involved in PTTG cellular
transformation and tumorigenesis remains to be elucidated. In this
study, by generating cell lines with tightly regulated inducible
expression of PTTG and expression profiling using DNA arrays
(24, 25), I have identified a down-stream target that is likely to
contribute to the function of PTTG as a oncogene. Induction
of PTTG expression in HeLaS3 cells resulted in increased
cell proliferation and colony formation. This effect involves induction
of another oncogene c-myc.
The product of the c-myc proto-oncogene is a critical
regulator of cellular proliferation (26). Overexpression of c-Myc contributes to the transformation of primary fibroblasts in culture (27) as well as development of neoplasia in a wide variety of tissues
(28). The c-Myc protein exhibits sequence-specific DNA binding when
dimerized with its partner Max (29-31). The interaction with Max is
necessary for most of the physiological effects of c-Myc (32). c-Myc
has been implicated in both activation and repression of transcription
(33-35). The expression of the c-myc gene is closely
correlated with proliferation. c-myc expression is absent in
quiescent cells but is rapidly induced upon the addition of growth
factor (36-38). The expression pattern of c-myc is similar to that of PTTG, which is also induced by mitogenic
treatment such as serum (8). Identification of c-myc as a
target for PTTG is therefore consistent with the potential role PTTG
plays in controlling cell proliferation. In my previous study, I have shown that PTTG is phosphorylated by MAP kinase and that activation of
this signal transduction pathway by growth factors such as epidermal
growth factor enhances PTTG transactivation function (11). c-Myc is
also a target of MAP kinase cascade and is phosphorylated on
Ser62 by MAP kinase (39, 40). Phosphorylation at this site
is associated with enhanced transactivation function of c-Myc (40-42).
In this study, I have identified MEK1 as another potential PTTG target. Taken together, these results suggest a functional pathway that includes MAP kinase cascade and c-Myc for PTTG-mediated effects on cell proliferation.
The identification of c-myc as an endogenous gene that is
rapidly induced after PTTG expression provides a physiological target to study the potential role of PTTG in transcriptional regulation. Our
results showed that transient transfection of a PTTG expression plasmid
led to activation of the proximal c-myc promoter, whereas expression of a transactivation-defective PTTG mutant had no effect on
c-myc promoter activity. In addition, in the cell line that expresses transactivation-defective PTTG, c-myc expression
was not induced after induction of mutant PTTG expression. These
results indicate that activation of c-myc expression
requires PTTG transactivation function.
I have provided evidence that PTTG transactivates c-myc by
directly interacting with its promoter. DNaseI protection assays showed
that nuclear proteins from HeLaS3 cell overexpressing PTTG interact
with the c-myc gene between
20 and +28. Results from gel
mobility shift assays using oligonucleotides containing various mutations of this region indicated that the DNA-protein complexes contain at least two proteins. One of these proteins is the ubiquitous basic helix-loop-helix-leucine zipper transcription factor USF1 that binds to the initiator element between
4 and +7 (InrA). Although
USF was shown to bind to Inr of the AdMLP and activates transcription through specific cooperation with TFII I (20, 21), the
data presented here show for the first time that USF1 also binds to the
Inr in c-myc promoter. The DNA-binding site for PTTG was
located between +4 and
20, and the nucleotides important for PTTG
binding include the residues between
3 and
5 as well as between
15 and
20. Importantly, mutations within these nucleotides not only
prevented PTTG from binding DNA but also abolished PTTG-mediated transcriptional activation of the c-myc gene. It is possible
that binding of PTTG and USF to adjacent sites on c-myc
promoter could result in cooperative binding of both factors to DNA, or
alternatively, transcriptional synergy may occur by the two
transcriptional activators contacting the general transcriptional
machinery simultaneously. These exciting possibilities will be explored
in the future studies.
A typical transcription activator protein is a modular protein composed
of DNA binding and activation domains (43). PTTG does not contain any
common DNA binding motifs such as homeodomain, zinc fingers, basic
leucine zipper (bZIP), and basic helix-loop-helix (44). Combining
mutagenesis with gel mobility assay, I have mapped the DNA binding
domain of PTTG to a region between amino acids 61 and 118 adjacent to
the transactivation domain (11). The amino acid sequence in this region
is rich in basic residues containing 12 lysines and arginines. PTTG DNA
binding domain may represent a new motif for DNA-binding proteins. Our
results showed that the DNA binding domain is required for PTTG
transactivation, for mutant-containing deletion of this domain was
unable to bind to DNA and activate c-myc promoter.
Interestingly, the DNA binding domain deletion mutant inhibited
transcriptional activation by wild type PTTG. This inhibition may occur
through sequestration of general transcription factors by the mutant protein.
In summary, the data presented in this study have established the
important role of PTTG in the regulation of transcription. Although the
definitive requirement for PTTG in c-myc activation will
need to be confirmed once PTTG-null cells are available, the data
presented here clearly indicate that c-myc is likely a
physiological target for PTTG and, thus, links PTTG to a functional pathway involved in cellular proliferation and transformation.