From the Cancer Therapy and Research Center and
¶ Department of Cellular and Structural Biology, The University of
Texas Health Science Center, San Antonio, Texas 78229 and the
§ Fels Institute for Cancer Research and Molecular Biology,
Temple University School of Medicine,
Philadelphia, Pennsylvania 19140
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ABSTRACT |
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The tumor suppressor protein p53 and the adenoviral 12 S E1A oncoprotein are both known to elicit their biological effects mainly by regulating the transcription of important cellular genes. The human proliferating cell nuclear antigen (PCNA) gene is a transcriptional target of both p53 and E1A. We have analyzed the effects of p53 and 12 S E1A, separately as well as together, on PCNA gene transcription. Our results showed that whereas both p53 and 12 S E1A separately activated PCNA transcription, 12 S E1A repressed p53-mediated transcriptional activation. Thus, 12 S E1A uses a dual strategy of transcriptional activation and repression to take control of the cellular PCNA gene regulation. The cyclic AMP-response element in the PCNA core promoter, besides being crucial for basal transcription, synergizes with p53 to activate transcription. The cyclic AMP response element-binding protein (CREB)-binding protein (CBP) is an essential component of both the transcriptional activation and repression by E1A. Our data demonstrate for the first time that E1A can modulate CBP function to activate PCNA transcription, while at the same time repressing p53-mediated activation by disrupting CBP interaction with p53, thereby uncoupling PCNA transcription from the regulatory effects of p53.
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INTRODUCTION |
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It is becoming increasingly evident that the tumor suppressor protein p53 elicits its biological effects as a cell cycle checkpoint molecule mainly by regulating transcription of important genes (1, 2). Similarly, the adenovirus 12 S E1A oncoprotein is a multifunctional protein whose cellular effects are mediated principally through its ability to modulate the transcriptional activity of various target genes (3). E1A is known to activate certain cellular genes, while repressing others. The human proliferating cell nuclear antigen (PCNA)1 gene is activated by both 13 and 12 S E1A (4, 5) oncoproteins.
PCNA plays an essential cellular role as a component of the DNA replication and repair machinery. From recent studies, it appears that PCNA may function as a communication link between important cellular processes such as DNA replication, DNA repair, and cell cycle control (6). Importantly, p21, another cell cycle regulatory protein, when up-regulated at the transcriptional level by p53 in response to DNA damage, associates with PCNA and inhibits DNA replication while allowing DNA repair to proceed (6, 7). Thus p21 and PCNA are components of the cell cycle checkpoint controlled by p53. 12 S E1A inhibits the activation of p21 by p53, thereby compromising the G1 arrest (8, 9). However, the effect of E1A on the transcriptional activation of PCNA by p53 remains unknown. The studies reported here address how 12 S E1A affects transcriptional activation of PCNA by p53.
The NH2-terminal region of E1A, together with a subdomain of conserved region 1 (CR1), targets the p300/CBP family of cellular proteins, and this interaction is sufficient to promote entry into the S phase of the cell cycle (10). Furthermore, it is well documented that this interaction severely compromises the transcriptional coactivator function of p300/CBP (11-13). E1A proteins have been reported to inhibit transcriptional activation by p53 (9), and E1A elicits this inhibitory effect through its p300/CBP-interacting region (8). Cellular p53 is known to activate PCNA gene transcription (14, 15), whereas high levels of p53 inhibit transcription (14-16), probably by titrating out an important cofactor(s). Recently, using other promoter systems, p300/CBP was shown to interact with p53 and function as an important cofactor in p53-mediated transcriptional activation; 12 S E1A, by virtue of its ability to bind p300/CBP, can disrupt the transcriptional function of p53 (17-20). On the other hand, using GAL4 reporter constructs, it was shown that 12 S E1A, through its interaction with p300, activates transcription by relieving transcriptional repression by YY1 (21). However, it was unclear how 12 S E1A would affect p53-mediated transcriptional regulation of a natural p53 target gene known to be positively regulated by E1A.
To investigate the mechanisms by which E1A interferes with p53-regulated transcription of a cellular gene, we analyzed transcriptional regulation of the human PCNA gene promoter, known to be activated by both E1A and p53. Consistent with earlier results, we found that both 12 S E1A and p53 separately activated PCNA gene transcription in Saos-2 cells. Surprisingly, in the presence of 12 S E1A, p53-mediated transcriptional activation was reduced to the much lower level of activation produced by E1A itself. Furthermore, we show that CBP is involved both in E1A-mediated and p53-mediated transcriptional activation of the PCNA gene. Moreover, CBP is at least one of the factors targeted by E1A in the process of repressing p53-activated PCNA transcription. We have also identified a synergism between CRE, a PCNA core promoter element, and p53 to activate transcription.
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EXPERIMENTAL PROCEDURES |
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Plasmids--
1265 PCNA luc. was constructed by ligating the
1265/+62 XhoI-HindIII fragment from
1265 PCNA
chloramphenicol acetyltransferase (4) to the pGL3-Basic vector
(Promega) linearized with XhoI and HindIII. The
CRE mutant PCNA promoter was made by the "QuikChange" site-directed
mutagenesis method (Stratagene). Complementary oligonucleotides used
were 5'-GGACAGCGTGtTGgatcCGCAACGCG-3' and
5'-CGCGTTGCGgatcCAaCACGCTGTCC-3', where the mutant bases are in
lowercase. The wild type human p53 expression constructs, pRc/CMV hp53
and the mutant p53 plasmid pRc/CMV hp53-22/23 (22), were obtained from
A. J. Levine. The CBP expression plasmid pRc/RSV-CBP (23) was from R. Goodman. pSR
-antisense CBP and pSR
plasmids (24) were kindly
provided by S. Ishii. The E1A expression constructs (12 S wild type,
2-36, and YH47/928) (10), and 12 S E1A.FS (25), were described
previously.
Cell Culture-- Saos-2 cells were obtained from American Type Culture Collection (ATCC) and maintained in Dulbecco's modified Eagle's medium (Cellgro/Mediatech) supplemented with 10% fetal bovine serum (Life Technologies, Inc.) at 37 °C under a humidified atmosphere of 5% CO2.
Transcription Assay in Vivo--
For transient expression
assays, Saos-2 cells were transfected by the calcium phosphate
coprecipitation procedure as described (26). 1.2 × 105 cells on each of the six-well flat bottom culture
plates (Falcon) were transfected with 1265 PCNA luc. reporter (1 µg) along with various expression constructs, as noted in the legends
for Figs. 1-4, after making up the total DNA concentration to 2.5 µg
with pUC119. Cells were washed, and fresh medium was added at 20 h after transfection. Cells were harvested 44 h posttransfection. Luciferase assay was performed according to the protocol of the supplier (Promega). Cells on each well were lysed in 1 ml of lysis buffer, and 10 µl of the lysate was used for assaying the luciferase activity in a TD-20/20 luminometer (Turner Designs). Several
independent transfections were performed to ensure reproducibility and,
of these, results from three typical transfections are depicted in figures. To confirm that the transcripts were properly initiated, RNA
isolated from parallel transfection samples was subjected to RNase
protection assay as described previously (26), using riboprobes
abutting the transcriptional start site of the PCNA promoter.
Protein Analysis-- 4 × 105 transfected cells were washed with phosphate-buffered saline, pelleted, and resuspended in 2 × SDS sample buffer; one-third of the sample was heated for 10 min at 100 °C and subjected to SDS-polyacrylamide gel electrophoresis, followed by Western blotting with the ECL (Amersham Pharmacia Biotech) method. E1A monoclonal antibody, M73, was described previously (27). p53 monoclonal antibody (DO-1) and CBP polyclonal antibody were from Santa Cruz Biotechnology, Inc., Santa Cruz, CA.
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RESULTS AND DISCUSSION |
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We have analyzed the mechanism of transcriptional regulation of
the human PCNA gene by p53 and adenoviral 12 S E1A proteins. Consistent
with earlier reports (4, 5, 14, 15), E1A and p53 individually activated
PCNA gene transcription (3- and 9-fold, respectively; Fig.
1A). This study is the first attempt to compare the
mechanisms underlying the effect of these proteins acting individually
as well as together on PCNA gene transcription. In previous studies,
either E1A effects on PCNA gene transcription were examined in cell
lines known not to contain much functional p53 protein or the effect of
p53 on E1A function was not addressed (4, 5). Likewise, in studies
where the effect of p53 on PCNA transcription was analyzed (14, 15),
the effect of E1A on p53 function was not tested. Experiments using
Saos-2 (p53/
) cells and the PCNA gene promoter enabled
us to analyze the transcriptional effects of these two proteins
individually as well as in the presence of each other.
Role of CBP in Differential Effect of 12 S E1A on PCNA
Transcription in Presence and Absence of p53--
Our experiments
revealed that CBP is an essential component of both p53-mediated and 12 S E1A-mediated transcriptional activation of the PCNA promoter (Figs.
1-3). First, when wild type 12 S E1A was cotransfected with p53,
transactivation by p53 was reduced about 3-fold. That the 12 S 2-36
mutant, which is unable to interact with p300/CBP, failed to repress
transcriptional activation by p53 (Fig. 1B) suggests that
p300/CBP is involved in activation of the PCNA promoter by p53. This
conclusion is further supported by the observation that another E1A
mutant, 12 S YH47/928, deficient in interacting with members of the pRB
protein family, did retain partial repression of activation by p53
(Fig. 1C). On the other hand, in the absence of p53, wild
type 12 S E1A activated PCNA transcription, whereas 12 S E1A
2-36
was not as effective (Fig. 1, A and B).
12 S E1A.FS, a control plasmid which does
not produce any E1A protein because of a frameshift mutation, produced
neither activation nor repression.2 When a plasmid
expressing p53 with mutations in the amino-terminal transcriptional
activation domain (p53-22/23) was transfected, there was no increase
in PCNA transcription.2 To
ensure that the repressive effect of 12 S E1A on p53-mediated transcriptional activation was not because of an effect of E1A on p53
protein expression, extracts from transfected cells were subjected to
Western blot analysis using p53 and E1A antibodies. The results (Fig.
1, D-F) showed that p53 protein expression remains constant
regardless of the presence of wild type or mutant E1A proteins. The
ability of E1A to repress transcriptional activation of the PCNA
promoter by p53 is consistent with earlier results obtained with other
promoters (17-20). Second, increased expression of CBP by transfection
could relieve 12 S E1A-mediated repression of PCNA transcriptional
activation by p53 (Fig. 2B). Interestingly, however,
increased amounts of CBP failed to fully restore transcriptional activation to the level elicited by p53 in the absence of E1A, suggesting that CBP may be functioning in concert with some other factor(s) in transcriptional activation by p53. High amounts of CBP
caused only a modest increase in the already high PCNA transcriptional activation produced by p53. These results (Fig. 2A)
demonstrated that although the amount of endogenous CBP in Saos-2 cells
is sufficient for functioning as a coactivator for transcriptional activation by p53. The amount of endogenous CBP becomes limiting when
p53 and 12 S E1A are present together (Fig. 2B). Third, when endogenous CBP was depleted by transfecting a CBP antisense construct, there was considerable dose-dependent reduction in
transcriptional activation by p53 (Fig. 3A). Finally,
transfection of antisense CBP plasmid suppressed p53-independent, 12 S
E1A-dependent activation of PCNA transcription to the basal
level (Fig. 3B) showing that CBP is an important component
of 12 S E1A-mediated transcriptional activation of native PCNA
promoter. This observation is consistent with an earlier report showing
that GAL4-CBP, in the presence of 12 S E1A, activated a synthetic
GAL4-PCNA promoter (25). When concentration of the antisense CBP
construct, but not the control plasmid, was increased, CBP protein
levels were considerably reduced (Fig. 3, C and
D, compare lanes 5-8 with lane 1).
Moreover, inhibition of the p53-mediated as well as the E1A-mediated
transcriptional activation of the PCNA promoter coincided with
the reduction in CBP protein levels (i.e. when 240 fmol or
more of antisense CBP plasmid were transfected; Fig. 3, compare
A and B with C), further demonstrating
direct involvement of CBP in the transcriptional activation of the PCNA
promoter by p53 as well as by 12 S E1A.
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Role of CRE in the Transcriptional Activation of PCNA by p53 and
E1A--
p300/CBP can interact with certain members of the CREB/ATF
and AP1 family of transcription factors, which bind to the CRE. Thus
CRE can be a major "recruiter" of CBP to a gene promoter. We wanted
to test whether the 53 GTGACGTCG
44 region in the PCNA promoter
homologous to the CRE consensus 5'-GTGACGT(A/C)(A/G)-3' (28) is
involved in transcriptional activation by p53. The PCNA promoter region
containing the wild type CRE, but not the mutant site
53 tTGgatcCG
44, is capable of specifically binding CREB-1 and ATF-12
and thereby recruiting CBP to the PCNA promoter (25). We analyzed the
transcriptional response of wild type and CRE mutant (
53 tTGgatcCG
44) PCNA promoters to p53 as well as to E1A. The results shown in
Fig. 4 have demonstrated for the first
time that CRE, which recruits CBP to the PCNA core promoter, synergizes
with p53 to activate transcription (Fig. 4). CRE is a core PCNA
promoter element, because mutation of this element severely debilitates (about 7-fold) the basal transcription of the promoter. Our results are
consistent with the reported importance of this element for PCNA basal
transcription as well as for full activation by 12 S E1A (29); however,
it is clear that this element is not uniquely required for activation
by E1A, but is also important for transcriptional activation by
cellular p53. Indeed, the effect of CRE and p53 in transcriptional
activation of the PCNA promoter is synergistic, whereas the effect of
CRE and 12 S E1A on PCNA transcription appears to be additive.
Therefore, regulation of PCNA transcription by p53 is intimately linked
to the basal transcription machinery by the core promoter CRE. These
results suggest that the role of CRE may not be the same for
p53-mediated as for E1A-mediated transcriptional regulation of the PCNA
gene. Future studies should reveal the specific CRE-binding factors
responsible for these effects.
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ACKNOWLEDGEMENTS |
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We thank J. Atencio for excellent technical
assistance. We are thankful to R. Goodman (RSV-CBP), S. Ishii
(pSR-anti CBP and pSR
), A. Levine (CMV-p53 wt and CMV-p53/22, 23 mut), and G. Morris (-1265 PCNA) for clones; and E. Harlow for the E1A
antibody (M73). We thank Phil Tucker for discussions and N. Elango for
providing access to the luminometer.
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FOOTNOTES |
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* This work was supported in part by American Cancer Society Institutional Grant IRG-116RAC from the University of Texas Health Science Center at San Antonio and NCI Cancer Center Support Grant P30 CA54174 from the San Antonio Cancer Institute.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: Cancer Therapy & Research Center, Gene Regulation Laboratory, 8122 Datapoint Dr., Suite
700, San Antonio, TX 78229. Tel.: 210-616-5876; Fax: 210-692-7502.
1 The abbreviations used are: PCNA, proliferating cell nuclear antigen; CRE, cyclic AMP response element; CREB, CRE-binding protein; CBP, CREB-binding protein.
2 S. Karuppayil and G. Das, unpublished observations.
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REFERENCES |
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