From the Department of Molecular Biology, Lerner
Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio
44195 and the § Department of Molecular Microbiology and
Immunology, and K. Norris, Jr. Comprehensive Cancer Center, University
of Southern California, Los Angeles, California 90033-1034
Received for publication, June 13, 2000, and in revised form, October 11, 2000
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ABSTRACT |
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p53 represses the transcription of
cdc2 and cyclin B1, causing loss of Cdc2
activity and G2 arrest. Here we show that the region The p53 tumor suppressor protects mammals from neoplasia by
eliminating cells containing damaged DNA through apoptosis or cell
cycle arrest (1, 2). p53-dependent arrest in response to
DNA damage occurs in both G1 and G2 phases of
the cell cycle (3-6). p53 also controls progression through S phase
when nucleotide pools are out of balance, thus avoiding DNA damage, and
inhibits entry into mitosis when DNA synthesis is blocked (7-9).
Damaged or unreplicated DNA, imbalances in nucleotide pools, or hypoxia cause the p53 protein to accumulate and activate its ability to bind to
promoters, activating transcription of the genes that control several
cellular responses to stress (1, 2, 9, 10). For example, p53 causes
G1 arrest in part by stimulating transcription of the
cyclin-dependent kinase
(CDK)1 inhibitor p21/waf1,
thus reducing the activities of CDK2, 4 and 6, required for progression
from G1 into S phase (11-14).
Whereas p53 is sufficient for G1 arrest, multiple
mechanisms mediate G2 arrest in response to DNA damage (3,
15). G2 arrest occurs in cells lacking p53, possibly
because of activation of the phosphatidylinositol 3-kinase family
member Atm, which phosphorylates and activates Chk1 and Chk2 (16, 17),
which in turn phosphorylate the phosphatase Cdc25 (18). Cdc25 normally activates Cdc2, the CDK required for entry into mitosis, by
dephosphorylating tyrosine 15 and threonine 14 (19, 20), the sites of
inhibitory phosphorylations catalyzed by Wee1 and Myt1, respectively
(21-25). Phosphorylation of Cdc25 by Chk kinases creates a binding
site for 14-3-3 proteins (18), which in fission yeast and
Xenopus embryos anchor Cdc25 in the cytoplasm, thus blocking
its ability to activate Cdc2 (26, 27).
G2 arrest is not maintained in irradiated cells lacking
either p53 or p21/waf1 (6). Several effects of p53 probably ensure prolonged arrest in G2. Overexpression of p53 arrests
fibroblasts in G2 (4, 5) with low levels of Cdc2 activity
(28). Cdc2 is normally regulated by phosphorylation, by its binding to
Cyclin B, and by nuclear localization (29, 30). p53 can inhibit the CDK-activating kinase, which activates Cdc2 by phosphorylating threonine 161 (31, 32). p21/waf1 associates with Cdc2/Cyclin B1 in
cells arrested by p53 in G2, suggesting an additional layer of inhibition (28). In addition, Gadd45 induced by p53 can inhibit Cdc2
activity by disrupting its binding to Cyclin B1 (33, 34). In epithelial
cells, p53 induces the expression of 14-3-3 Repression of the transcription of several genes by p53 contributes to
G2 arrest. Topoisomerase II orchestrates the higher order
compaction of chromatin that is required to form mitotic chromosomes
(37). Inhibition of topoisomerase II blocks cells in G2
because chromatin is not condensed and decatenated (38). p53 represses
the topoisomerase II promoter, which may contribute to G2
arrest (39, 40). Repression of the cyclin B1 promoter by p53
is an important factor in the inhibition of Cdc2 (28, 41). Furthermore,
in some cell types overexpression of Cyclin B1 alone can abrogate
p53-induced G2 arrest (41). p53 also represses the
transcription of cdc2 (15, 28), and the CCAAT element is
involved in this process (42). Also, down-regulation of the level of
Cdc2 protein in response to p53 requires p21/waf1 and can be abrogated
by the E7 protein of human papilloma virus (43). E7 binds to several
cellular proteins, including those of the Rb family (44-48). In the
present study, we demonstrate that p53 represses the cdc2
promoter through a mechanism involving induction of p21/waf1 and
binding of the Rb family protein p130 and the transcription factor E2F4
to the CDE and CHR elements within the R box of the cdc2 promoter.
Cell Lines and Culture Conditions--
Cells were grown in
Dulbecco's minimal essential medium (Life Technologies, Inc.),
supplemented with antibiotics and 10% fetal bovine serum (Life
Technologies, Inc.) in a humidified atmosphere containing 10%
CO2. The TR9-7 cell line, expressing a
tetracycline-regulated p53 cDNA, was derived from MDAH041 cells
(4). Cells were arrested in G2 in response to p53 as
described by Taylor et al. (28). Briefly, mimosine (Sigma),
which arrests cells reversibly at the beginning of S-phase (49), was
added at a final concentration of 200 µM to
asynchronously growing TR9-7 cells for 48 h. The cells were
released from the mimosine block and simultaneously deprived of
tetracycline to induce p53-mediated arrest, mainly in G2
(4). Recombinant adenoviruses, provided by Joseph Nevins (Duke
University, Durham, NC; Ref. 50), were amplified by infection of HEK293
cells (American Type Culture Collection, Manassas, VA). Cell
supernatant fluids were collected 48 h later, and debris was
removed with a 0.2-µm filter. Viral titers were determined by
infecting confluent monolayers of HEK293 cells for 2.5 h, removing the medium that contains the virus, and overlaying the cells with a
solution of 1% low melting agarose (FMC Bioproducts, Rockland, ME) in
Dulbecco's modified Eagle medium plus 10% fetal bovine serum.
Infected cells were identified 72 h later by immunofluorescence with an antibody specific for the adenovirus hexon protein (Biodesign International, Kennebunk, ME).
Western Analysis--
Extracts were prepared by lysing cells in
50 mM HEPES, pH 7.0, 250 mM NaCl, 0.1%
Nonidet P-40, 10% glycerol, 1 mM phenylmethanesulfonyl fluoride (PMSF), 2 µg/ml aprotinin, 25 µg/ml leupeptin, 5 µg/ml pepstatin A, and 1 mM dithiothreitol (DTT). Extracts
containing equal quantities of proteins, determined by the Bradford
method (Bio-Rad), were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (12.5% acrylamide) and
transferred to polyvinylidene difluoride membranes (Millipore, Bedford,
MA). Membranes were probed with monoclonal antibodies specific for p53
(DO-1), actin (C-2), and rabbit polyclonal antibodies specific for Cdc2
(C-19), p130 (C-20), Rb (C-15), p107 (C-18), or E2F4 (C108), all from Santa Cruz Biotechnology (Santa Cruz, CA). Bound antibodies were detected with goat anti-mouse or goat anti-rabbit antibodies conjugated to horseradish peroxidase (Hoffman-La Roche, Basel, Switzerland), using
enhanced chemiluminescence (DuPont) (4).
Reporter Constructs--
cdc2 promoter activities
were determined by measuring luciferase activity in pools of TR9-7
cells stably transfected with constructs containing regions of the
cdc2 promoter driving the expression of luciferase (51).
Chimeric constructs were described by Sugarman et al. (51).
Mutations were generated using the polymerase chain reaction and
confirmed by DNA sequencing. Luciferase activity in extracts of stable
pools was corrected for total protein concentrations in each lysate,
determined by the Bradford method. Luciferase activity in transient
transfections was corrected for transfection efficiency, determined by
cotransfection of a plasmid constitutively expressing
Gel Mobility Shift Analysis--
Complexes capable of binding to
a consensus E2F site were detected as described by Nevins et
al. (52). Briefly, cells were lysed in 10 mM HEPES, pH
7.9, 60 mM KCl, 1 mM EDTA, 0.1 mM
EGTA, 0.075% Nonidet P-40, 1 mM NaVO4, 5 mM NaF, 1 mM DTT, 1 mM PMSF, 2 µg/ml aprotinin, 25 µg/ml leupeptin, and 5 µg/ml pepstatin A for
15 min on ice. Nuclei, collected by centrifugation at 1300 × g at 4 °C for 15 min, were lysed in 50 mM
HEPES, pH 7.9, 250 mM KCl, 0.2 mM EDTA, 0.2 mM EGTA, 0.1% Nonidet P-40, 10% glycerol, 4 mM NaVO4, 4 mM NaF, 1 mM DTT, 1 mM PMSF, 2 µg/ml aprotinin, 25 µg/ml leupeptin, and 5 µg/ml pepstatin A for 30 min on ice. Debris
was removed by centrifugation at 16,000 × g at 4 °C
for 30 min. Protein concentrations were determined by the Bradford method. Binding reactions contained 2 µl of lysate (3-4 µg of protein), 1 µl of sonicated salmon sperm DNA (0.5 mg/ml), 1.4 µl of
5× Shift buffer (100 mM HEPES, pH 7.9, 200 mM
KCl, 30 mM MgCl2, 0.5% Nonidet P-40, 5 mM DTT, and 5 mM PMSF), 1.5 µl of bovine
serum albumin (1 mg/ml), 3.8 µl of H2O, and 0.3 µl of
probe end-labeled with T4 polunucleotide kinase and
[
To detect proteins binding to the cdc2 R box, gel mobility
shift assays were carried out as described previously, with an oligonucleotide spanning this region (54). Briefly, cells were lysed
for 20 min on ice in buffer I, containing 10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.3 M sucrose, 0.1 mM EGTA, 0.5% Nonidet P-40, 0.5 mM DTT, 0.5 mM PMSF, 1 mM
NaVO4, 2 µg/ml aprotinin, 25 µg/ml leupeptin, and 5 µg/ml pepstatin A. Nuclei were collected by centrifugation at
3000 × g for 10 min at 4 °C. Cells were resuspended in buffer I without Nonidet P-40 and immediately centrifuged at 3000 × g for 10 min at 4 °C. Nuclei were lysed for
20 min on ice in buffer II, containing 20 mM HEPES, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.1 mM EGTA 0.5 mM DTT, 0.5 mM PMSF, 1 mM
NaVO4, 2 µg/ml aprotinin, 25 µg/ml leupeptin, and 5 µg/ml pepstatin A. Lysates were centrifuged at 16,000 × g at 4 °C for 30 min and dialyzed against 50 volumes of
buffer III, containing 20 mM HEPES, pH 7.9, 20% glycerol,
100 mM KCl, 0.2 mM EDTA, 0.5 mM
DTT, and 0.5 mM PMSF. Lysates were again centrifuged at
16,000 × g at 4 °C for 30 min and frozen in
aliquots at Repression of the cdc2 Promoter by p53 Requires a Region Proximal
to the Start of Transcription--
We have studied regulation of the
cdc2 promoter by p53 using the TR9-7 cell line, which
contains tetracycline-regulated p53 (4). Luciferase constructs linked
to regions of the cdc2 promoter were transfected into TR9-7
cells, and pools of clones in which the reporter constructs had stably
integrated into the genome were analyzed. A reporter construct
containing cdc2 promoter sequences from
To test whether the R box is important for repression by p53, several
chimeric constructs were stably transfected into TR9-7 cells (Fig.
1). Cells were arrested in G2
before analysis of luciferase activity (28). The region
The R box contains several DNA sequence elements that regulate the
transcription of the cdc2 and other promoters. The cell cycle-dependent element (CDE) is 4 bp upstream from the
cell cycle gene homology region (CHR) (55). Mutation of these elements in the cdc25c, cyclin A, and cdc2
promoters relieves the repression that normally occurs upon arrest of
the cells in G0 (55). The CDE in the cdc2
promoter overlaps a putative E2F-binding site. Binding of p130/E2F4 to
this element was correlated with repression of the promoter in
G0-arrested cells (54). The factor CDF-1 can also bind to
the CDE/CHR elements of the cdc2 promoter (56). We tested
the importance of these elements for repressing the cdc2
promoter by p53. Constructs containing mutations in either the CDE or
CHR were transfected into TR9-7 cells, and stable pools were incubated
in the presence or absence of tetracycline to induce expression of p53.
The wild-type promoter was repressed 5.6-fold by p53 (Fig.
2A). Mutation of either the
CDE or CHR elements substantially inhibited repression by p53. The
promoter with mutations in the CDE was repressed 2.3-fold by p53 (Fig.
2A). Similarly, mutations in the CHR reduced repression to
2.2-fold (Fig. 2A). A comparison of the sequences in the
chimeric and mutant promoters revealed several important points.
Replacement of the first 5 bp of the putative E2F site, without
altering the CDE, did not substantially affect repression (Figs. 1,
A and B, and 2B, construct
PCP), whereas mutations in the CHR that maintain the putative E2F
site caused a loss of repression (Figs. 1A and 2,
constructs CPC and mCHR). Mutations in the CDE
that disrupt the putative E2F site also caused a loss of repression
(Fig. 2, construct mCDE). These analyses localized
repression of the cdc2 promoter by p53 to two distinct elements.
The R Box of the cdc2 Promoter Can Mediate
p53-dependent Repression of Heterologous Promoters--
We
placed the region Role of p21/waf1 in Repressing the cdc2 Promoter in Response to
p53--
Ionizing radiation causes down-regulation of cdc2
mRNA in a p53- and p21/waf1-dependent manner (57, 58).
Our results show that overexpression of p53 without exogenous
activation can repress the cdc2 promoter, suggesting that
the down-regulation of cdc2 mRNA caused by DNA damage is
due to repression of its promoter by p53. We tested whether promoter
repression was dependent on p21/waf1 by using a p21/waf1-null HCT116
colorectal tumor cell line, which contains wild-type p53 (59). Parental
and p21/waf1-null cells were transfected transiently with a construct
containing cdc2 promoter sequences up to
Constructs containing either the wild-type cdc2 promoter
(CC) or the prolactin promoter chimera (PP) were transfected stably into parental and p21/waf1-null HCT116 cells. cdc2 promoter
activity was reduced 2.6-fold upon treatment of the parental cells with adriamycin, a DNA damaging agent known to induce functional p53 in
these cells (Fig. 4B and Ref. 59). cdc2 promoter
activity was reduced only 1.5-fold upon treatment of p21/waf1-null
cells with adriamycin (Fig. 4B). Transcription driven by the
prolactin chimera was not affected substantially after treatment of
either cell type with adriamycin (Fig. 4B). Therefore,
repression of the cdc2 promoter in response to DNA damage
requires p21/waf1. We tested whether p21/waf1 was sufficient for
repression of the cdc2 promoter by using a recombinant
adenovirus in which the expression of p21/waf1 is driven by a
constitutive cytomegalovirus promoter (50).2 A pool of
TR9-7 cells transfected stably with a construct containing the region
up to E2F4/p130 Binds to the R Box of the cdc2 Promoter to Mediate
Repression by p53--
We next investigated which downstream targets
of p53 and p21/waf1 might mediate repression of the cdc2
promoter. p130 and E2F4 bind to the putative E2F site that overlaps the
CDE of this promoter and have been implicated in the repression
observed in G0 and G1 (Ref. 54 and Fig.
2B). p21/waf1 can inhibit the ability of Cyclin/CDK
complexes (12) to phosphorylate the Rb family members pRb, p107 and
p130 (60, 61). Complexes of Rb family members with E2F/DP heterodimers
repress the transcription of genes containing E2F-binding sites (62).
Phosphorylation of Rb family members by CDKs blocks their interaction
with E2Fs (60). Therefore, p53 might repress the cdc2
promoter by inducing p21/waf1 which, by inhibiting CDKs, causes Rb/E2F
complexes to accumulate. These might bind to the R box and repress
transcription. We tested the requirement of various Rb family members
in down-regulating Cdc2 in response to DNA damage. Mouse embryo
fibroblasts (MEFs), derived from mice in which Rb, p107, p130, or both
p107 and p130 were inactivated by gene targeting (63), were treated
with adriamycin for 24, 48, or 72 h to induce functional p53.
Western analysis of Cdc2 protein levels indicated that Cdc2 was
down-regulated efficiently in MEFs from wild-type, heterozygous, and
p107- or p130-null mice (Fig. 5).
However, the down-regulation of Cdc2 was less efficient in Rb-null
cells and was highly defective in cells lacking both p107 and p130
(Fig. 5). Therefore, either Rb or p107 or p130 are required for
efficient Cdc2 down-regulation in response to DNA damage in MEFs.
To test the importance of Rb family members and E2F proteins in
repressing the human cdc2 promoter, we analyzed TR9-7 cells for expression of the relevant proteins. Rb, p107, and p130 were detected in TR9-7 cells by Western blotting and the electrophoretic mobility of all three proteins was increased following induction of p53
(Fig. 6A), consistent with
inhibition of CDKs in response to p53 overexpression, because
phosphorylation of Rb family proteins by CDKs decreases their mobility
(for example see Ref. 64). Similarly, removal of serum, which reduces
CDK activity, also increased the mobility of Rb, p107, and p130 (Fig.
6A). Removal of serum caused a reduction in the level of Rb
and p107 but an increase in the amount of p130 compared with the levels
in asynchronously growing cells (Fig. 6A). TR9-7 cells also
contain E2F4, which has been implicated in suppressing cdc2 promoter
activity (Fig. 6A and Ref. 54). Removal of serum caused an
increase in the intensity of more slowly migrating bands, detected with
the E2F4 antiserum. These bands may represent phosphorylated species
(65). Gel mobility shift analysis using a probe containing a consensus E2F-binding site was used to characterize E2F-binding complexes in
TR9-7 cells. We focused on E2F4 and p130, given their role in
regulating the human cdc2 promoter (54). Several complexes containing p130 and E2F4 in TR9-7 cells were detected before and after
induction of p53 (Fig. 6B). p53 induction changed the
complement of E2F complexes, consistent with CDK inhibition and changes
in the phosphorylation of Rb family members and in their affinities for
E2Fs (Fig. 6B). Thus, TR9-7 cells contain E2F4 and p130
complexes capable of binding to a well defined E2F site.
Gel mobility shift analysis with a probe derived from the R box was
used to test whether Rb/E2F complexes were capable of binding to the
putative E2F site in the cdc2 promoter. p53 induced the
binding of a complex to the R box that could be supershifted with
antibodies to either E2F4 or p130 (Fig.
7, A and B). The antibody to p130 cross-reacts with p107. However, an antibody to p107
did not supershift the p53-induced complex, suggesting that the complex
contains p130 and not p107 (Fig. 7B). An antibody to pRb did
not supershift the p53-induced complex (Fig. 7B). The p53-induced complex was reduced by a 100-fold molar excess of wild-type
unlabeled probe. However, unlabeled probes containing mutations in
either the CDE or CHR that impaired repression by p53 could not
efficiently compete with the labeled probe (Figs. 2A and
7C). These results suggest that p53 induces a complex on the
cdc2 promoter containing p130 and E2F4 and that formation of
this complex requires the CDE and CHR elements.
The arrest of mammalian cells in G2 in response to
damaged DNA is ensured by multiple p53-dependent and
-independent pathways (15, 28). p53 represses the transcription of
cyclin B1 and cdc2 and induces p21/waf1, Gadd45,
and 14-3-3 p53 Represses Transcription by Several Distinct
Mechanisms--
Repression of the transcription of a large number of
genes by p53 occurs through several distinct mechanisms. p53 can bind to TATA-binding protein-associated factors to activate the
transcription of genes containing p53-binding sites (67). At the same
time, high levels of p53 bound to TATA-binding protein-associated
factors block their interaction with the TATA regions of genes that do not contain p53-binding elements, resulting in transcriptional repression (68-70). This squelching mechanism can also affect genes that do not contain TATA regions, presumably because TATA-binding protein-associated factors are still required for the transcription of
those genes (71). Similarly, p53 can bind to TFIIIB, a TATA-binding protein-containing complex that is specifically required by RNA polymerase III, resulting in repression of the transcription of the
tRNA, 5 S rRNA, and U6 snRNA genes (72). Also, repression of AP1-driven
transcription by p53 requires binding of p53 to the p300 histone
acetyltransferase. This event blocks the binding of p300 to
c-Jun at the AP1 site, which is normally required for transcriptional activation (73).
Repression can also involve interference by p53 with sequence-specific
transcriptional activators. The Repression of the cdc2 Promoter by p53 Requires the CDE and CHR
Elements--
p53 represses the cdc2 promoter by inducing
p21/waf1, which inhibits CDKs, resulting in the binding of a complex
containing p130/E2F4 to the cdc2 promoter. Repression of
cdc2 requires the CDE and CHR elements. The
cdc25c, cyclin A, and cdc2 genes
contain CDE and CHR elements and are repressed during G0
and G1 (55). Repression during G0 and
G1 correlates with the binding of the factor CDF-1 to the
CDE and CHR elements (55). Mutations in CDE or CHR cause loss of CDF-1
binding and abolish repression (56). Thus, it has been postulated that
CDF-1 is a repressor of transcription. Supershift analyses suggest that
CDF-1 does not contain E2F or Rb proteins (56). Purification and
cloning of CDF-1 will allow assessment of the mechanism used to repress transcription. The factor CHF can bind to the CHR element of the cyclin A but not cdc2 nor cdc25c
promoters. The relationship of CHF to CDF-1 is not known (79). We have
found that repression of the cdc2 promoter requires both the
CDE and CHR elements, which mediate the binding of a p53-inducible
complex containing E2F4 and p130. Consistent with our results, DNA
damage leads to repression of the cdc2 promoter in a manner
dependent on both the CDE and CHR elements (80). Repression was
observed in WI38, HT1080, and HCT116 cells, all of which contain
wild-type p53, but not in HeLa cells in which p53 is inactivated by the
human papilloma virus E6 protein. Our results suggest that the
repression of cdc2 by DNA damage depends on p53 and not on
other factors that are defective in HeLa cells. We observed that the
cdc2 promoter was partially repressed even after mutation of
the CDE or CHR elements. These mutations may not completely abolish
E2F4/p130 binding in vivo, or p53 may target other elements
in the cdc2 promoter to cause complete repression.
p21/waf1 and Rb Family Members Are Essential for Repression of cdc2
by p53--
Mouse fibroblasts lacking p130 show normal down-regulation
of Cdc2 protein after DNA damage. However, Cdc2 was not down-regulated in cells after deletion of both p130 and p107, and down-regulation was
less efficient in cells lacking Rb. These results suggest that all
three Rb family members play overlapping roles in the repression of
cdc2 by p53 in mouse cells. Using gel mobility shift analysis with extracts of human cells, we did not detect p107 or Rb
bound to the R box, suggesting that the p53-dependent
repression of the human promoter may be more specific in its
requirement for p130. Alternatively, the interaction of Rb and p107
with the human cdc2 promoter may not be detectable by gel
mobility shift analysis under the conditions employed. It is also
possible that Rb and p107 contribute to Cdc2 down-regulation by a
mechanism that does not involve their binding to the cdc2 promoter.
When CDKs are inactive, Rb family proteins are hypophosphorylated. In
this state they can bind to E2F/DP complexes, which target them to
E2F-binding sites. Binding to Rb family members converts E2F/DP from a
sequence-specific transcriptional activator to a sequence-specific
repressor (62). Repression is at least partly dependent on the ability
of Rb proteins to recruit histone deacetylases, which inhibit
transcription through their effects on chromatin condensation (81, 82).
Trichostatin A, an inhibitor of histone deacetylases, only partially
relieved repression of the cdc2 promoter by
p53,2 suggesting that other mechanisms may also be
important and consistent with the ability of p130 to interact with the
CtIP/CtBP corepressor that has been postulated to function
independently of histone deacetylases (83). E2F/Rb complexes have been
implicated in the cell cycle-regulated expression of cdc25c.
The E2F-binding site mediating this effect is distinct from the CDE/CHR
element (84).
We found that the CDE of the cdc2 promoter is required for
repression by p53 and for binding of E2F4/p130. E2F sites contain the
consensus TTTGCGCGC sequence, whereas in the cdc2 CDE
region, the T triplet is immediately followed by A (85). van der
Waal's contacts between tyrosine 124 of E2F4 and the cytosine after
the T triplet stabilize binding of E2F4/DP2 to DNA (85). Replacement of
cytosine with adenine is expected to disrupt these interactions and may
impair DNA binding. Furthermore, we found that replacement of the T
triplet with the sequence AAA reduced repression by p53 only slightly
(Fig. 2B). The T triplet interacts with arginine 17 of E2F4,
suggesting that the replacement we have made might also impair the
binding of an E2F/DP heterodimer to this site (85). These results
suggest that E2F4/p130 might bind relatively weakly to the CDE.
Furthermore, mutations in the CHR 6 bp downstream of the putative E2F
site caused a loss of E2F4/p130 binding and greatly reduced repression
by p53. E2F4/DP2 appears not to make specific base contacts at this
location downstream from the core binding site (85). These results
suggest that other proteins perhaps bound to the CHR might stabilize
binding of E2F4/p130 to the noncanonical E2F site in the CDE region of
the cdc2 promoter.
The R box mediates repression by p53 when placed downstream of SP1 or
AP1 sites, suggesting that the E2F4/p130-containing complex induced by
p53 does not specifically target NF-Y. However, interference of p53
with SP1 or AP1 could also contribute to the repression of these
constructs. We also observed that repression mediated by the R box
depended on p21/waf1, consistent with the ability of p21/waf1 to
inhibit CDKs, which is expected to stimulate the formation of E2F4/p130
complexes. cdc2 mRNA is suppressed upon overexpression
of p21/waf1 (86), consistent with our result that p21/waf1
down-regulates the cdc2 promoter. p53 was much more efficient in repressing the cdc2 promoter in experiments
using transient transfection compared with experiments using pools of cells with stably integrated cdc2 reporter constructs and
tetracycline-regulated p53. This effect may be due to higher levels of
p53 expression and the failure of the reporter construct to integrate
into the chromosomes in the transient assay, which may make it more
susceptible to repression. Using transient transfections we also
observed that a cdc2 chimeric construct lacking the R box
was still repressed, although at a lower level. This effect was
dependent on p21/waf1, suggesting that high levels of p21/waf1 may
affect the cdc2 promoter independently of the R box.
Physiological Relevance of Repression of cdc2 by p53--
The
down-regulation of Cdc2 protein by p53 is delayed compared with the
down-regulation of Cyclin B1, suggesting that the repression of
cdc2 may contribute only partially to the initial arrest
(28). However, the down-regulation of Cdc2 may be more important for
the stability of the arrest. For example, cell cycle checkpoints in
yeast that block entry into mitosis in response to DNA damage (87)
eventually become attenuated, and the cells eventually do enter
mitosis, presumably by reactivating Cdc2, without having repaired the
damaged DNA (88). Continued proliferation of unicellular organisms may
be more important than eliminating all cells with damaged DNA (88). In
mammals, a single cell with damaged DNA may develop into a neoplasm.
One way to ensure that damaged mammalian cells do not continue to
proliferate even if checkpoint pathways become attenuated is to
eliminate the proteins required for entry into mitosis. Mammals may
utilize p53 to repress the cdc2 promoter for this reason.
22
to
2 of the cdc2 promoter called the R box is required for repression by p53 but not for basal promoter activity. The R box
confers p53-dependent repression on heterologous promoters and binds to p130/E2F4 in response to overexpression of p53. R box-dependent repression requires p21/waf1, and
overexpression of p21/waf1 also represses the cdc2
promoter. These observations suggest that p53 represses the
cdc2 promoter by inducing p21/waf1, which inhibits
cyclin-dependent kinase activity, enhancing the binding of
p130 and E2F4, which together bind to and repress the cdc2 promoter.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, which anchors Cdc2 in
the cytoplasm, where it is unable to phosphorylate substrates
required for entry into mitosis (35, 36).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase.
-32P]ATP. The probe was ATTTAAGTTTCGCGCCCTTTCTCAA.
Protein-DNA complexes were separated by electrophoresis at 4 °C in
gels composed of 4% acrylamide:bisacrylamide (29:1), 0.5× TBE (45 mM Tris-HCl, 44 mM boric acid, and 1 mM EDTA), and 5% glycerol (53).
80 °C. Binding reactions contained 8 µl of lysate
(8-10 µg of protein), 1 µl of sonicated salmon sperm DNA (0.5 mg/ml), 8 µl of buffer III with 1.5 mM MgCl2,
1.0 µl of end-labeled probe and 7 µl H2O. The probe was
CCGGGGCCCTTTAGCGCGGTGAGTTTGAAACTGCT. Protein-DNA complexes were
separated by electrophoresis at 4 °C using gels composed of 6%
acrylamide:bisacrylamide (75:1), 0.5× TAE (20 mM Tris
hydrochloride, 10 mM acetic acid, and 1 mM
EDTA), and 5% glycerol (53).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
94 to +75 was
repressed ~3-fold by p53.2
However, a construct extending from
74 to +75 was not sufficient for
basal promoter activity and was not repressed by p53 (28). A CCAAT
element between
94 and
74 binds to the NF-Y transcriptional activator (42), suggesting that repression of the cdc2
promoter by p53 requires NF-Y. Because the CCAAT element is also
required for basal activity of this promoter, it was possible that loss of repression by deletion of the CCAAT element might be due to a loss
of basal promoter activity. For example, the construct extending to
74 might not be repressed by p53 because basal level activity is
~40-fold lower, with repression obvious only when promoter activity
is higher (28, 42). The
94 to +75 region of the cdc2
promoter contains an additional regulatory region, called the R box
(51), immediately upstream of the start of transcription. The R box is
required for repression of the cdc2 promoter by phorbol
ester (51).
36 to +25
containing the R box was replaced with a region from the prolactin
promoter from the same position relative to the start of transcription
(51). Whereas the wild-type cdc2 promoter construct CC was repressed
4.4-fold, replacement of the R box region with prolactin sequences (the PP construct) completely eliminated repression by p53 (Fig. 1, A and B). Importantly, this replacement did not
substantially affect the basal activity of the promoter, suggesting
that the swapped region is required for repression specifically (Fig.
1A). Construct PCP generated by inserting 21 bp of the
cdc2 promoter into the prolactin region, was repressed
3.8-fold by p53 (Fig. 1, A and B). However,
construct CPC in which 10 bp from the prolactin promoter were inserted
into the cdc2 promoter, showed a smaller repression of
2.0-fold (Fig. 1, A and B). These experiments
localized the minimal region required for repression to the R box,
located at nucleotides
22 to
2.
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Fig. 1.
Repression of cdc2/prolactin
promoter chimeras by p53. A, luciferase activity after
G2 arrest induced by p53 overexpression. Stable pools of
cells containing the promoter chimeras shown in B and
C were released from a mimosine block in the presence or
absence of tetracycline (TET). Removal of tetracycline
induces the expression of high levels of p53, which causes cell cycle
arrest mainly in G2. Luciferase activity was measured
48 h after removal of mimosine. Rlu: relative light units.
Standard errors are shown by the bars. B,
schematic diagrams of chimeras and average fold repression from two
independent experiments (data shown in A). C,
sequences of chimeric promoter constructs in the region of the R
box.
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Fig. 2.
Effects of mutations in the CDE and CHR
elements on repression of the cdc2 promoter by
p53. A, fold repression of wild-type and mutant
promoters. Mutations were generated in a cdc2 promoter
fragment extending to 94 relative to the start of transcription.
Wild-type and mutant reporter constructs were stably transfected into
TR9-7 cells. Asynchronously growing pools of cells were incubated in
the presence or absence of tetracycline for 72 h, followed by
measurement of luciferase activity. Standard errors were less that 10%
of the mean in all experiments. mCDE, mutant CDE;
mCHR, mutant CHR. B, details of mutations in the
cdc2 promoter and comparison with the relevant regions of
the chimeric promoters shown in Fig. 1.
74 to +75 of the cdc2 promoter, which
contains the R box downstream of two different elements capable of
stimulating transcription. Protein(s) bound to the R box might interact
specifically with NF-Y bound to the CCAAT box between
74 and
94,
thereby blocking the ability of NF-Y to activate transcription.
Alternatively, the R box might bind to a repressor capable of blocking
several different types of upstream activators. TR9-7 cells were stably
transfected with constructs containing the R box downstream of binding
sites for either AP1 or SP1, two efficient activators of transcription.
AP1- and SP1-driven transcription was reduced by 2.5- and 3.0-fold by
p53, respectively (Fig. 3). Wild-type
cdc2 promoter sequences extending to
94 were used as a
positive control. Luciferase activity driven by the cdc2
promoter was reduced by 3.3-fold by p53 (Fig. 3). These observations
suggest that NF-Y is not specifically targeted by the protein(s) bound to the R box. Instead, the repressor recruited to the R box in response
to p53 blocks transcription induced by three different transcriptional
activators. Also, the ability of the R box to inhibit transcription
driven by upstream activators may explain why deletion of the CCAAT box
eliminates repression (28, 42).
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Fig. 3.
Effects of the R box from the cdc2
promoter on different transcriptional activators. The R box
was placed downstream of either three AP1 sites or one SP1 site, and
TR9-7 cells were stably transfected with these constructs.
cdc2 promoter sequences extending to 94 were used as a
positive control. Luciferase activity was measured after incubation of
asynchronously growing pools of cells in the presence or absence of
tetracycline for 48 h. Standard errors are shown by the
bars.
245 linked to
luciferase (Fig. 1B, CC) and a plasmid expressing
p53 under the control of the constitutive cytomegalovirus promoter. As
a negative control, we used a reporter construct in which prolactin
sequences replaced the R box (Fig. 1B, PP).
Overexpression of p53 repressed the cdc2 promoter by
60-70-fold in parental HCT116 cells (Fig.
4A). Deletion of p21/waf1
substantially impaired the ability of p53 to repress the
cdc2 promoter; however, repression by 10-fold was still
observed (Fig. 4A). The prolactin promoter chimera was
repressed by 10-fold in parental cells but not in p21/waf1-null cells
when transfected with 0.5 µg of the plasmid expressing p53 (Fig.
4A). The fold repression of cdc2 by p53 in
transient assays was much higher than in pools of TR9-7 cells stably
transfected with cdc2 reporter constructs, probably because
of differences in the levels of expression of p53 and differences in
chromatin structure of the reporter construct.
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Fig. 4.
Role of p21/waf1 in repression of the
cdc2 promoter by p53. A, HCT116 cells
in which p21/waf1 was inactivated by gene targeting were transfected
transiently with various amounts of a p53 expression plasmid and either
the cdc2 promoter (CC) or prolactin chimeric
(PP) reporter constructs described in Fig. 1. Luciferase
activity measured 48 h after transfection was corrected for
transfection efficiency by using a cotransfected -galactosidase
construct. A representative experiment is shown. B, role of
p21/waf1 in repression of the cdc2 promoter in response to
DNA damage. HCT116 cells with or without p21/waf1 were transfected
stably with the CC or PP reporter constructs. Stable pools of cells
were treated with adriamycin to induce DNA damage, followed by
measurement of luciferase activity 48 h later. Standard errors
were less than 15% of the mean in all experiments. C,
effects of overexpression of p21/waf1 on the cdc2 promoter.
A pool of TR9-7 cells stably transfected with a reporter construct
extending up to
94 of the cdc2 promoter was infected at
the indicated multiplicities with an adenovirus to overproduce
p21/waf1. Luciferase activity was measured 72 h after infection.
Standard errors are shown by the bars.
94 of the wild-type cdc2 promoter was incubated in
the presence of tetracycline to maintain low levels of p53. Infection
with adenovirus expressing p21/waf1 repressed the cdc2 promoter by 2.1- and 7.1-fold at multiplicities of infection of 20 or
100, respectively (Fig. 4C). These results suggest that the
elevated levels of p21/waf1 resulting from overexpression of p53 are
important for repressing the cdc2 promoter.
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Fig. 5.
Effect of DNA damage on the level of Cdc2
protein in MEFs lacking Rb family proteins. The Cdc2 protein in
MEFs incubated in the presence or absence of 0.2 µg/ml adriamycin
(ADR) for 24, 48, or 72 h was detected by Western
analysis. MEFs were derived from mouse embryos in which the indicated
proteins were eliminated by gene targeting.
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Fig. 6.
Detection of E2F4 and Rb family members in
TR9-7 cells. A, Western analysis of TR9-7 cells with
antibodies to p53, Rb, p107, p130, and E2F4 before and after induction
of p53. Asynchronously growing TR9-7 cells were incubated for 48 h
in the presence or absence of tetracycline (TET) to induce
p53 expression. Cells incubated for 48 h in 0.25% serum and then
stimulated for 24 h with 10% serum are also shown. B,
gel mobility shift analysis using a consensus binding site for
E2F-containing complexes. TR9-7 cells were released from a mimosine
block in the presence or absence of tetracycline to induce
p53-dependent G2 arrest. Nuclear lysates were
incubated with a radioactively labeled probe in the presence or absence
of antibodies to p130 or E2F4.
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Fig. 7.
Gel mobility shift analysis of the
cdc2 promoter. The experiments were carried out
with a radioactively labeled probe derived from the R box of the human
cdc2 promoter. TR9-7 cells released from a mimosine block
and incubated in the presence or absence of tetracycline
(TET) for 72 h were lysed and analyzed. A,
effect of p53 expression on complexes bound to the R box region of the
human cdc2 promoter. The analysis was carried out in the
presence or absence of an antibody to p130. B, supershift
analysis of a p53-induced complex with antibodies to E2F1, E2F4, and Rb
family members. Lysates from cells arrested in G2 by
overexpression of p53 were used for analyses in the presence or absence
of the indicated antibodies. C, competition analysis of a
p53-induced complex. Binding reactions were carried out in the absence
or presence of the indicated competitor probes, added at a 100-fold
molar excess over the labeled probe. Competition was carried out with
probes with a wild-type sequence or with mutations in either the CDE or
CHR, as shown. Mutated bases are shown in lowercase letters.
The mutations are the same as used in Fig. 2.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
to inhibit residual Cdc2/Cyclin B1 (15, 28, 34, 35). p53
affects proteins other than Cdc2 to contribute to G2
arrest. For example, it causes the down-regulation of topoisomerase II
(39, 40), required for entry into mitosis, and induces the expression
of B99 (66), which can induce G2 arrest, possibly because
of its interaction with the microtubule cytoskeleton. Several cell
types lacking p53 still arrest in G2 in response to DNA
damage, suggesting that p53 provides a redundant level of control (3,
15). Under some circumstances, the loss of p53 can affect the duration
of G2 arrest, clearly implicating p53 as a significant
factor in the control of the G2/M transition (6).
p53-dependent mechanisms of G2 control are
probably important in maintaining genomic stability and in suppressing neoplasia.
-fetoprotein gene contains a
p53-binding site in its promoter (74) and p53 represses expression of
the
-fetoprotein gene in hepatocytes that contain the HNF-3
transcriptional activator, which also binds to the
-fetoprotein promoter (74). p53 bound to the
-fetoprotein promoter may interfere with the ability of HNF-3 to activate transcription. p53 also binds to
the bcl2 promoter and interferes with the ability of the
simultaneously bound Brn-3a transcription factor to drive the
transcription of this gene (75). Repression of transcription of the
map4 gene also requires p53, which binds to the corepressor mSin3a, recruiting histone deacetylases that cause repression (76).
Consistent with this mechanism, the repression of stathmin and map4 expression by p53 can be reversed by inhibiting
histone deacetylases with trichostatin A (76). p53 can also repress transcription by inhibiting the binding of sequence-specific
transcriptional activators to DNA. The binding of p53 to the SP1
transcriptional activator blocks its ability to bind to promoters (77).
This mechanism is responsible for the repression of the Werner helicase gene by p53 (78). Repression of the cyclin B1 promoter by
p53 maps to a region between
287 and
123, suggesting that the
effects of p53 on transcriptional activators bound to this region are involved (28).
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ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge Joe Nevins (Duke University, Durham, North Carolina) for adenoviruses, Nick Dyson (Massachusetts General Hospital Cancer Center, Boston, MA) for MEFs, and Bert Vogelstein and Todd Waldman (Johns Hopkins, Baltimore, MD) for p21/waf1-null HCT116 cells.
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FOOTNOTES |
---|
* This work was supported by Grant GM 49345 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Lerner Research Inst., Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-3900; Fax: 216-444-3279; E-mail: starkg@ccf.org.
Published, JBC Papers in Press, October 13, 2000, DOI 10.1074/jbc.M005101200
2 W. R. Taylor and G. R. Stark, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: CDK, cyclin-dependent kinase; DTT, dithiothreitol; MEF, mouse embryo fibroblast; PMSF, phenylmethanesulfonyl fluoride; bp, base pair(s); CDE, cell cycle-dependent element; CHR, cell cycle gene homology region.
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