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SV40 Large T Antigen Transactivates the Human cdc2 Promoter by
Inducing a CCAAT Box Binding Factor*
(Received for publication, January 22, 1996, and in revised form, April 1, 1996)
Haifeng Chen‡§, Judith Campisi¶, and R. Padmanabhan‡i
From the ‡Department of Biochemistry and Molecular Biology, the University of Kansas Medical Center, Kansas City,
Kansas 66160-7421, and ¶Department of Cancer Biology, Berkeley National Laboratory, University of California,
Berkeley, California 94720
Cyclin-dependent protein kinases (Cdks) play a key
role in the cell division cycle of eukaryotic cells. Cdc2,
the first mammalian Cdk that was discovered, is ex-
pressed in S phase and functions in the G
2
to M phase
transition. By transfecting segments of the human cdc2
promoter linked to a reporter gene into monkey kidney
(CV-1) cells, we identified the region containing the Sp1,
E2F, and two CCAAT box binding sites as essential and
sufficient for basal transcription. SV40 large T antigen
(SV40-LT) is a viral oncoprotein that transactivates vi-
ral and cellular promoters and induces DNA synthesis in
quiescent cells. SV40-LT transactivated wild-type cdc2
promoter/reporter constructs in a dose-dependent man-
ner, coinciding with an increase in endogenous cdc2
mRNA. A mutant promoter from which the two CCAAT
box motifs were deleted was 8-fold less sensitive to SV40-
LT. Activation by SV40-LT did not require its ability to
bind the retinoblastoma or p53 tumor suppressor pro-
teins. SV40-LT induced a specific CCAAT box-binding
factor (CBF) in CV-1 and COS-7 cells, as judged by gel
shift and Southwestern analyses. Similar results were
obtained in human fibroblasts expressing a conditional
SV40-LT. The SV40-LT-inducible CBF appears to be
novel and differs from the CBF that activates heat shock
protein 70 gene expression.
The cyclin-dependent protein kinases (Cdks)
1
play an impor-
tant role in the control of cell division in eukaryotic cells. The
first Cdks identified were the products of the cdc2 gene in the
fission yeast Schizosacchomyces pombe and the CDC28 gene of
Saccharomyces cerevisiae. The mammalian homologue of cdc2
and CDC28 encodes a catalytic subunit, p34
cdc2
, which is now
known to be an important regulator of cell cycle progression
(Nurse and Bissett, 1981; Beach et al., 1982; Reed et al., 1985;
Simanis and Nurse, 1986; Draetta et al., 1987; Lee and Nurse,
1987) (for reviews, see Nurse (1990), Draetta (1990), and Pines
and Hunter (1990)). p34
cdc2
, like other Cdks, requires associa-
tion with a cyclin regulatory subunit for kinase activity (re-
viewed by Morgan (1994) and Pines (1994)). p34
cdc2
is primar-
ily required for progression from G
2
to M (Riabowol et al., 1989;
Fang and Newport, 1991; Murray, 1992), whereas other mam-
malian Cdks (e.g. p33
cdk2
) have been implicated in the initia-
tion of DNA replication (Fang and Newport, 1991; Tsai et al.,
1991; Pagano et al., 1993) (for reviews, see Morgan (1994),
Pines (1994), and references therein).
Expression of the mammalian cdc2 mRNA, as well as p34
cdc2
protein, depends on the cellular growth state, declining signif-
icantly when cells undergo growth arrest, differentiation, or
development (Draetta et al., 1987; Lee et al., 1988; Krek and
Nigg, 1989; D’Urso et al., 1990; McGowan et al., 1990; Wang et
al., 1991; Dalton, 1992), and increasing when quiescent cells
enter the cell cycle. The human cdc2 promoter contains con-
sensus binding sites for several cellular transcription factors,
including ATF, c-Myb, Sp1, E2F, and CCAAT box binding
factor (Dalton, 1992; Ku et al., 1993). The product of the c-myb
protooncogene (Ku et al., 1993) and the Rb tumor suppressor
protein (Dalton, 1992; Yamamoto et al., 1994) have been shown
to regulate the cdc2 promoter through c-myb and E2F binding
elements, respectively. Some members of the E2F transcription
factor family interact with the Rb protein, whereas other mem-
bers interact with the Rb-related protein p107 and p130 (Bag-
chi et al., 1991; Chittenden et al., 1991; Bandara and La
Thangue, 1991; Chellappan et al., 1992; Shirodkar et al., 1992;
Lees et al., 1993; Beijersbergen et al., 1994; Ginsberg et al.,
1994; Hijmans et al., 1995; Sardet et al., 1995). Since E2F is
important for the transcription of several cell cycle-regulated
genes such as c-myc (Hiebert et al., 1989; Thalmeier et al.,
1989) and genes expressed at the G
1
/S boundary (De Gregori et
al.1995), the Rb-E2F interaction is an important negative reg-
ulator of the expression of these genes. The interaction of Rb or
the related proteins, p107 and p130, with E2F is disrupted by
several viral oncoproteins. These include SV40-LT, adenovirus
E1A, and papilloma virus E7 (Whyte et al., 1988; De Caprio et
al., 1988; Dyson et al., 1989; Chellappan et al., 1992). This is
consistent with the finding that the Rb-mediated repression of
cdc2 promoter can be reversed by coexpression of adenovirus
E1A protein (Dalton, 1992).
SV40-LT induces DNA synthesis in human fibroblasts that
are quiescent and/or senescent (Tsuji et al., 1983; Gorman and
Cristofalo, 1985; Wright et al., 1989; Shay et al., 1991; Saka-
moto et al., 1993; Dimri and Campisi, 1994), and express low or
undetectable levels of cdc2 mRNA and p34
cdc2
protein (Richter
et al., 1991; Stein et al., 1991). These findings suggest that
SV40-LT might activate, directly or indirectly, one or more
* This research was supported in part by National Institutes of
Health Grants CA33099 (to R. P.) and AG09909 and AG11658 (to J. C.),
and by National Science Foundation Grant MCB-9507034 (to R. P.).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
§ Supported in part by a postdoctoral fellowship from the Marrion
Merrell Dow Foundation.
iTo whom correspondence should be addressed: Dept. of Biochemis-
try and Molecular Biology, University of Kansas Medical Center, 3901
Rainbow Blvd., Kansas City, KS 66160-7421. Tel.: 913-588-7018; Fax:
913-588-7440; E-mail: rpadmana@kumc.edu.
1
The abbreviations used are: Cdk, cyclin-dependent protein kinase;
E1A, adenovirus early region 1A-encoded protein(s); CAT, chloram-
phenicol acetyltransferase; CBF, CCAAT box binding factor; pCMV,
plasmid encoding cytomegalovirus early promoter; EMSA, electro-
phoretic mobility shift analysis; HSP70, heat shock protein 70; Rb,
retinoblastoma protein; RSV-
b
-gal, E. coli lacZ gene encoding
b
-galac-
tosidase under the control of Rous sarcoma virus long terminal repeat
promoter; LTR, long terminal repeat; PAGE, polyacrylamide gel elec-
trophoresis; SV40-LT, SV40 large T antigen; tk, thymidine kinase;
YY-1, yin-yang-1 factor; bp, base pair(s).
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 24, Issue of June 14, pp. 13959–13967, 1996
© 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
13959
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growth regulatory genes such as cdc2. Here, we examine
whether SV40-LT activates the human cdc2 promoter and, if
so, whether the E2F binding sites are required for this activa-
tion. We show that, for basal activity of the human cdc2 pro-
moter in monkey kidney (CV-1) cells, the region containing the
Sp1, E2F, and two CCAAT box binding elements (between
2171 and 228) are essential and sufficient. This activity was
transactivated severalfold by SV40-LT, and only the two
CCAAT box binding motifs were required for optimal transac-
tivation. In addition, we show that the ability of SV40-LT to
bind and inactivate the retinoblastoma (Rb) or p53 tumor sup-
pressor proteins is not necessary for this transactivation. Fi-
nally, we show that SV40-LT induces a novel CCAAT box
binding factor in cycling CV-1 cells and SV40-transformed
COS-7 cells, and in human fibroblasts that are reversibly se-
nescent due to a conditional SV40-LT.
EXPERIMENTAL PROCEDURES
Materials—All enzymes involved in the cloning procedures were
purchased from New England Biolabs, Inc. Dulbecco’s modified Eagle’s
medium, Trizol, and Lipofectin were obtained from Life Technologies,
Inc. Defined/supplemented bovine calf serum and fetal bovine serum
were purchased from Hyclone Laboratories (Logan, UT) or Sigma. An-
tibiotics, dexamethasone, CsCl, ethidium bromide, n-butyryl-coenzyme
A, and o-nitrophenyl-
b
-D-galactopyranoside were purchased from
Sigma. Bradford reagent for protein estimation was from Bio-Rad.
Baker Si500F TLC plate-silica gel was from J.T. Baker, Inc. [
14
C]Chlor-
amphenicol (49 mCi/mmol) was from Moravek Biochemicals, Inc.
[
g
-
32
P]ATP (3000 Ci/mmol), GeneScreen membrane, and NEF-495 x-ray
film were from DuPont NEN. Poly(dI-dC) was purchased from Boeh-
ringer Mannheim.
Construction of cdc2 Promoter Deletions—Methods for plasmid con-
structs were carried out according to standard procedures (Sambrook et
al., 1989). To construct cdc2 promoter deletions, cdc2-PstI, and cdc2-7
clones (kindly provided by Dr. Bruno Calabretta; Ku et al., 1993) were
used. The 431-bp TaqI fragment of cdc2-7 containing the promoter
sequence from nucleotide 2359 to nucleotide 164 was ligated to the
AccI site of pCAT-Basic (Promega) to obtain cdc2-TaqI (Fig. 1). cdc2-
TaqI was digested by SmaI and ApaI to remove a 81-bp fragment, and
the vector fragment was blunt-ended by treatment with Escherichia coli
DNA polymerase (Klenow), which upon religation gave rise to cdc2-
TaqDSA. cdc2-TaqI was digested by SmaI and BamHI, and the SmaI-
BamHI fragment was ligated to HindIII-digested pCAT-Basic, blunt-
ended by DNA polymerase (Klenow), and then digested by BamHI to
yield cdc2-SmaI. To construct cdc2-PstDNA, cdc2-PstI was digested by
NarI, blunt-ended by DNA polymerase (Klenow), and then digested by
HindIII to isolate the 766-bp fragment. This fragment was cloned into
cdc2-TaqI, which had been digested with ApaI, blunt-ended by DNA
polymerase (Klenow), digested with HindIII, and then gel-purified to
remove the HindIII-ApaI fragment. To obtain pTI-CCAAT-CAT, the
pTI-CAT plasmid containing a basic promoter (Shi et al., 1991) was
digested with BglII, blunt-ended by DNA polymerase (Klenow), and
ligated to the SmaI-ApaI fragment containing the two CCAAT box
binding sites of the cdc2 promoter. In all these constructs, the cdc2
promoter fragments were fused to the reporter gene, CAT, as shown in
Fig. 1.
Cell Culture, DNA Transfections, and CAT Assays—Monkey kidney
cells (CV-1) and COS-7 cells (constitutively expressing SV40-LT) were
cultured at 37 °C, 5% CO
2
in Dulbecco’s modified Eagle’s medium
containing 50
m
g/ml each of streptomycin and penicillin, and 10% de-
fined/supplemented bovine calf serum. IDH4 cells were cultured in
Dulbecco’s modified Eagle’s medium, 10% fetal calf serum, and 10
26
M
dexamethasone as described (Wright et al., 1989). IDH-4 cells were
plated at 3–5 310
3
/cm
2
in 100-mm or 35-mm dishes. Twenty-four hours
after plating, dexamethasone-deprived cells were washed and shifted to
medium lacking dexamethasone and phenol red but containing char-
coal-stripped serum. Cells were grown for 7 days, and dexamethasone-
added cells were grown in the continual presence of dexamethasone.
For dexamethasone-added/deprived cells, the cells were first dexa-
methasone-deprived for 7 days and then grown in the presence of
dexamethasone for 24 h. Cells were harvested and fractionated into
cytoplasmic and nuclear fractions as described (Chen et al., 1994).
Plasmid DNAs were purified by CsCl-ethidium bromide equilibrium
density gradients (Sambrook et al., 1989). For transfection, cells were
grown to 50–70% confluence and plasmid DNAs were transfected using
Lipofectin as follows. A cdc2 promoter-CAT reporter plasmid (3
m
g) and
the RSV-
b
-gal plasmid (1
m
g;, E. coli lacZ under control of the RSV-
LTR) as an internal control, were transfected into CV-1 cells. Cells were
harvested 45 h post-transfection, and cell extracts were prepared by
three cycles of freeze-thawing. CAT assays were carried out at 37 °C for
indicated intervals in a total volume of 125
m
l containing 250 mM
Tris-HCl (pH 7.8), 25
m
g of butyryl-coenzyme A, and 0.25
m
Ci of
[
14
C]chloramphenicol (Moravek Biochemicals, Inc.) according to the
protocol supplied by Promega and as described by Gorman et al. (1982).
Acetylated [
14
C]chloramphenicol derivatives separated by thin-layer
chromatography were scraped and counted in a liquid scintillation
counter. Each experiment was repeated at least twice with different
DNA preparations. E. coli
b
-galactosidase activity was determined us-
ing the substrate o-nitrophenyl-
b
-D-galactopyranoside as described
(Craven et al., 1965). Transfection experiments with reporter plasmids
were carried out in duplicate, and results with more than 30% variation
between duplicates were discarded and the experiments repeated.
Mutagenesis of CCAAT Box Binding Sites in cdc2-TaqI Plasmid—
Two overlapping oligodeoxynucleotides (shown below) containing
CCAAT 3GGCCT mutations in the consensus CCAAT box binding
sites in the SmaI-ApaI fragment were annealed.
59-GGGAAGCCTACCCAGCGTAGCTGGGCTCTGAggccCTGCTTTGAAAG-39
59-CCGGATTCAggccTCGGGTAGCCCGTAGACTTTCAAAGCAGggccTC-39
(Lowercase letters indicate the mutated CCAAT box, and the underline
indicates the overlap between the oligonucleotides.) The annealed oli-
gonucleotides were treated with E. coli DNA polymerase (Klenow) to
fill-in the 59-protruding ends and cloned into cdc2-TaqI, which had been
digested with SmaI and ApaI, blunt-ended, and gel purified to remove
the SmaI-ApaI fragment, to yield the plamid cdc2-TaqMutCCAAT.
Northern (RNA) Analysis—Total RNA was isolated using Trizol,
fractionated on a 0.8% agarose-formaldehyde gel by electrophoresis and
transferred to a GeneScreen membrane following the manufacturer’s
protocol. Antisense cdc2 mRNA probe was prepared from a cDNA clone
using the SP6 RNA polymerase for in vitro transcription as described
(Sambrook et al., 1989). The 18 and 28 S RNAs were visualized by
staining the membrane with 0.02% methylene blue and 0.5 MNaAc
(pH5.2), and destaining in 20% methanol. After photography, the meth-
ylene blue was removed from the gel by soaking in a solution containing
0.2 3SSPE (Sambrook et al., 1989) and 1% SDS. The membrane was
prehybridized at 65 °C in hybridization solution containing 1 MNaCl,
0.05 g/ml dextran sulfate, 50
m
g/ml yeast tRNA, 40% (v/v) formamide,
and 1% SDS for 3 h and then hybridized overnight at 65 °C in hybrid-
ization solution containing
32
P-labeled cdc2 RNA probe. The mem-
branes were rinsed twice with 1 3SSC, 0.1% SDS and washed in this
buffer at 65 °C for 1 h. The blots were then rinsed twice with 0.3 3SSC,
0.1% SDS at room temperature, and once in this buffer at 65 °C for 1 h.
The blots were subjected to autoradiography (NEF-495 x-ray film) at
270 °C.
Gel Mobility Shift Assay—cdc2-TaqIorcdc2-TaqMutCCAAT was
digested by SmaI and XbaI. The 180-bp SmaI-XbaI fragment contain-
ing the wild-type or mutated CCAAT box binding sites was dephospho-
rylated, and labeled with [
g
-
32
P]ATP by T4 polynucleotide kinase. Gel
mobility shift assays were carried out as described (Chen et al., 1994).
Briefly, nuclear extracts were mixed with labeled probe in 20
m
lof
DNA-binding reaction buffer containing 25 mMHEPES-KOH (pH 7.9),
5m
MKCl, 0.5 mMEDTA, 1
m
g/ml bovine serum albumin, 10% glycerol,
0.25 mMdithiothreitol, and 0.1
m
g/ml poly(dI-dC) and incubated at
37 °C for 30 min. The reaction was stopped by adding 2
m
l of stop
solution containing 50 mMEDTA, 0.05% bromphenol blue, 0.05% xylene
cyanole, and 5% glycerol. The DNA-binding complexes were separated
on a 4% polyacrylamide gel. The gel was dried and exposed to NEF-495
film at 270 °C.
Southwestern Analysis—The interaction between the CCAAT box
motif and nuclear proteins was analyzed essentially as described (Silva
et al., 1987). Nuclear proteins were separated on a 8% SDS-polyacryl-
amide gel and transferred to a nitrocellulose membrane. After renatur-
ing, the proteins on the membrane were probed with radiolabeled DNA
fragment containing CCAAT box binding sites. The membrane was
then washed to remove unbound probe and exposed to NEF-495 film at
270 °C.
Western Analysis—Cells were washed with phosphate-buffered sa-
line, lysed in 150
m
lof23Laemmli sample buffer (Laemmli, 1970)
lacking
b
-mercaptoethanol and tracking dye, and frozen at 280 °C.
Radiolabeled cells were processed for autoradiography, as described
(Seshadri and Campisi, 1990). Lysates were supplemented with
b
-mer-
captoethanol and tracking dye and heated to 95 °C for 5 min. Proteins
SV40 T Antigen Transactivates Human cdc2 Promoter13960
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were separated by SDS-polyacrylamide gel electrophoresis (8%; Lae-
mmli (1970)) and transferred by electrophoresis to a polyvinylidene
difluoride membrane in 20 mMTris (pH 8.3), 100 mMglycine, 0.5% SDS,
20% methanol. The membrane was preincubated in 5% powdered milk
in blocking solution (20 mMTris (pH 8.0), 150 mMNaCl, 0.05% Tween
20), incubated with primary antibody (1:1000 in blocking solution),
washed, incubated with secondary antibody (1:1000 in blocking solu-
tion), and washed. Secondary antibody binding was detected by a
chemiluminescence kit (ECL, Amersham Corp.). The primary antibod-
ies were a mouse monoclonal against SV40 large T antigen (ammonium
sulfate enriched supernatant from PAB101; American Type Culture
Collection) and a mouse monoclonal against
b
-tubulin (Oncogene Sci-
ence, Ab-1). The secondary antibody was a horseradish peroxidase-
conjugated sheep anti-mouse IgG from Amersham.
RESULTS
Characterization of cdc2 Promoter—An analysis and charac-
terization of control elements in the human cdc2 promoter that
bind the c-Myb protein as well as the transcription factor E2F
has been described (Dalton, 1992; Ku et al., 1993; Yamamoto et
al., 1994). However, the functions of other control elements
have not been studied. To further characterize the human cdc2
promoter, we constructed a series of deletions in the cdc2
promoter linked to the reporter gene CAT (Fig. 1). Monkey
kidney (CV-1) cells were transfected with these reporter plas-
mids along with RSV-
b
-gal, which encodes the E. coli
b
-galac-
tosidase and serves as an internal control for transfection effi-
ciency. CAT and
b
-galactosidase activities were determined in
extracts prepared 45 h post-transfection.
As shown in Fig. 2 (Aand B), the cdc2-PstI (parent) plasmid
exhibited significant promoter activity, as reported previously
(Ku et al., 1993). In contrast, cdc2-PstDNA, having a deletion
between the NarI and ApaI sites (2171 and 228 nucleotides
upstream of the transcription start site) showed only 2% of the
parent promoter activity. This result indicates that the Sp1,
E2F, and the two inverted CCAAT box binding elements are
essential for basal cdc2 promoter activity. Deletion of se-
quences upstream of the TaqI site (2359; cdc2-TaqI; Fig. 1)
reproducibly increased promoter activity over that shown by
the parent cdc2-PstI plasmid (see Fig. 2, Aand B). This finding
suggests that there may be a negative regulatory element
upstream of the TaqI site. Deletion of sequences between the
SmaI and ApaI sites, which contain the two CCAAT box motifs
(cdc2-TaqDSA in Figs. 1 and 2 (Aand B)), decreased promoter
activity by 5-fold. When sequences upstream of the SmaI site
(2109) were deleted (cdc2-SmaI, containing only the two
CCAAT box binding sites, shown in Figs. 1 and 2 (Aand B)),
promoter activity was reduced about 2.5-fold compared with
the cdc2-TaqI construct. These results suggested that although
the Sp1, E2F, and the CCAAT- box motifs all contribute to cdc2
promoter activity, the two CCAAT box motifs play an essential
and major role.
To explore the activity of the two CCAAT box binding sites in
the cdc2 promoter, the SmaI-ApaI fragment was cloned up-
stream of the TATA box (BglII site) in the basic promoter/
reporter plasmid pTI-CAT (Shi et al., 1991). The resultant
plasmid, pTI-CCAAT-CAT, was transfected into CV-1 cells and
CAT activity was determined. Fig. 3 (Aand B) shows that
pTI-CAT, which contains only the TATA element, displayed
low promoter activity, as reported (Shi et al., 1991). By con-
trast, when the two cdc2 CCAAT box binding sites were present
upstream of the TATA element in pTI-CAT, there was a 14-fold
increase in CAT activity. These data further demonstrate that
the CCAAT box motifs in the cdc2 promoter are functional, not
only in the cdc2 promoter, which lacks a TATA element, but
also in a basic promoter containing only a TATA element.
Transactivation of cdc2 Promoter by SV40 T Large Antigen
through CCAAT Box Binding Sites—We next sought to deter-
FIG.1.Schematic representation of the human cdc2 promoter-
CAT fusion plasmids. The relative positions of the putative cis-acti-
vating regulatory elements are indicated. Constructs were generated by
standard DNA cloning techniques as described under “Experimental
Procedures.”
FIG.2.Analysis of basal activity of human cdc2 promoter us-
ing deletion mutants. A, autoradiography of a thin-layer chromatog-
raphy plate from a representative CAT assay (Gorman et al., 1982)
using extracts prepared from CV-1 cells transiently transfected with a
cdc2 promoter/CAT construct shown in Fig. 1 (3
m
g each) and RSV-
b
-gal
plasmid (1
m
g; Zhao and Padmanabhan (1988)) as internal control. The
CAT activity assay was carried out by incubation of the extracts with
labeled [
14
C]chloramphenicol at 37 °C for 2 h. B, the results in Awere
quantitated by liquid scintillation counting of the radioactivity in each
sample, and are presented as a bar graph.
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mine whether the cdc2 promoter is a target for transactivation
by SV40-LT. CV-1 cells were cotransfected with a cdc2-pro-
moter construct and a SV40-LT expression plasmid pCMV-TAg
(Sakamoto et al., 1993; Dimri and Campisi, 1994; Hara et al.,
1996). In control experiments, an equal amount of the pCMV
vector plasmid (without an insert) was included instead of
pCMV-TAg. The results shown in Fig. 4 (Aand B) indicate that
expression of SV40-LT efficiently induced transcription from
the parent cdc2-PstI plasmid (compare lanes with and without
SV40-LT). Deletion of sequences upstream of the TaqI site
(2359 as in cdc2-TaqI; compare lanes with and without SV40-
LT) or upstream of the SmaI site (2109 as in cdc2-SmaI;
compare lanes with and without SV40-LT) did not appreciably
affect the level of transactivation by SV40-LT.
These results indicate that the CCAAT box binding sites
alone contribute 73% of the level of SV40-LT-mediated trans-
activation achieved with the cdc2-TaqI construct. This con-
struct contains the ATF, c-Myb, Sp1, and E2F, as well as the
CCAAT box motifs. Deletion of sequences between the SmaI
(2109) and ApaI(228) sites, which contains the two CCAAT
box binding sites, dramatically reduced the SV40-LT-mediated
transactivation (Fig. 4, cdc2-TaqDSA, lanes with and without
SV40-LT). The cdc2-PstDNA promoter showed low promoter
activity, which could not be transactivated by SV40-LT (com-
pare lanes with and without SV40-LT). Thus, the elements
upstream of the Sp1 site, notably the ATF and c-myb binding
sites, were unresponsive to SV40-LT-mediated transactivation.
The low level of transactivation by SV40-LT observed with this
construct is consistent with an earlier study showing that
SV40-LT can activate transcription from a promoterless re-
porter gene (Rice and Cole, 1993). Taken together, these results
show that the CCAAT box binding motifs are the major target
for SV40-LT-mediated activation.
One mechanism by which SV40-LT may activate the cdc2
promoter is by disrupting transcriptionally inactive E2F-Rb or
Rb-related protein complexes. SV40-LT interacts directly with
Rb or Rb-related proteins, thereby releasing transcriptionally
active E2F, which participate in the transcription of E2F-de-
pendent genes (Chellappan et al., 1992; Dyson et al., 1990;
Ludlow et al., 1989; for a review, see Nevins (1992)). Our
results indicate that the E2F binding site in the human cdc2
promoter does not contribute significantly to SV40-LT-medi-
ated activation (compare cdc2-TaqI, cdc2-TaqDSA, and cdc2-
SmaI in Fig. 4). Alternatively, SV40-LT, which also interacts
with p53, may relieve p53-mediated repression of cdc2 pro-
moter. To investigate these possibilities and determine
whether the Rb- or p53-binding regions of SV40-LT are re-
quired for activation of the cdc2 promoter, CV-1 cells were
cotransfected with the cdc2-TaqI promoter containing the E2F
binding sites along with pCMV-TAg (wild-type SV40-LT),
pCMV-TAg-Rb
2
or pCMV-TAg-p53
2
(mutant SV40-LT defec-
tive in Rb- or p53- binding, respectively; see Tevethia et al.
(1988), Sakamoto et al. (1993), Dimri and Campisi (1994), and
Hara et al. (1996)). The results of these experiments shown in
Fig. 5 indicated that transactivation of the cdc2 promoter by
SV40-LT was independent of its ability to bind to Rb or p53.
To confirm that activation of cdc2 promoter by SV40-LT was
FIG.3.Activation of a heterologous basic promoter/reporter
pTI-CAT by the CCAAT box binding motifs of the cdc2 promoter.
The CCAAT box binding elements of the cdc2 promoter were excised
and cloned into the basic promoter pTI-CAT as described under “Ex-
perimental Procedures.” Transfection and CAT activity assay were
carried out as described in Fig. 2. Panels A and Brepresent the auto-
radiography and the bar graph as in Fig. 2.
FIG.4. Transactivation of various deletion mutants of cdc2
promoter by SV40 T antigen. CV-1 cells were transfected with 3
m
g
ofacdc2 promoter-CAT reporter plasmid (Fig. 1) with or without 2
m
g
of pCMV-TAg (Sakamoto et al., 1993). In the absence of pCMV-TAg
plasmid, an equal amount of pCMV vector plasmid was used for trans-
fections to equalize the total amount of plasmid DNA in each transfec-
tion. The CAT activity assay was carried out at 37 °C for 1 h. Panels A
and Brepresent the autoradiography and the bar graph as in Fig. 2.
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mediated through CCAAT box binding sites, increasing
amounts of the SV40-LT expression plasmid (pCMV-TAg) and
fixed amounts of two cdc2 promoter constructs, cdc2-TaqI and
cdc2-TaqDSA, were cotransfected into CV-1 cells. These plas-
mids have the same upstream sequences except that cdc2-
TaqDSA lacks the two CCAAT box binding sites. The results
are shown in Fig. 6. As the amount of pCMV-TAg was in-
creased, CAT expressed from the cdc2-TaqI plasmid increased
correspondingly (up to 1
m
g). By contrast, CAT expressed from
the cdc2-TaqDSA plasmid increased only moderately (up to
0.01
m
g) with increasing dosage of pCMV-TAg. Higher amounts
of pCMV-Tag (1 and 2
m
g) had no effect on activation. The peak
level of activation obtained with cdc2-TaqI (containing wild-
type CCAAT box motif) was 8-fold higher than that obtained
with the mutant cdc2-TaqDSA construct (in which the two
motifs were deleted; Fig. 6).
It still remained possible that differences in the reporter
expression driven by various cdc2 promoter segments were due
to differences in the distance between the transcription factor
binding sites and the transcription start sites created by the
deletions we introduced in the promoter. To rule out this pos-
sibility, and further confirm that the CCAAT box binding mo-
tifs were the prime targets for SV40-LT-mediated transactiva-
tion, we introduced point mutations into the two CCAAT box
binding sites in cdc2-TaqI by site-directed mutagenesis. The
mutant cdc2-TaqMutCCAAT construct was transfected with or
without pCMV-TAg into CV-1 cells, and cell extracts were
assayed for CAT activity. The results, shown in Fig. 7, demon-
strate that the response of the mutant cdc2-TaqMutCCAAT
promoter to SV40-LT activation was significantly reduced, al-
though it retained detectable basal promoter activity. These
results presented in Figs. 6 and 7, taken together, support our
conclusion that the two CCAAT box binding sites in the human
cdc2 promoter contribute significantly to transactivation by
SV40-LT.
The results of the above experiments measured SV40-LT
transactivation of the cdc2 promoter in transient transfection
assays. To determine whether SV40-LT also activates the en-
dogenous cdc2 gene, we analyzed cdc2 mRNA levels in CV-1
cells mock-transfected or transfected with increasing amounts
of pCMV-TAg plasmid, as well as the level of mRNA tran-
scribed from the endogenous cdc2 gene in SV40-transformed
COS-7 cells. At 45 h post-transfection, total RNA were ex-
tracted from the cells. As shown in Fig. 8, Northern hybridiza-
tion using an antisense cdc2 RNA as a probe indicated that the
endogenous cdc2 mRNA levels increased in a dose response
manner with increasing amounts of SV40-LT (lanes 1–7). The
highest level of cdc2 mRNA induced by SV40-LT was about
3-fold greater than the level in mock-transfected cells (compare
lane 1 with lane 6). Because transfection is well below 100%
efficient, these results suggest that SV40-LT can induce sub-
stantial levels of the endogenous cdc2 mRNA.
The results thus far indicate that SV40-LT activation of the
cdc2 promoter is mediated by the CCAAT box binding motifs.
We therefore asked whether SV40-LT induces and/or interacts
with a CCAAT box-binding factor (CBF). We performed elec-
trophoretic mobility shift assays (EMSA) to analyze the DNA-
protein complexes formed from such interactions using radio-
labeled wild-type and mutant CCAAT box binding elements in
the cdc2 promoter. As shown in Fig. 9, nuclear extracts from
CV-1 cells transfected with pCMV-TAg (lane 2) or from SV40-
LT-expressing COS-7 cells (lane 3) formed a specific DNA-
protein complex, whereas extracts from mock-transfected CV-1
cells did not form this complex (lane 1). Furthermore, when a
mutant CCAAT box motif was used for EMSA, this specific
DNA-protein complex was not formed in all three extracts
(lanes 4–6). These results demonstrate that expression of a
specific CBF was augmented by SV40-LT in CV-1 cells in
FIG.5.Transactivation of cdc2 promoter by wild-type and mu-
tant SV40-LTs lacking pRb or p53 binding domains. The plasmids
encoding wild-type or mutant SV40-LTs were individually cotrans-
fected with cdc2-PstI into CV-1 cells, and the CAT activity was meas-
ured as described in Fig. 4, except that the enzyme reaction was carried
out at 37 °C for only 30 min.
FIG.6.Dose response of CCAAT box binding motifs to trans-
activation by SV40 T antigen. CV-1 cells were cotransfected with 3
m
gofcdc2-TaqIorcdc2-TaqDSA promoter-CAT reporter plasmid (Fig.
1) and with increasing amounts of pCMV-TAg plasmid. CAT activity
assay was carried out at 37 °C for 1 h. Panels A and Brepresent the
autoradiography and the bar graph as in Fig. 2. The bar graph shows
the CAT activities (cpm) versus the amount of pCMV-TAg plasmid used
for transfections (
m
g).
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transient transfection assays and in stably transformed COS-7
cells.
Lum et al. (1990) reported that a specific CBF of 114 kDa
binds to the CCAAT box motif of the HSP70 promoter and is
responsible for the activation of HSP70 gene expression. To
determine whether the CBF that binds the CCAAT box binding
motif in the cdc2 promoter is the same as that involved in the
activation of the HSP70 promoter, an antibody to the HSP70-
CBF was used for supershift analysis in EMSA. As shown in
Fig. 9 (lanes 7–9), the HSP70-CBF antibody had no effect on
the mobility of the SV40-LT-inducible cdc2 promoter CCAAT
box motif-specific DNA protein complex. To further confirm
this result, we asked whether the HSP70-CBF can activate the
cdc2 promoter in a transient transfection assay. As shown in
Fig. 10, expression of HSP70-CBF in CV-1 cells activates the
HSP70 promoter as expected (Agoff et al., 1993). However,
HSP70-CBF had no effect on the cdc2 promoter. These results
suggest that a novel CBF, which is involved in activation of
cdc2 promoter, may be induced by SV40-LT.
To further characterize the CBF that interacts with the cdc2
promoter, Southwestern analysis was performed. Nuclear ex-
tracts from mock-transfected cells, pCMV-TAg-transfected
CV-1 cells, and COS-7 cells were fractionated by SDS-PAGE.
The proteins were transferred to a nitrocellulose membrane,
renatured, and probed with a
32
P-labeled DNA fragment con-
taining the CCAAT box binding motif. Proteins bound to the
labeled probe were detected by autoradiography. The results,
shown in Fig. 11, indicate that a protein of about 110 kDa
specifically bound to the CCAAT box binding sites in the cdc2
promoter. In mock-transfected CV-1 cells, the level of this
protein was low (lanes 1–3), whereas SV40-LT, whether tran-
siently expressed in CV-1 cells (lanes 4–6) or constitutively
expressed in stably transformed COS-7 cells (lanes 7 and 8),
greatly enhanced the expression of this CBF. Probing the mem-
brane with the anti-HSP70/CBF antibody showed that the
HSP70/CBF protein migrated more slowly than the radiola-
beled SV40-LT-inducible protein (data not shown).
We also tested the ability of SV40-T to induce a 110-kDa
CBF/cdc2 protein in human fibroblasts that are reversibly im-
mortalized by an inducible SV40-LT (IDH4 cells; Wright et al.
(1989)). IDH4 cells were grown in the absence or presence of
dexamethasone (Fig. 12, 2dex,1dex, and 2/1dex), and ex-
tracts were prepared 7–8 days later for Western and South-
western analyses. Under these conditions, the cells expressed
high levels of SV40-LT in the presence of dexamethasone (rel-
ative to the tubulin control), and very low levels of SV40-LT in
the absence of dexamethasone (Fig. 12). The same extracts also
showed high and low levels of the 110-kDa CBF/cdc2 protein,
respectively (Fig. 13). When dexamethasone-deprived cultures
expressing low levels of T antigen and CBF/cdc2 were restim-
ulated with dexamethasone (Fig. 12, 2/1dex), both T antigen
and CBF/cdc2 were induced substantially within 24 h (Figs. 12
and 13), and the CBF/cdc2 was enriched in the nuclear fraction
(Fig. 13). These results support our data on the induction of a
CBF/cdc2 protein by SV40-LT in CV-1 and COS-7 cells. To-
gether, the data suggest that SV40-LT induces the expression
of a potentially novel 110-kDa CBF/cdc2 in monkey kidney cells
as well as in human fibroblasts.
FIG.7.SV40-LT mediated transactivation of the wild-type and
mutant CCAAT box binding motifs. The CCAAT box binding motifs
in the cdc2-TaqI promoter/reporter plasmid were mutated specifically
by point mutagenesis as described under “Experimental Procedures.”
Transfections and CAT assays were carried out as described in Fig. 4,
except that the enzyme reaction was carried out at 37 °C for only 30 min
to ensure that the substrates were not used up at the end of the
incubation period.
FIG.8.cdc2 mRNA levels in transfected CV-1 cells and COS-7
cells. Total RNA was isolated from the cells using Trizol reagent and 10
m
g of total RNA was loaded onto each lane. The 18 and 28 S RNA was
stained by methylene blue, and cdc2 mRNA levels were detected by
32
P-labeled RNA probe as described under “Experimental Procedures.”
Lane 1, mock-transfected CV-1 cells; lanes 2–7, CV-1 cells transfected
with 0.0001, 0.001, 0.01, 0.1, 1.0, and 2.0
m
g of pCMV-TAg; lane 8,
COS-7 cells.
FIG.9.DNA-binding activity of nuclear extracts from mock-
transfected and pCMV-TAg-transfected CV-1 cells and COS-7
cells. Nuclear extracts were prepared and gel mobility shift assays
were performed as described under “Experimental Procedures.” Lanes
1,4, and 7, extracts from mock-transfected CV-1 cells. Lanes 2,5, and
8, extracts from pCMV-TAg-transfected CV-1 cells. Lanes 3,6, and 9,
extracts from COS-7 cells. Lanes 1–3 and 7–9, extracts were probed with
the wild-type CCAAT box binding site. Lanes 4–6, extracts were probed
with the mutant CCAAT box binding site. Lanes 7–9, monoclonal anti-
body against HSP70-CBF (Agoff et al., 1993) was added.
SV40 T Antigen Transactivates Human cdc2 Promoter13964
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DISCUSSION
This study further dissects the cis-regulatory elements in the
human cdc2 promoter that contribute to basal transcription,
and reveals the CCAAT box binding motifs as major targets for
transactivation mediated by SV40-LT. Studies carried out in
other laboratories on the human cdc2 promoter revealed mul-
tiple regulatory elements, suggesting complex regulation of
cdc2 gene expression. The human cdc2 promoter does not con-
tain a classical TATA-like element, although two TTTGAAA
elements, located between 66 and 10 bp upstream of the tran-
scription start site, are present (Dalton, 1992; Ku et al., 1993).
We found that the basal activity of cdc2-TaqI(2359) was
reproducibly higher than that of cdc2-PstI(2722) in cycling
CV-1 cells. This suggests that the region upstream of 2359
nucleotides may have a negative regulatory element such as
the one that is highly homologous to the yin-yang-1 (YY-1; Shi
et al., 1991) binding site (AAAATGT). The YY-1 site at position
2593 to 2587 in the cdc2 promoter lies in a region that is
responsive to cooperative induction by c-Myc and Ha-Ras
v-12
(Born et al., 1994). Further mutational analysis of the cdc2
promoter in our study revealed that, within the 2359 region,
the Sp1, E2F, and two CCAAT box binding motifs are impor-
tant for overall basal activity.
Growth-dependent expression of the human cdc2 gene sug-
gests that it is a likely target for tumor suppressor proteins Rb
and p53 which are negative regulators of proliferation. It is also
a likely target for positive regulators of proliferation such as
cellular protooncogene products. Rb or the Rb-related proteins
p107 and p130 interact physically with the components of the
transcription factor E2F (Bagchi et al., 1991; Chellappan et al.,
1991; Chittenden et al., 1991; Nevins, 1992). The Rb-E2F in-
teraction inactivates the transcription of E2F-dependent genes,
many of which are needed for DNA synthesis. These genes
include those encoding dihydrofolate reductase, thymidine ki-
nase, c-Myc, DNA polymerase
a
, and Cdc2 kinase.
A major breakthrough regarding the function of Rb in the
control of proliferation came from the discoveries that the
transforming proteins of certain DNA tumor viruses target Rb
by physical interaction and inactivate its function. For exam-
ple, SV40-LT (De Caprio et al., 1988), adenovirus E1A (Whyte
et al. 1988), and HPV-E7 (Dyson et al., 1989) proteins bind and
inactivate Rb. E1A induces cell proliferation and the endoge-
nous cdc2 mRNA and p34
cdc2
protein (Draetta et al., 1988;
Wang et al., 1991; Dalton, 1992). Since the E1A protein encoded
by the 12 S mRNA has no transactivation function (for a re-
view, see Moran and Matthews (1987)), but forms a stable
complex with Rb (Whyte et al., 1988) and disrupts Rb-E2F
complexes in vitro (Bagchi et al., 1990; Bandara and La
Thangue, 1991), it was thought that activation of cdc2 pro-
moter by E1A occurred via disruption of Rb-E2F complexes
(Dalton, 1992). SV40-LT and HPV-E7 proteins also act via E2F
binding sites in promoters, and the Rb-binding domains of
SV40-LT and HPV-E7 are critical for this activity (Phelps et al.,
1988; Loeken and Brady, 1989). These results suggest that
E1A, SV40-LT, and HPV-E7 proteins may have a common
FIG. 12. Conditional expression of SV40-LT in human diploid
fibroblasts. IDH-4 cells were grown in the absence and presence of
dexamethasone (2dex and 1dex, respectively) and the lysates were
prepared for Western blot analyses as described under “Experimental
Procedures.” After 7 days, cells grown in the absence of dexamethasone
were induced for 24 h with dexamethasone (2/1dex). The extracts
were fractionated by SDS-PAGE (8%) and subjected to Western blotting
using a mouse monoclonal antibody against SV40-LT or against
b
-tu-
bulin as control. The secondary antibody (horseradish peroxidase-con-
jugated sheep anti-mouse IgG from Amersham Corp.) binding was
detected by a chemiluminescence kit as described under “Experimental
Procedures.”
FIG. 13. Southwestern blot analysis of SV40-LT-inducible
CCAAT box binding factor in IDH-4 cells. The growth conditions of
IDH-4 cells are same as in Fig. 12. Nuclear (N) and cytoplasmic (C)
fractions were prepared as described under “Experimental Procedures.”
Lysates of CV-1 and COS-7 cells were used as negative and positive
controls, respectively.
FIG. 10. The effect of HSP70-CBF on cdc2 promoter. CV-1 cells
were transfected with the plasmid pMT2-CBF (expression plasmid en-
coding HSP70-specific CBF or pCMVex vector plasmid; Agoff et al.
(1993)) and cdc2-PstI (Fig. 1) or HSP70-CAT and the CAT activity
assays were carried out as described in Fig. 4, except that the enzyme
reaction was carried out at 37 °C for 2 h.
FIG. 11. Southwestern analysis of binding of nuclear proteins
from CV-1 cells to CCAAT box motif of cdc2 promoter. Nuclear
proteins were separated on a 8% SDS-PAGE and transferred to nitro-
cellulose membrane. The proteins were renatured and probed with
32
P-labeled CCAAT box motif as described under “Experimental Proce-
dures.” Lanes 1–3,1,2,and3
m
g of total protein from mock-transfected
CV-1 cell lysate. Lanes 4–6,1,2,and3
m
g of proteins from pCMV-TAg-
transfected CV-1 cell lysate. Lanes 7 and 8, 1.4 and 2.8
m
g of COS-7 cell
lysate, respectively.
SV40 T Antigen Transactivates Human cdc2 Promoter 13965
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pathway in transactivation of promoters containing E2F bind-
ing sites.
Our results indicate that the two CCAAT box binding motifs
in the human cdc2 promoter not only contribute significantly to
the overall basal activity of the promoter but also are the major
target for transactivation by SV40-LT. Contrary to our expec-
tations, our results also clearly show that the region containing
the Sp1 and E2F binding sites are not responsive to a signifi-
cant extent to transactivation by SV40-LT in cycling CV-1 cells.
Furthermore, we found little difference between the wild-type
and a mutant SV40-LT defective in Rb binding in their ability
to stimulate cdc2 promoter constructs containing E2F binding
sites. At least one other study showed that E2F-1 failed to
activate a cdc2 promoter-reporter (CAT) gene in human glio-
blastoma (T98G) and osteosarcoma (SAOS-2) cells (Sala et al.,
1994). Moreover, the Rb binding domain of SV40-LT was not
required to immortalize rat lung epithelial cells or elevate
p34
cdc2
and cyclin A levels (Oshima et al., 1993). Our results
show that neither the Rb or p53 binding domains are critical for
SV40-LT activation of the cdc2 promoter.
Previous studies have characterized a nuclear factor that
bound the two inverted CCAAT box binding elements (2146 to
2121 and 282 to 256) present in the Rous sarcoma virus long
terminal repeat (RSV-LTR) promoter, and addition of serum to
serum-deprived rat 3Y1 fibroblast cells increased the level of
this factor (Dutta et al., 1990; Maity et al., 1988). This nuclear
factor also bound the CCAAT box elements in the promoters of
the human heat shock protein 70 (HSP70), c-Ha-ras, and his-
tone H1 genes, but not the CCAAT box in the adenovirus type
2/5 origin of DNA replication (which is recognized by NFI/CTF
family; see Santoro et al. (1988)), or the SV40 enhancer core
sequence (recognized by CCAAT enhancer-binding protein
C/EBP; Landschultz et al., 1988). The CCAAT box binding
factor (CBF) that controls transcription from the HSP70 pro-
moter has been cloned; it encodes a 114-kDa polypeptide that
binds the CCAAT box element (Lum et al., 1990). From the
results of Dutta et al. (1990), it appears that the serum-induced
factor that recognizes the CCAAT box elements in RSV-LTR,
c-Ha-ras, and histone H1 promoters is either related to, or
likely to be the same as, the 114-kDa CBF that binds the
HSP70 promoter (CBF/HSP70).
Our evidence suggests that CBF/cdc2 differs from CBF/
HSP70. On the other hand, CBF/cdc2 may be related to the
factor (CCAAT box-binding protein; CBF/tk) that is induced by
serum in presenescent human diploid fibroblast (IMR-90) cells
but absent in senescent cells (Pang and Chen, 1993). CBF/tk
recognizes two inverted CCAAT box motifs in the human thy-
midine kinase (tk) gene (Pang and Chen, 1993). The biochem-
ical properties of this factor (CBF/tk), such as sensitivity to
heat, metal chelating agents, and protein synthesis inhibitors,
suggest that it is a distinct member of the CCAAT box-binding
protein family that could play a role in the cell cycle- and
senescence-dependent regulation of TK gene expression (Pang
and Chen, 1993). Interestingly, SV40-LT extends the limited
proliferative life span of human diploid fibroblasts, and induces
DNA synthesis in quiescent and senescent cells without induc-
ing mitosis (Gorman and Cristofalo, 1985; Shay and Wright,
1989; Wright et al., 1989) (for reviews, see Goldstein (1990),
Peacocke and Campisi (1991), Campisi et al. (1996), and refer-
ences therein). SV40-LT has also been shown to induce DNA
synthesis, p34
cdc2
kinase, and Rb phosphorylation in quiescent
baby rat kidney cells (Wang et al., 1991). The results of our
study indicate that SV40-LT mediates its activation of the
human cdc2 gene through induction of a specific CBF (CBF/
cdc2) in immortal monkey kidney cells (CV1, COS) and revers-
ibly immortal human fibroblasts (IDH4), which is distinct from
the CBF/HSP70. Therefore, it will be interesting to determine
the levels of the CBF/cdc2 in senescent and quiescent human
diploid fibroblasts.
Acknowledgments—We thank Dr. Bruno Calabretta for providing the
cdc2-PstI and cdc2-7 plasmids, Dr. Bruce Howard for providing the
pCMV-TAg and pCMV-TAg-Rb
2
plasmids, Dr. Thomas Shenk for pro-
viding the pTI-CAT plasmid, and Dr. Barbara Wu for providing the
CBF/HSP70 cDNA, anti-CBF/HSP70, and HSP70 promoter-CAT
plasmid.
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SV40 T Antigen Transactivates Human cdc2 Promoter 13967
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Haifeng Chen, Judith Campisi and R. Padmanabhan
CCAAT Box Binding Factor Promoter by Inducing acdc2SV40 Large T Antigen Transactivates the Human
doi: 10.1074/jbc.271.24.13959
1996, 271:13959-13967.J. Biol. Chem.
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