Department of Biochemistry, University of
California, Riverside, California 92521
Received 19 January 2001/Returned for modification 26 February
2001/Accepted 7 March 2001
Simian virus 40 large T antigen has been shown to inhibit
p53-mediated transcription once tethered to p53-responsive promoters through interaction with p53. In this study we report that p53 stimulates transcription by enhancing the recruitment of the basal transcription factors, TFIIA and TFIID, on the promoter (the DA complex) and by inducing a conformational change in the DA complex. Significantly, we have demonstrated that T antigen inhibits
p53-mediated transcription by blocking this ability of p53. We
investigated the mechanism for this inhibition and found that DA
complex formation was resistant to T-antigen repression when the
TFIID-DNA complex was formed prior to addition of p53-T antigen
complex, indicating that the T antigen, once tethered to the promoter
by p53, targets TFIID. Further, we have shown that the p53-T antigen
complex prevents the TATA binding protein from binding to the TATA box.
Thus, these data suggest a detailed mechanism by which p53 activates
transcription and by which T antigen inhibits p53-mediated transcription.
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INTRODUCTION |
The tumor suppressor p53 protein
responds to DNA damage, resulting in cell cycle arrest or apoptosis
(15, 27). The biochemical activity of p53 that is required
for this relies on its ability to function as a sequence-specific DNA
binding transcription factor (26). A number of
oncoproteins have been discovered to transform cells by binding to p53
and inactivating its transcriptional activity. The inactivation can be
achieved by a mechanism in which the transactivation domain of p53 is
inhibited while DNA binding is unaffected (21, 28, 29,
31). In the case of the adenovirus early 1B (E1B) 55K protein,
once it is brought to the promoter through interaction with p53 the E1B
55K protein can function as an active repressor to inhibit
p53-responsive genes (31) and inhibit acetylation of p53
by PCAF (20). In the case of the cellular oncoprotein MDM2, once it is tethered to the promoter MDM2 can conceal the activation domain of p53 from the transcription machinery
(22) and function as an active repressor
(29).
Simian virus 40 (SV40) T antigen has been shown to bind to p53 and
inhibit p53-dependent transcription via two mechanisms. In the first, T
antigen prevents human p53 binding to its cognate DNA binding sequence
(1). Recently we and others proposed a second mechanism by
which T antigen forms a DNA binding complex with p53 that is
transcriptionally inactive (28). Because this mechanism
occurs in mouse cells but not in human cells, we proposed a model for
T-antigen transformation (28). In human cells, latent p53
and T antigen form a complex that is unable to bind to DNA. Upon
activation of p53, a possible conformational change in p53 allows p53
to dissociate from the p53-T complex and bind to DNA. Therefore,
p53-activated transcription that is required for growth arrest and
apoptosis would not be lost. In mouse cells, again latent p53 and T
antigen form a complex that cannot bind DNA. Upon activation, however,
the p53-T complex binds DNA as a transcriptionally inactive complex.
p53-responsive promoters therefore would be completely blocked. Because
SV40 T antigen is known to cause tumors in rodents but has not been
shown to be a complete carcinogen in humans (3, 4), we
speculate that our model may provide an explanation at the molecular
level. Therefore, it will be of interest to elucidate the mechanism by
which T antigen negatively affects p53-mediated transcription once
tethered to the promoter.
Transcription initiation by RNA polymerase II involves the assembly of
TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (23). One of
the first steps in preinitiation complex assembly is the association of
TFIID with the TATA element. This is followed by the binding of the
general transcription factor TFIIA to form the TFIIA-TFIID-promoter
(DA) complex. The formation of the DA complex can be a major
rate-limiting step in gene expression (5, 13, 17, 30). The
DA complex assembly activity can be assayed in vitro by an
electrophoretic mobility shift assay (EMSA) using Mg-agarose gels
(Mg-EMSA). Using this assay system, several activators, such as the
herpes simplex virus VP16 and the Epstein-Barr virus Zta, have been
found to stimulate polymerase II transcription at least in part by
promoting DA complex formation (6, 14).
In the present study we show that, like Zta and VP16, p53 also
stimulates DA complex formation. Analysis of a transcriptionally inactive mutant form of p53 (a deletion at amino acids 1 to 92 [
92]) reveals that the binding of the DA complex to the TATA box
region is necessary but not sufficient for transcription activation. It
appears that a conformational change in the DA complex is also required
for transactivation. Importantly, we have demonstrated that the
transcriptionally inactive p53-T complex blocks the formation of the DA
complex by targeting TFIID. Further, we have found that the p53-T
complex is capable of preventing the TATA binding protein (TBP) from
binding to the TATA box. Taken together, these results suggest that p53
stimulates transcription at least in part by enhancing the recruitment
of the basal transcription factors, TFIIA and TFIID, and by inducing a
conformational change in the DA complex. The transcriptionally inactive
p53-T complex inhibits p53-mediated DA complex formation by preventing
TBP from binding to the TATA element.
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MATERIALS AND METHODS |
Protein purification.
The TFIID complex was affinity
purified from the LTR
3 cell line, a HeLa cell line that expresses
hemagglutinin (HA)-tagged TBP, as described previously
(18). Recombinant HA-tagged TBP was prepared essentially
as described previously (19). The TFIIA
and TFIIA
subunits were expressed in Escherichia coli BL21 cells from
the plasmids pET-mycTFIIA (13) and pQIIA-
(24), respectively, and recombinant TFIIA containing the
fused subunit and the subunit was purified and renatured
essentially as described previously (24).
p53-T complex was immunopurified from scid cell nuclear extracts as
described previously (28). Briefly, 1 ml of scid nuclear extract (7 mg of protein/ml) was incubated with 100 µl of packed protein A-Sepharose beads to which Pab 421, a monoclonal antibody specific for p53, was covalently linked. Beads were then washed and p53
was eluted with 100 µl of Pab 421 peptide (KKGQSTSRHKK) at
a 1-mg/ml concentration in 0.1 M KCl D buffer (20 mM HEPES [pH 7.9],
20% glycerol, 0.2 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride). To purify mouse p53 in the absence of T
antigen (p53-T-T), Pab 421 anti-p53 beads were incubated with nuclear
extract. T antigen was eluted from the bound p53 by being washed with 2 M urea in 0.1 M KCl D buffer. The beads were then washed overnight with
0.1 M KCl D buffer, after which p53 was eluted as described above.
Recombinant T antigen was prepared from SF21 insect cells infected with
baculovirus expressing T antigen (a gift from C. Prives, Columbia
University) as described by Bargonetti et al. (1). To
purify p53 and N92, HeLa cells were infected with recombinant
vaccinia viruses expressing either an HA-tagged p53 (30)
or an HA-tagged N92 (N92 [18]). The p53 proteins
were affinity purified from the nuclear extract of infected cells by
being bound to a matrix of anti-HA antibody, 12CA5, and eluted with HA
epitope oligopeptide as described previously (18).
All purified proteins were analyzed by electrophoresis on sodium
dodecyl sulfate (SDS)-polyacrylamide gels followed by Western blotting
or silver staining.
DA complex assembly assays.
The radiolabeled 250-bp DNA
fragment was prepared from plasmid p5RGCE4CAT (18), which
contains five consecutive p53 binding sites and the adenovirus E4 TATA
box. EMSA was performed in a total volume of 12.5 µl in 12.5 mM HEPES
(pH 7.9), 60 mM KCl, 12.5% glycerol, 5 mM MgCl2, 1.6 mg of
bovine serum albumin per ml, and 6 fmol of the radiolabeled probe
essentially as described previously (14). The amounts of
protein used in different reactions were as follows: 1 µl of TFIID
that contained 2 to 4 ng of TBP, 20 ng of TFIIA, 10 ng of TBP, 50 ng of
p53, 50 ng of N92, or 3 µl of p53-T complex that contained 100 ng
of p53. The reaction mixtures were incubated at 30°C for 30 min and
analyzed on a 1.4% low EEO-agarose gel (Fisher) containing 5 mM MgAc
as described previously (17). The DNA-protein complexes
were visualized with a PhosphorImager using Adobe Photoshop software.
In the supershift experiments, 100 ng of anti-TBP antibody (N-12; Santa
Cruz, Santa Cruz, Calif.) was added at completion of DNA binding (30 min at 30°C). Samples were incubated for another 30 min at 30°C,
and the reactions were stopped by loading the samples on the gel.
The DNase I footprinting assays were performed in a final volume of 25 µl, with reaction mixtures containing 6 fmol of the radiolabeled
fragments one-end labeled. Binding reactions were performed as
described above. Digestion reactions were performed with 1 or 2 ng of
DNase I (BRL) for 60 s. The reactions were stopped by the addition
of 50 µl of stop solution (200 mM NaCl, 30 mM EDTA, 1% SDS, 100 mg
of yeast tRNA per ml), phenol-chloroform extracted twice, and ethanol
precipitated in the presence of carrier RNA. The pellets were washed
with 70% ethanol, dried, and resuspended in 3 µl of formamide
loading buffer. The samples were then resolved on a 6% denaturing
polyacrylamide gel.
E1B TATA-oligo probe (top; 5'-TCGACTTAAAGGGTATATAATGCGCCGTG-3';
bottom, 5'-TCGACACGGCGCATTATATACCCTTTAAG-3') was
labeled in the presence of [-32P]dGTP using Klenow
enzyme. Binding reactions were performed as described above. The
reaction mixtures were analyzed on a 3% polyacrylamide gel. Mutant E1B
TATA-oligo probe (5'-TCGACTTAAAGGGGAGAGAATGCGCCGTG-3') was
included in some EMSA reactions as indicated.
In vitro protein-protein interaction assays.
Approximately
20 µl of glutathione-agarose beads were incubated with 2 µg of
glutathione S-transferase (GST) or GST-TFIIA and then washed
with 0.1 M KCl D buffer. p53 and N92 were in vitro transcribed and
translated in the presence of [35S]methionine. p53-T
complex was radiolabeled in vivo by incubating scid cells in 1 ml of
low-methionine medium containing 0.1 mCi of
[35S]methionine for 3 h. After being labeled, the
cells were washed with phosphate-buffered saline and lysed with radio
immunoprecipitation assay buffer (10 mM NaPO4 [pH 7.2],
150 mM NaCl, 1% NP-40, 0.05% SDS, 0.5 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride). p53-T complex was purified using Pab
421-conjugated beads as described above. The radiolabeled p53 proteins
were incubated with the GST and GST-TFIIA beads for 1 h at room
temperature in a total volume of 0.2 ml in 0.1 M KCl D buffer. The
beads were then washed with buffer D containing 500 mM KCl and 0.2%
NP-40 and then with buffer D containing 100 mM KCl. The bound proteins
were eluted by boiling the beads in SDS-polyacrylamide gel
electrophoresis (PAGE) loading buffer, resolved on a SDS-10% PAGE
gel, and visualized by autoradiography. GST-TBP protein interactions
were performed using the same procedure described above.
TFIID in 250 µl of nuclear extract was immobilized on 20 µl of
packed protein A-Sepharose beads using 2 µl of TAFII250
antibody (6B3; Santa Cruz). The protein pull-down assays were performed as described above.
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RESULTS |
p53 stimulates the TFIID-TFIIA-promoter complex formation.
To
better understand how T antigen inhibits p53-mediated transcription
once brought to the promoter, we studied the transcription function of
p53 at the molecular level. As several sequence-specific DNA binding
transcription factors have been shown to function by stimulating
TFIID-TFIIA-promoter complex (the DA complex) formation, we initially
used the in vitro Mg-agarose gel shift assay (Mg-EMSA) to determine
whether this is also true for p53. When a DNA fragment bearing five RGC
p53 binding sites upstream of a high-affinity TATA box (Fig.
1C) was incubated with increased amounts
of affinity-purified TFIID complex, a retardation of only a small
fraction of the probe in a complex with TFIID was observed (Fig. 1A,
lanes 2 and 3). Addition of purified p53 and TFIIA, however, resulted
in a marked stimulation in the shifted TFIID-DNA complex (lanes 8 and
9). Under these conditions, neither p53 (lanes 4 and 5) nor TFIIA alone
(lanes 6 and 7) significantly stimulated the TFIID-DNA complex assembly. Using identical conditions to those employed with wild-type p53, we have shown that a transcription-inactive mutant of p53, N92
(18 and Fig. 1C), resulted in reduced DA complex formation (Fig. 1B, lanes 6 and 7). These results suggest that p53 activates transcription at least in part by stimulating DA complex formation. Of
note, the addition of N92 alone appeared to stimulate TFIID-DNA complex assembly in the absence of TFIIA in Mg-EMSA (Fig. 1B, lanes 8 and 9). The significance of this observation is presently unknown.
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FIG. 1.
p53 stimulates TFIID-TFIIA-promoter complex assembly.
(A) Mg-EMSA of p53-induced DA complex formation. Purified TFIID
complex, recombinant TFIIA (rTFIIA), and p53 (as indicated above each
lane) were incubated with the 5RGCE4T probe for 30 min at 30°C. The
amount of each factor added to the Mg-EMSA reactions corresponds to 0.5 or 1 µl of TFIID, 20 ng of rTFIIA, and 100 ng of p53. The specific
position of the DA complex is indicated on the right. (B) N92
(92) fails to induce DA complex formation. The amount of each factor
added to the Mg-EMSA reactions corresponds to 0.5 or 1 µl of TFIID,
20 ng of rTFIIA, 100 ng of p53, or 100 ng of N92. The position of
the DA complex is indicated on the right. (C) Schematic diagram of the
5RGCE4T template (transcription initiation site designated by the
right-angle arrow), p53, and N92 used Act, activation domain; NLS,
nuclear localization signal; Tet, tetramerization; Reg, regulatory
domain.
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The conformational change of the DA complex is necessary for p53
transactivation.
To further study the stimulation of DA complex
formation by p53, in vitro DNase I footprinting was carried out using
the same promoter (Fig. 2A). In the
absence of p53, the purified TFIID complex resulted in a weak
protection of the TATA box region as expected (Fig. 2A, lane 4).
Addition of purified p53 alone slightly enhanced protection of the TATA
box (lane 5). A less complete protection of the p53 binding sites,
however, was observed in lane 5. This may be due to a cooperative
binding between p53 and TFIID. When all three proteins were present
simultaneously, a significant protection of the TATA box was observed
(lane 6). In addition to the alteration of the protection of the TATA
box, at least two hypersensitive sites downstream of the transcription initiation site were generated (lane 6). The appearance of the hypersensitive cleavage sites indicates that p53 induced a
conformational change over the promoter region, which may lead to
enhanced assembly of the rest of the transcription machinery. Taken
together, these results suggest that p53 may result in a more stable
and active TFIIA-TFIID-DNA complex.
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FIG. 2.
The conformational change of the DA complex is necessary
for p53 transactivation. (A) p53, TFIIA, and TFIID cooperate to form a
stable TFIID footprint on the promoter. In vitro DNase I footprint
reactions were carried out as described in Materials and Methods. The
amount of each factor added to the DNase I footprint reactions (as
indicated above each lane) corresponds to 5 µl of TFIID, 100 ng of
recombinant TFIIA, and 500 ng of p53. The positions of the p53 binding
sites, the TATA box, and the transcription (Txn) start site are
indicated on the right. Arrows denote hypersensitive sites. (B) N92
(92) fails to induce the conformational change of the DA complex on
the promoter. The amount of each factor added to the DNase I footprint
reactions (as indicated above each lane) corresponds to 5 µl of
TFIID, 100 ng of recombinant TFIIA, 500 ng of p53, or 500 ng of N92.
The positions of the p53 binding sites, the TATA box, and the
transcription start site are indicated on the right.
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Compared to p53, N92 was impaired in its ability to stimulate DA
complex formation as expected (Fig. 2B, lane 6). However, in agreement
with Mg-EMSA results (Fig. 1B, lanes 8 and 9), the addition of N92
in the absence of TFIIA stimulated TFIID-DNA complex assembly (Fig. 2B,
lane 7). Despite the enhanced TFIID-DNA complex assembly, no
hypersensitive cleavage sites were observed in the downstream region in
DNase I footprinting. Thus, a combination of recruitment and
conformational change of the DA complex appears to be necessary for p53 transactivation.
p53-T complex is incapable of stimulating DA complex assembly.
Having established that p53 induces DA complex formation, we next
tested if T antigen affects this function of p53. Because p53-T complex
purified from scid cells has been demonstrated previously to bind to
DNA as a transcriptionally inactive complex (28), the
effect of T antigen on the ability of p53 to stimulate DA complex
formation was tested using this complex. When the p53-T complex was
incubated with the same DNA template as that used with the wild-type
p53 assays, it produced a slowly migrating p53-T-DNA complex (Fig.
3A, lane 9). This result suggested that the p53-T complex could bind to the 5RGCE4T probe in the Mg-EMSA assay.
However, by comparison to p53 (lanes 3 and 4) shows that p53-T complex
failed to stimulate DA complex assembly (lanes 5 and 6) at the expected
shift position. The amounts of p53-T complex have been tested over a
range of concentrations corresponding from one half to eight times the
amount of p53 used in lane 3 of Fig. 3A. At each concentration tested,
no stimulation of DA complex assembly was observed (data not shown).
These results indicated that T antigen affected the ability of p53 to
stimulate DA complex formation.
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FIG. 3.
p53-T complex (p53/T) is incapable of stimulating DA
complex assembly. (A) p53-T complex fails to induce DA complex
formation. Mg-EMSA reactions were carried out as described in Materials
and Methods. The amount of each factor added to the Mg-EMSA reactions
corresponds to 1 µl of TFIID, 20 ng of recombinant TFIIA (rTFIIA), 50 or 100 ng of p53, and 3 or 6 µl of p53-T complex (containing 100 or
200 ng of p53). The positions of the DA complex, p53-T-DNA, and
p53-DNA are indicated on the right. (B) Experiments were performed as
for panel A but in the presence of 100 ng of anti-TBP antibody
(-TBP, N-12). (C) p53 purified from mouse cells is capable of
stimulating DA complex assembly. Mg-EMSA reactions were carried out as
described. The amount of each factor added to the Mg-EMSA reactions (as
indicated above each lane) corresponds to 1 µl of TFIID, 20 ng of
rTFIIA, 100 ng of p53, and 100 ng of p53-T without T antigen (p53/T
 T). The position of the DA complex is indicated on the left. (D) A
silver-stained SDS-PAGE gel is shown. Lane 1, 200 ng of HA-tagged human
p53; lane 2, 100 ng of HA-tagged N92; lane 3, 1 µl of purified
p53-T antigen complex corresponding to 30 ng of p53; lane 4, 1 µl of
purified p53-T complex without T antigen corresponding to 30 ng of p53.
The sizes (in kilodaltons) of molecular mass standards are indicated on
the left.
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We considered the possibility that the shifted DA complex might exist
but that it migrated at a different position. This notion, however,
cannot be tested using DNase I footprinting assays because of technical
difficulties in obtaining concentrated p53-T and TFIID complexes. To
exclude the possibility that the shifted DA complex might migrate at
the same positions as the p53-T-DNA complex, we carried out Mg-EMSA in
the presence of an anti-TBP antibody, N-12. While incubation of
anti-TBP antibody resulted in a partial inhibition of TFIID binding to
DNA either alone (Fig. 3B, lane 3) or in the presence of p53 and TFIIA
(Fig. 3B, lane 5), there was no alteration of the gel shift pattern at
the p53-T-DNA complex position. These results support the view that T
antigen, when in a complex with p53 and DNA, affects the ability of p53
to stimulate DA complex formation.
To determine if these results were specific for the mouse p53 protein,
a further Mg-EMSA was performed with mouse p53 purified from scid cells
without T antigen (p53-T-T [see Materials and Methods for
purification]) (Fig. 3D). Results are shown in Fig. 3C. The addition
of p53-T-T resulted in a generation of a shifted complex (lane 4) at a
position similar to that of the human p53-DNA complex. Importantly, the
addition of p53-T-T also resulted in a stimulation of DA complex
formation (lane 6), similar to that of human p53 (lane 3). Notably, we
consistently observed that mouse p53 was less efficient than human p53
in promoting DA complex formation under these conditions (cf. lanes 3 and 6). This may be due to a number of reasons. For instance, human p53
may interact with human transcription factors better. Alternatively,
mouse p53 may partially lose its activity during purification. The fact that the purified mouse p53 protein without T antigen is less active in
an in vitro transcription assay (28) supports this hypothesis. Nevertheless, the mouse p53 protein is capable of stimulating the DA complex, and therefore it can be concluded that it
is the associated T antigen that inhibits DA complex assembly.
T antigen targets TFIID to inhibit DA complex assembly.
On the
basis of our results, it seemed probable that TFIIA and/or TFIID were
likely targets with which p53 interacted to promote DA complex
assembly. Therefore, to characterize the components required for
T-antigen inhibition, we performed protein-protein interaction
experiments to test if T antigen can affect the interaction of p53 with
TFIID or TFIIA.
As several activators, such as VP16 and Zta, have been shown to
stimulate DA complex formation through direct interaction with TFIIA
(6, 14), we first determined if this was so for p53.
GST-TFIIA binding assays were carried out with 35S-labeled
p53, and an interaction of p53 with TFIIA was observed (Fig.
4, upper left panel). Next, we asked if
the p53-T complex and N92 that severely reduced DA complex assembly
would alter their ability to interact with TFIIA. Our results show that
under identical conditions, although p53-T complex greatly reduced its interaction with TFIIA, N92 binds efficiently to TFIIA (bottom left
panel). Therefore, interaction with TFIIA may be required, but is not
sufficient, for the inhibition of DA complex assembly by p53-T antigen
complex. Of note, p53-T antigen complex in the absence of T antigen (as
shown in Fig. 3D) restores the ability of p53 to bind to TFIIA (data
not shown).
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FIG. 4.
Interaction with TFIIA is not sufficient for the
inhibition of DA complex assembly. (A) p53 and N92 (92) were
translated in vitro with [35S]methionine, and p53-T
complex (p53/T) was radiolabeled in vivo in the presence of
[35S]methionine and purified with Pab 421 antibody as
described in Materials and Methods. The labeled proteins were either
incubated with GST or GST-TFIIA immobilized on glutathione Sepharose
beads (left panel) or with TFIID which was immobilized on protein
A-Sepharose beads using TAFII250 antibody (6B3, right
panel, TFIID IP). After being washed, bound proteins were analyzed by
SDS-PAGE followed by autoradiography of the proteins retained on the
beads. (B) Experiments were performed as for panel A but using GST-TBP.
The relative amount of bound protein was quantitated using a
PhosphorImager and ImageQuant software and is presented as a
percentage of the total radiolabeled protein.
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In earlier work, a direct interaction between p53 and the TFIID complex
was found (19). We next addressed the possibility that p53
was able to stimulate DA complex formation through direct interaction
with TFIID. We therefore compared the interaction of p53, N92, and
p53-T complex with TFIID complex by immunoprecipitation assays with an
anti-TAFII250 antibody, 6B3. Results of representative immunoprecipitation assays are shown in Fig. 4A, right panels. As
expected, p53 interacted with TFIID. Mutant N92 and p53-T complex,
however, appeared to interact with TFIID indistinguishably from p53.
These results suggest that interaction with TFIID may not be sufficient
to promote DA complex assembly. Previously, we also found a direct
interaction between p53 and TBP (19). To test the
possibility that p53-T complex may affect the interaction with TBP, we
performed GST-TBP pull-down experiments and showed that p53-T complex
interacts with TBP to an extent at least similar to that of p53 (Fig.
4B).
Although similar binding between TFIID and p53, N92, or p53-T
complex was observed, we considered the possibility that the interaction between p53-T complex and TFIID may affect the ability of
TFIID to bind DNA. To test this, we performed the Mg-EMSA (Fig. 5A), which showed that when increased
amounts of p53-T complex were included in the reactions, the TFIID DNA
binding was reduced (lanes 5 and 6). However, under identical
conditions the addition of p53 (lanes 3 and 4) and p53-T-T (lanes 7 and 8) did not affect the ability of TFIID to bind DNA. To further
confirm these results, we carried out order-of-addition experiments in
which p53-T complex was added to the Mg-EMSA either during or after the
assembly of the TFIID-DNA complex (Fig. 5B). Our results showed that
TFIID binding became partially resistant to inhibition by p53-T complex if the DNA template was first incubated with TFIID followed by addition
of p53-T complex (lanes 5 and 6). In contrast, when the p53-T complex
was introduced during the assembly of the TFIID-DNA complex, inhibition
of binding was observed (lanes 3 and 4). Collectively, these data
demonstrate that preincubation of TFIID and the DNA template prevents
p53-T complex repression, thereby suggesting that T antigen, once
brought to the promoter through interaction with p53, inhibits DA
complex assembly via TFIID.
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FIG. 5.
T antigen targets TFIID to inhibit DA complex assembly.
(A) p53-T complex (p53/T) prevents TFIID from binding to the promoter.
Mg-EMSA reactions were carried out with TFIID complex, p53, and p53-T
complex (as indicated above each lane) using the 5RGCE4T probe. (B) A
preassembled complex containing DNA and TFIID is partially resistant to
p53-T complex inhibition. TFIID was incubated with the probe for 30 min
at 30°C in either the presence or absence of increasing amounts of
p53-T complex. p53-T complex was then added to the reaction mixtures in
lanes 5 and 6 and was incubated for an additional 30 min prior to the
Mg-EMSA. (C) A preassembled complex containing DNA and TBP is not
resistant to p53-T complex inhibition. Experiments were performed as
for panel A but with 10 ng of TBP instead of TFIID. All preincubation
Mg-EMSA reactions were carried out for 60 min.
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T antigen prevents TBP from binding to the TATA box.
To extend
the above studies regarding the effect of T antigen on the assembly of
TFIID on a promoter, we next wanted to determine whether T antigen
functions via TBP in the TFIID complex, as TBP makes direct contact
with the DNA template. To address this, TFIID was replaced by
bacterially expressed and purified TBP (Fig. 5C). TBP bound to the
template and produced a retarded TBP-DNA complex at a position similar
to that of TFIID-DNA in the Mg-EMSA (lane 2). When p53-T complex was
included in the reactions, the ability of TBP to bind DNA was repressed
(lanes 3 and 4). An equivalent amount of p53, however, had no effect on
TBP binding (data not shown). This result argues strongly that p53-T
complex targets TBP to inhibit DA complex formation, although it does
not rule out the possibility that p53-T complex might make additional
contacts with one or more TAFs. In contrast to TFIID, however, TBP DNA binding was also repressed by the later addition of p53-T complex (lanes 5 and 6), suggesting that preincubation of TBP and the DNA
template is unable to prevent p53-T complex repression. Thus, our data
provide evidence that TAFs function to stabilize TBP binding, and their
presence reduces the inhibition of TBP binding by T antigen.
The data described above suggest a model for the inhibition of DA
complex formation by p53-T complex in which p53-T complex contacts TBP
and inhibits TBP binding to DNA. If this model is correct, addition of
p53-T complex should also inhibit TBP in a probe lacking p53 binding
sites. To test this possibility, we examined the effect of p53-T
complex on TBP DNA binding with a probe containing the E1B TATA
element. As expected, TBP bound to the E1B probe and produced a
retarded TBP-DNA complex (Fig. 6A, lane
2). Addition of a 100-fold excess of cold wild-type, but not mutant
(lane 3), E1B probe was sufficient to inhibit its formation (data not
shown). This TBP-specific DNA binding was largely reduced when p53-T
complex was included in the reaction mixture (lanes 6 and 7). Addition
of an equivalent amount of p53 or p53-T-T (Fig. 6A, lanes 4 and 5, and
6C, lanes 3 to 6), however, had no effect on TBP DNA binding. These
results suggest that p53-T complex inhibits the ability of TBP to bind
to the TATA element.
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FIG. 6.
T antigen prevents TBP from binding to the TATA box. (A)
EMSA reactions were carried out with 10 ng of TBP, 10 or 20 ng of p53,
and 0.3 or 0.6 µl of p53-T complex corresponding to 10 or 20 ng of
p53 for 30 min at 30°C. The reaction mixtures were analyzed on a 3%
polyacrylamide gel. The position of the TBP-DNA complex is indicated on
the left. Mut Olig, E1B TATA-oligo probe. (B) T antigen alone has no
effect on TBP binding. Experiments were performed as for panel A but
with 10 or 20 ng of T antigen. (C) Mouse p53 alone has no effect on TBP
binding. Experiments were performed as for panel A but with 10 or 20 ng
of mouse p53 (p53/TT). (D) A preassembled complex containing DNA,
TFIIA, and TBP is partially resistant to p53-T complex inhibition. TBP,
TFIIA, and the E1B probe were preincubated for 5, 15, or 30 min before
the addition of p53-T complex. The time at which p53-T complex (p53/T)
was added is indicated above. All preincubation EMSA reactions were
carried out for 60 min. The arrows on the left indicate the positions
of the TBP-DNA and TBP-IIA-DNA complexes.
|
|
The observation that p53-T complex inhibits TBP DNA binding, thereby
affecting the ability of p53 to stimulate the DA complex, raised the
question of whether this inhibition relies on p53-T configuration. T
antigen has been previously demonstrated to stabilize TBP-TFIIA complex
formation on the TATA element (10). We therefore determined if T antigen could affect TBP DNA binding under our assay
conditions. Our results showed that in contrast to p53-T complex (Fig.
6B, lanes 3 and 4), T antigen alone was unable to prevent TBP from
binding to the TATA element (lanes 5 and 6). Taken together, our data
suggest that p53-T complex prevents TBP from binding to the TATA
element and thus inhibits p53-mediated transcription.
| |
DISCUSSION |
Although SV40 T antigen from mouse cells is known to form a
transcription-inactive complex with p53 on p53-responsive promoters (28), the molecular mechanisms by which this occurs
remained elusive. In this work, we present biochemical evidence that
p53 stimulates transcription, at least in part, by enhancing assembly of the DA complex on the promoter and by inducing a conformational change. Importantly, we have shown that T antigen, once tethered to the
promoter through interaction with p53, represses transcription by
inhibiting DA complex assembly. Furthermore, the repression of DA
complex formation by T antigen was largely reduced when the TFIID-DNA
complex was formed prior to addition of T antigen. These results
indicate that the T antigen targets TFIID to inhibit transcription. In
support of this theory, we have found that the p53-T complex is capable
of preventing TBP from binding to the TATA element. A model summarizing
these results is shown in Fig. 7. On the
basis of these results, we propose that p53-T complex inhibits DA
complex formation by disrupting the activity of the general
transcription factor TBP.
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|
FIG. 7.
A model for how p53-T complex inhibits p53-mediated
transcription. (Top) p53 stimulates the TFIID-TFIIA-promoter complex
formation. (Bottom) T antigen represses transcription when tethered to
the promoter by p53. This could occur through a conformational change
of TBP within the TFIID-TFIIA-promoter complex or could be due to the
exclusion of the ability of TFIIA to function as an antirepressor.
|
|
Several activators have been found to stimulate the formation of the DA
complex through a direct interaction with the general transcription
factor TFIIA (13, 14). Our results suggested that p53,
like other transcription factors, interacted with TFIIA. However, we
have observed that a deletion mutant of p53 lacking the activation
domain, N92, which did not stimulate DA complex assembly, bound to
TFIIA to an extent similar to that of p53 (Fig. 4). Furthermore, we
failed to detect a correlation between the ability of p53 to interact
with TFIID and its ability to promote DA complex assembly (Fig. 4).
These results indicated that interaction with TFIIA or TFIID may be
necessary but is not sufficient for the stimulation of DA complex
formation. This view is supported by the demonstration that p53, but
not N92, induced a conformational change in the DA complex, as
indicated by the formation of hypersensitive cleavage sites detected in
DNase I footprinting assays. These data are consistent with the idea
that the formation of the isomerized DA complex is necessary and
sufficient for gene activation (5, 11). Thus, the
conformational change and/or isomerization of the DA complex mediated
by p53 is probably required for efficient assembly of a functional
transcription complex.
We have shown that the p53-T complex inhibits the TBP-TATA interaction
in the absence of p53 binding sites. Thus, the p53-T complex inhibits
DA complex formation probably by disrupting the activity of the general
transcription factor TBP. In this regard, T antigen, once tethered to
the p53-responsive promoters, appears to function in a manner similar
to that of the Drosophila melanogaster transcriptional
repressor, Eve (16). Eve represses transcription by
directly binding TBP in such a way that it blocks the interaction between TBP and DNA. However, Eve seems to repress transcription by
competing with the TATA box for TBP binding. p53-T complex is distinct
from Eve in that it does not specifically bind to the TATA element
(Fig. 6). It may be similar in this regard to SAGA subunits, Spt3 and
Spt8, which can downregulate TBP function through interaction with TBP
(2). Alternatively, TBPs have been reported to exist as
dimers, which must dissociate to bind to DNA (6-8).
Perhaps p53-T complex prevents this required dissociation, thereby
inhibiting stable association of TBP with the promoter. Indeed, we have
observed that TBP binds to p53-T complex more stably than transcription
activator p53 (Fig. 4B). Although the relevance of the interaction in
regulating TBP dimers remains uncertain, the interaction of p53-T
complex with TBP appears to play a role in the inhibition of TBP
binding. It is possible that p53-T complex, like several other
characterized repressors, may function by more than one mechanism
(reviewed in reference 12). Nevertheless, the efficiency
of TBP-mediated repression suggests that this is an important aspect of
p53-T complex activity. Of note, despite the ability of p53-T complex
to inhibit TBP on promoters without p53 binding sites, specific
inhibition of transcription is likely to occur on the promoters
containing p53 binding sites in cells.
SV40 T antigen has been previously shown to stabilize the
TBP-TFIIA-promoter complex and to activate transcription
(10). Thus, T antigen appears to play both negative and
positive roles in transcription regulation. When bound to p53 and
tethered to p53-responsive promoters, T antigen inhibits TBP-DNA
interaction and represses transcription from p53-activated genes. When
not bound to p53, however, T antigen may stabilize the
TBP-TFIIA-promoter complex and activate transcription. Interestingly, T
antigen has been reported to function like TAFII250
(9), which prevents TBP from binding to the TATA element
(25). The question that arises is whether TFIIA can
prevent p53-T complex inhibition of TBP-DNA binding, since TFIIA has
been shown to counteract a number of TBP inhibitors (reviewed in
reference 23), including TAFII250 (25). We therefore tested T-antigen inhibition in DNA
binding reactions with or without TFIIA (Fig. 6D). Our results reveal that simultaneous addition of p53-T complex and TFIIA to TBP resulted in a reduction of DNA binding (Fig. 6D, lane 4). However, after 30 min
of preincubation of TFIIA, TBP, and DNA, a stable complex formed that
was partially resistant to p53-T inhibition (lane 6). Thus, TFIIA
appears to compete with p53-T complex for interaction with TBP,
resulting in a stable association of TBP with the promoter.
SV40 is known to cause tumors in rodents (3, 4) but has
not been proven to do so in humans. In a recent study, we and others
demonstrated that T antigen present in transformed mouse cells, but not
in human cells, forms a DNA binding complex with p53 that is
transcriptionally inactive (28). In the present study, we
have defined a detailed mechanism of transcription repression of p53 by
T antigen in mouse cells. These studies have extended our understanding
of T-antigen inhibition of transcription from p53-activated genes. A
number of cellular proteins have also been shown to bind to p53 and
negatively affect its transcriptional activity. It will be interesting
to determine whether the mechanism of T-antigen inhibition could
resemble that of other cellular p53-interacting proteins.
We are very grateful to A. Berk, N. Kobayashi (University of
California, Los Angeles), P. Lieberman (Wistar), J. Ross, and B. Dynlacht (Harvard) for many helpful discussions. We thank A. Berk and
N. Kobayashi for providing TFIIA expression vectors and anti-TFIIA
antibody, L. Anton for LTR3 nuclear extracts, and E. Martinez for
valuable comments on the manuscript.
This work was supported by grants from the National Cancer Institute
(CA75180) and the U.S. Army Breast Cancer Research Program (DAMD17-96-6076) to X.L. S.I.C is supported by a postdoctoral fellowship from the California Cancer Research Program.
| 1.
|
Bargonetti, J.,
I. Reynisdottir,
P. Friedman, and C. Prives.
1992.
Site-specific binding of wild-type p53 to cellular DNA is inhibited by SV40 T antigen and mutant p53.
Genes Dev.
6:1886-1898[Abstract/Free Full Text].
|
| 2.
|
Belotserkovskaya, R.,
D. E. Sterner,
M. Deng,
M. H. Sayre,
P. M. Lieberman, and S. L. Berger.
2000.
Inhibition of TATA-binding protein function by SAGA subunits Spt3 and Spt8 at Gcn4-activated promoters.
Mol. Cell. Biol.
20:634-647[Abstract/Free Full Text].
|
| 3.
|
Carbone, M.,
P. Rizzo, and H. I. Pass.
1997.
Simian virus 40, poliovaccines and human tumors: a review of recent developments.
Oncogene
15:1877-1888[CrossRef][Medline].
|
| 4.
|
Carbone, M.,
P. Rizzo,
P. M. Grimley,
A. Procopio,
D. J. Y. Mew,
V. Shridhar,
A. de Bartolomeis,
V. Esposito,
M. T. Giuliano,
S. M. Steinberg,
A. Levine,
A. Giordano, and H. I. Pass.
1997.
Simian virus-40 large T-antigen binds p53 in human mesotheliomas.
Nat. Med.
3:908-912[CrossRef][Medline].
|
| 5.
|
Chi, T.,
P. Lieberman,
K. Ellwood, and M. Carey.
1995.
A general mechanism for transcription synergy by eukaryotic activators.
Nature
377:254-257[Medline].
|
| 6.
|
Chi, T., and M. Carey.
1996.
Assembly of the isomerized TFIIA-TFIID-TATA ternary complex is necessary and sufficient for gene activation.
Genes Dev.
10:2540-2550[Abstract/Free Full Text].
|
| 7.
|
Coleman, R. A.,
A. K. Taggart,
S. L. R. Benjamin, and B. F. Pugh.
1995.
Dimerization of the TATA binding protein.
J. Biol. Chem.
270:13842-13849[Abstract/Free Full Text].
|
| 8.
|
Coleman, R. A.,
A. K. P. Taggart,
S. Burma,
J. J. Chicca II, and B. F. Pugh.
1999.
TFIIA regulates TBP and TFIID dimers.
Mol. Cell
4:451-457[CrossRef][Medline].
|
| 9.
|
Damania, B., and J. C. Alwine.
1996.
TAF-like function of SV40 large T antigen.
Genes Dev.
10:1369-1381[Abstract/Free Full Text].
|
| 10.
|
Damania, B.,
P. L. Lieberman, and J. C. Alwine.
1998.
Simian virus 40 large T antigen stabilizes the TATA-binding protein-TFIIA complex on the TATA element.
Mol. Cell. Biol.
18:3926-3935[Abstract/Free Full Text].
|
| 11.
|
Emami, K. H.,
A. Jain, and S. T. Smale.
1997.
Mechanism of synergy between TATA and initiator: synergistic binding of TFIID following a putative TFIIA-induced isomerization.
Genes Dev.
11:3007-3019[Abstract/Free Full Text].
|
| 12.
|
Hampsey, M.
1998.
Molecular genetics of the RNA polymerase II general transcriptional machinery.
Microbiol. Mol. Biol. Rev.
62:465-503[Abstract/Free Full Text].
|
| 13.
|
Kobayashi, N.,
T. G. Boyer, and A. J. Berk.
1995.
A class of activation domains interacts directly with TFIIA and stimulates TFIIA-TFIID-promoter complex assembly.
Mol. Cell. Biol.
15:6465-6473[Abstract].
|
| 14.
|
Kobayashi, N.,
P. J. Horn,
S. M. Sullivan,
S. J. Trizenberg,
T. G. Boyer, and A. J. Berk.
1998.
DA-complex assembly activity required for VP16C transcriptional activation.
Mol. Cell. Biol.
18:4023-4031[Abstract/Free Full Text].
|
| 15.
|
Levine, A. J.
1997.
p53, the cellular gatekeeper for growth and division.
Cell
88:323-331[CrossRef][Medline].
|
| 16.
|
Li, C., and J. L. Manley.
1998.
Even-skipped repressed transcription by binding TATA binding protein and blocking the TFIID-TATA box interaction.
Mol. Cell. Biol.
18:3771-3781[Abstract/Free Full Text].
|
| 17.
|
Lieberman, P. M., and A. J. Berk.
1994.
A mechanism for TAFs in transcriptional activation: activation domain enhancement of TFIID-TFIIA-promoter DNA complex formation.
Genes Dev.
8:995-1006[Abstract/Free Full Text].
|
| 18.
|
Liu, X., and A. J. Berk.
1995.
Reversal of in vitro p53 squelching by both TFIIB and TFIID.
Mol. Cell. Biol.
15:6474-6478[Abstract].
|
| 19.
|
Liu, X.,
C. W. Miller,
H. P. Koeffler, and A. J. Berk.
1993.
The p53 activation domain binds the TATA box-binding polypeptide in holo-TFIID, and a neighboring p53 domain inhibits transcription.
Mol. Cell. Biol.
13:3291-3300[Abstract/Free Full Text].
|
| 20.
|
Liu, Y.,
A. L. Colosimo,
X.-J. Yang, and D. Liao.
2000.
Adenovirus E1B 55-kilodalton oncoprotein inhibits p53 acetylation by PCAF.
Mol. Cell. Biol.
20:5540-5553[Abstract/Free Full Text].
|
| 21.
|
Martin, M. E. D., and A. J. Berk.
1999.
Corepressor required for adenovirus E1B 55,000-molecular weight protein repression of basal transcription.
Mol. Cell. Biol.
19:3403-3414[Abstract/Free Full Text].
|
| 22.
|
Oliner, J. D.,
J. A. Pietenpol,
S. Thiagalingam,
J. Gyuris,
K. W. Kinzler, and B. Vogelstein.
1993.
Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53.
Nature
362:857-860[CrossRef][Medline].
|
| 23.
|
Orphanides, G.,
T. Lagrange, and D. Reinberg.
1996.
The general transcription factors of RNA polymerase II.
Genes Dev.
10:2657-2683[Free Full Text].
|
| 24.
|
Ozer, J.,
P. A. Moore,
A. H. Bolde,
A. Lee,
C. A. Rosen, and P. M. Lieberman.
1994.
Molecular cloning of the small () subunit of human TFIIA reveals functions critical for activated transcription.
Genes Dev.
8:2324-2335[Abstract/Free Full Text].
|
| 25.
|
Ozer, J.,
K. Mitsouras,
D. Zerby,
M. Carey, and P. M. Lierberman.
1998.
Transcription factor IIA derepresses TATA-binding protein (TBP)-associated factor inhibition of TBP-DNA binding.
J. Biol. Chem.
273:14293-14300[Abstract/Free Full Text].
|
| 26.
|
Pietenpol, J. A.,
T. Tokino,
S. Thiagalingam,
W. S. El-Deiry,
K. W. Kinzler, and B. S. Vogelstein.
1994.
Sequence-specific transcriptional activation is essential for growth suppression by p53.
Proc. Natl. Acad. Sci. USA
91:1998-2002[Abstract/Free Full Text].
|
| 27.
|
Prives, C.
1998.
Signaling to p53: breaking the Mdm2-p53 circuit.
Cell
95:5-8[CrossRef][Medline].
|
| 28.
|
Sheppard, H. M.,
S. J. Corneillie,
C. Espiritu,
A. Gatti, and X. Liu.
1999.
New insights into the mechanism of inhibition of p53 by simian virus 40 large T antigen.
Mol. Cell. Biol.
19:2746-2753[Abstract/Free Full Text].
|
| 29.
|
Thut, C. J.,
J. A. Goodrich, and R. Tijan.
1997.
Repression of p53-mediated transcription by MDM2: a dual mechanism.
Genes Dev.
11:1974-1986[Abstract/Free Full Text].
|
| 30.
|
Wang, W.,
J. D. Gralla, and M. Carey.
1992.
The acidic activator GAL4-AH can stimulate polymerase II transcription by promoting assembly of a closed complex requiring TFIID and TFIIA.
Genes Dev.
6:1761-1772.
|
| 31.
|
Yew, P. R.,
X. Liu, and A. J. Berk.
1994.
Adenovirus E1B oncoprotein tethers a transcriptional repression domain to p53.
Genes Dev.
8:190-202[Abstract/Free Full Text].
|
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Abstract
Introduction
Present address: Department of Biochemistry, University of
Leicester, Leicester LE1 7RH, United Kingdom.
