 |
INTRODUCTION |
In the eukaryotic nucleus, DNA exists in a highly organized
chromatin. The basic structural unit is the nucleosome, consisting of
DNA wrapped around an octamer of histones (two each of H2A, H2B, H3,
and H4). The packaged nucleosome is the natural barrier to most
regulatory proteins due to restriction of access to DNA (1-5).
Therefore, activation of transcription requires disruption of the
highly dense structure of chromatin to increase the accessibility of
DNA to transcription factors. It is believed that disruption of
chromatin structure depends on chromatin-modifying complexes, which
include two major groups, the ATP-dependent remodeling
complexes and the histone modification enzymes. Histone
acetyltransferase is one of the well characterized histone modification
enzymes that acetylates histone tails and introduces negative charge to histones, thereby reducing the interaction between histones and DNA and
promoting accessibility of DNA to the transcriptional machinery (3, 6).
Therefore, increased acetylation of histones on promoters often leads
to transcriptional activation. In mammalian cells the histone
acetyltransferase-containing transcription co-activators p300/CBP1 and PCAF acetylate
histones after being recruited by sequence-specific DNA binding
transcription factors (7). This is underscored by evidence that
p300/CBP can interact with many transcription factors, such as c-Jun,
JunB, JunD, MyoD, STATs (signal transducers and activators of
transcription), and p53, and increase the transcriptional activity of
these proteins (7).
The p53 tumor suppressor gene is the most frequent target
for genetic alterations in human cancers, with mutations occurring in
almost 50% of all human tumors (8, 9). In response to environmental
and intracellular stresses, including DNA damage, ionizing irradiation,
and hypoxia, p53 is rapidly stabilized and accumulated (10). The
activated p53 induces many target genes, including p21, Bax,
PUMA, and MCG10, which mediate p53-dependent cell
cycle arrest and/or apoptosis (11-15).
The p53 protein consists of two N-terminal activation domains (AD1
within residues 1-42 and AD2 within residues 43-63), a proline-rich
domain (PRD, within residues 64-93), a sequence-specific DNA binding
domain (within residues 102-292), and an extreme C-terminal basic
domain (BD within residues 364-393) (16). The transcription co-activators p300/CBP have been found to interact with p53 activation domains and enhance the ability of p53 to activate p21 or mdm2 (17-21). A dominant-negative CBP mutant can suppress the
p53-dependent induction of p21 (21). Mutant p53 with a
double point mutation in AD1 and AD2, each of which is deficient in
transactivation, is unable to interact with CBP in vitro
(21). In addition, the oncoprotein E1A suppresses
p53-dependent induction of p21, probably through disruption
of the interaction between CBP and p53 (20). All this evidence suggests
that the transcriptional activity of p53 depends on its
interaction with p300/CBP.
The C terminus of p53 has been shown to play a critical role in
regulation of p53 functions. For example, the DNA binding ability of
p53 is increased after deletion of the C-terminal domain, phosphorylation of serine residues in this domain, or binding of an
antibody specific to this domain (10). More recent studies show that
p300/CBP and PCAF can acetylate lysine residues within the
C-terminal domain in response to p53-activating agents (22-26). Acetylated p53 has a higher DNA binding affinity to short
oligonucleotides containing a p53 binding site from the p21 promoter
in vitro (23, 24) but not to a long DNA fragment (27).
In this study, we used stable cell lines to analyze differential
regulation of p21 by wild-type p53 or various mutants. We showed that
although all of the p53 mutants used in this assay are compromised in
inducing endogenous p21 gene, both wild-type p53 and various p53
mutants have an equivalent sequence-specific DNA binding ability. We
found that the ability of p53 mutants to increase acetylation of
histones H3 and H4 is impaired. We also found that the compromised
transcriptional activity of p53 mutants correlates with the acetylation
level of histones H3 and H4 on the region proximal to the TATA box. We
used the RNA interference technique to confirm and extend the previous
observation that p300/CBP is required for the transcriptional activity
of wild-type p53. Furthermore, we provided evidence that compromised
transcriptional activity of various p53 mutants correlates with their
decreased interaction with p300/CBP. Therefore, our data suggest that
the amount of p300/CBP recruited by p53 and the extent of acetylated histones bound to the proximal promoter can in part explain the differential transcriptional activity among wild-type p53 and various
p53 mutants.
 |
EXPERIMENTAL PROCEDURES |
Cell Lines--
H1299 cell lines, which can be induced to
express wild-type p53, p53(R249S), p53(AD1
),
p53(AD2
), p53(AD1
AD2
),
p53(
BD), and p53(
PRD), were described previously (28, 29). Except
p53(AD1
) and p53(R249S), wild-type p53 and the other four
mutants are tagged with an HA at the N terminus.
RNA Isolation and Northern Blot Analysis--
Total RNA was
isolated using Trizol reagent (Invitrogen). Northern blot analysis was
performed as described (30). The p21 and glyceraldehyde-3-phosphate
dehydrogenase probes were prepared as described previously (31).
Luciferase Assay--
The luciferase reporter under the control
of the p21 promoter with two p53-responsive elements was described
previously (32). 1.0 µg of the reporter vector was cotransfected into
H1299 cells with 1.0 µg of pcDNA3 control vector or a vector
expressing wild-type p53, p53(R249S), p53(AD1
),
p53(AD2
), p53(AD1
AD2
),
p53(
BD), or p53(
PRD). For an internal control, 25 ng of the Renilla luciferase vector, pRL-CMV (Promega, Madison, WI),
was cotransfected. Dual luciferase assays were performed in triplicate according to the manufacturer's instructions (Promega).
Protein Extraction and Immunoblotting--
Small portions of
cells, which were used for RNA isolation or luciferase assay, were
lysed with 2× SDS sample buffer (33). Protein was resolved by 8%
SDS-PAGE gel. The expression level of wild-type p53,
p53(AD2
), p53(AD1
AD2
),
p53(
BD), and p53(
PRD) in stable cell lines or transiently transfected cells was detected by anti-HA antibody (Santa Cruz). p53(R249S) and p53(AD1
) were detected by pAb1801 and
pAb421. Actin was also detected to ensure an equal loading.
Chromatin Immunoprecipitation Assay (ChIP)--
1 × 108 H1299 cells, which were uninduced (
) and induced (+)
to express p53, p53(R249S), p53(AD1
),
p53(AD2
), p53(AD1
AD2
),
p53(
BD), or p53(
PRD), were washed 3 times with cold
phosphate-buffered saline. Genomic DNA and proteins were cross-linked
by the addition of formaldehyde (1% final concentration) directly into
culture medium and incubated for 10 min at room temperature. The
cross-linked cells on plates were then washed with cold
phosphate-buffered saline three times and collected by scraping. The
cells were pelleted by centrifugation, resuspended with 1 ml of cold
lysis buffer (1% SDS, 10 mM EDTA, 50 mM
Tris-HCl, pH 8.1, proteinase inhibitor mixture), and sonicated to
generate 200-1000-bp DNA fragments. After clearance by centrifugation,
the supernatant was diluted 10-fold with a dilution buffer (1%
Triton-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH 8.1, proteinase inhibitor mixture) and
incubated with anti-p53, anti-acetylated histone H3, and
anti-acetylated histone H4, respectively, at 4 °C overnight. The
immunocomplexes were captured by protein A beads saturated with salmon
sperm DNA. The beads that contained various immunocomplexes were washed
sequentially with TSE buffer (0.1% SDS, 1% Triton-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1) containing 150 mM NaCl, TSE containing 500 mM NaCl, buffer III
(0.25 M LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1), and TE buffer
(10 mM Tris, pH 8.0, 0.1 mM EDTA). The
immunocomplexes were then eluted with the elution buffer (1% SDS, 0.1 M NaHCO3) for 15 min at the room temperature. The DNA-protein cross-links were reversed by heating at 65 °C for
4 h. DNA was purified by phenol/chloroform and precipitated by
ethanol and resuspended in 50 µl of TE. 2.5 µl of DNA sample was
used as a template for PCR amplification. To amplify the regions containing p53-responsive element 1 (RE1) and RE2 and the region proximal to TATA box in the p21 promoter, PCRs were performed with the
following pairs of primers: p53 RE1, forward primer, 5'-CAGGCTGTGGCTCTGATTGG-3', and reverse primer,
5'-TTCAGAGTAACAGGCTAAGG-3'; p53 RE2, forward primer,
5'-GGTCTGCTACTGTGTCCTCC-3', and reverse primer,
5'-CATCTGAACAGAAATCCCAC-3'; p21 TATA, forward primer, 5'-TATTGTGGGGCTGTTCTGGA-3', and reverse primer
5'-CTGTTAGAATGAGCCCCCTTT-3'.
Generation of Short Interference RNA (siRNA) Expression Plasmids
under the Control of the U6 Promoter--
The U6 RNA polymerase III
promoter was derived from cleavage of plasmid LS9/Hae/RA.2 (34) with
HincII. The fragment containing the U6 promoter was cloned
into the SmaI site of plasmid pBluescript to obtain an
EcoRI and a BamH1 site. The U6 promoter fragment was then excised with EcoRI and BamH1 and cloned
into plasmid pBabe. The resulting plasmid was named as pBabe-U6.
Selected siRNA oligos were then cloned into pBabe-U6 at
BamHI and XhoI sites for expression of siRNA
in vivo. Two pairs of siRNA oligos from both p300 and CBP
were synthesized and cloned into pBabe-U6, and the resulting plasmids
were designated as pBabe-U6-p300-1, pBabe-U6-p300-2, pBabe-U6-CBP-1,
and pBabe-U6-CBP-2, respectively. The siRNA oligos cloned in
pBabe-U6-p300-1 (shown in bold, derived from the p300 gene from +370 to
+389) are: sense,
5'-TCGAGGTCCGCCCAATGACACAGGCAGGCttcaagagaGCCTGCCTGTGTCATTGGGCTTTTTG-3', and antisense, 5'-GATCCAAAAAGCCCAATGACACAGGCAGGCtctcttgaa GCCTGCCTGTGTCATTGGGCGGACC-3'. The siRNA oligos cloned in pBabe-U6-p300-2 (derived from the p300 gene from +1008 to +1027) are:
sense,
5'-TCGAGGTCCGCTCATCCAGCAGCAGCTTGttcaagagaCAAGCTGCTGCTGGATGAGCTTTTTG-3', and antisense
5'-GATCCAAAAAGCTCATCCAGCAGCAGCTTGtctcttgaaCAAGCTGCTGCTGGATGAGCGGACC-3'. The siRNA oligos cloned in pBabe-U6-CBP-1 (derived from the CBP gene
from +3510 to +3528) are: sense,
5'-TCGAGGTCCGACATCCCGAGTCTATAAGttcaagagaCTTATAGACTCGGGATGTCTTTTTG-3', and antisense,
5'-GATCCAAAAAGACATCCCGAGTCTATAAGtctcttgaaCTTATAGACTCGGGATGTCGGACC-3'. The siRNA oligos cloned in pBabe-U6-CBP-2 (derived from the CBP gene
from +4430 to +4449) are: sense,
5'-TCGAGGTCCGTGAAGGAGATGATTACATCttcaagagaGATGTAATCATCTCCTTCACTTTTT-3', and antisense,
5'-GATCCAAAAAGTGAAGGAGATGATTACATCtctcttgaaGATGTAATCATCTCCTTCACGGACC-3'.
RNA Interference Assay--
H1299 cells were transfected with
the mixture of 0.5 µg of each of pBabe-U6-p300-1, pBabe-U6-p300-2,
pBabe-U6-CBP-1, and pBabe-U6-CBP-2 alone or together with 0.1 µg of
pcDNA3-HAp53. H1299 cells were also transfected with 2 µg of
control pBabe-U6 or pcDNA3 alone or together with 0.1 µg of
pcDNA3-HAp53. The cell extracts were collected on the second day
after transfection. The level of p300, CBP, p53, and p21 was detected
by anti-p300 (Santa Cruz), anti-CBP (Upstate Biotechnology), anti-p53
(pAb1801 and pAb421), and anti-p21 (Santa Cruz), respectively. Actin
was also detected to ensure an equal loading.
Co-immunoprecipitation--
Approximately 2 × 108 H1299 cells were induced to express wild-type p53,
p53(AD1
), p53(AD2
),
p53(AD1
AD2
), p53(
BD), or
p53(
PRD). Cells were washed 3 times with cold phosphate-buffered
saline and collected by scraping. The collected cells were resuspended
in 20 ml of hypotonic buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 × proteinase inhibitor mixture, 50 mM NaF) and placed on ice
for 8 min. Nonidet P-40 was then added to cell extracts to the final
concentration of 0.1%. Cell extracts were placed on ice for another 8 min. After centrifugation, the cell pellets were resuspended in 10 ml
of 0.1% Nonidet P-40 lysis buffer (0.1% Nonidet P-40, 50 mM Tris-HCl, 2 mM EDTA, 150 mM
NaCl) and frozen and thawed 3 times. The extracts were precleared by
centrifugation at 14,000 rpm for 10 min. Wild-type p53,
p53(AD2
), p53(AD1
AD2
),
p53(
BD), and p53(
PRD) were captured by incubation with 80 µl of
monoclonal anti-HA-conjugated agarose beads (Sigma) overnight at
4 °C. p53(AD1
) was captured by 80 µl of anti-p53
(pAb1801 and pAb421)-conjugated protein A beads. The beads were washed
5 times with 0.1% Nonidet P-40 lysis buffer, and p53 was eluted using
60 µl of 2× SDS sample buffer at 100 °C for 8 min. The amount of
CBP or p300 co-immunoprecipitated with p53 or various mutants was
detected by Western blot analysis.
 |
RESULTS |
p21 Is Differentially Regulated by Wild-type p53 and Various p53
Mutants--
In the process of delineating p53 functional domains, we
generated a number of cell lines that could be induced to express wild-type p53 or various p53 mutants. We found that the level of p21
induced by p53 in these cell lines varied (28, 29, 35). To
systematically analyze the induction of p21 in these cell lines, we
performed Northern blot analyses and compared the level of p21
induction. We found that p21 was strongly induced in the presence of
wild-type p53. In contrast, p53(R249S), which is mutated in the DNA
binding domain, was inert in up-regulation of p21 (Fig.
1A), consistent with the
previous finding (36). We also found that induction of p21 was
diminished by mutation of activation domain I (p53(AD1
))
or activation domain II (p53(AD2
)) (Fig. 1A),
suggesting that both AD1 and AD2 are important to, if not necessary
for, the transcriptional activity of p53. This was further supported by
the evidence that simultaneous double point mutations in both AD1 and
AD2 abrogated the induction of p21 (Fig. 1A). In addition,
p53(
BD), which lacks the C-terminal basic domain, and p53(
PRD),
which lacks the proline-rich domain, had a reduced activity in inducing
p21 (Fig. 1A), although both mutants are capable of inducing
cell cycle arrest (29, 35). To rule out the possibility that
differential transcriptional activity of wild-type p53 and various p53
mutants on p21 was due to different expression level of p53 in these
stable cell lines, we performed Western blot analysis and found that
the level of these p53 mutants was comparable with that of wild-type
p53 (Fig. 1B).

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Fig. 1.
Wild-type p53 and various mutants
differentially regulate p21. A, induction of p21 by
various p53 mutants was impaired. Northern blots were prepared using
total RNA isolated from H1299 cells that were uninduced ( ) and
induced (+) to express wild-type p53 or various mutants for 24 h.
The blots were probed with cDNAs derived from the p21 and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes,
respectively. B, the level of wild-type p53 and various p53
mutants in stable cell lines was comparable. Western blots were
performed using extracts purified from uninduced cells ( ) and cells
induced to express p53 (+) as in A. Except for p53(R249S) and
p53(AD1 ), which were detected by pAb1801 and pAb421,
other p53 proteins were detected by anti-HA antibody. The level of
actin was measured as an equal loading control. C, the p21
promoter was differentially activated by wild-type p53 and various
mutants. The luciferase reporter under the control of the p21 promoter
was co-transfected into H1299 cells with a pcDNA3 control vector or
a vector that expresses wild-type p53 or various mutants. The fold
increase in relative luciferase activity by wild-type p53 or various
mutants was calculated using an empty pcDNA3 vector as a control.
D, the level of transiently transfected wild-type p53 and
various p53 mutants was comparable in H1299 cells. Western blot was
performed using extracts purified from H1299 cells transfected with a
vector expressing wild-type p53 or various p53 mutants as in
C. Anti-p21 antibody was used to detect p21. Antibodies used
to detect p53 were the same as in B.
|
|
p53(
PRD) and p53(
BD), Both of Which Have an Impaired Activity
in Inducing Endogenous p21, Are as Potent as Wild-type p53
in Activating the Transiently Transfected p21 Promoter--
The
activity of luciferase under the control of a responsive element of a
transcription factor has been widely used to measure the
transcriptional activity (37). Here, we used a luciferase reporter
under the control of the p21 promoter containing two p53-responsive
elements to determine the transcriptional activity of wild-type p53 and
various p53 mutants. We found that p53(R249S) was inactive, and
p53(AD1
) and p53(AD1
AD2
)
showed a very weak transcriptional activity on the p21 promoter (Fig.
1C). However, p53(AD2
) significantly activated
the p21 promoter (Fig. 1C), which is different from the
result obtained by Northern blot analysis (Fig. 1A,
p21 panel). Additionally, we found that p53(
BD) and
p53(
PRD) had equivalent, if not more, transcriptional activity on
the p21 promoter compared with wild-type p53 (Fig. 1C). That
both mutants induced much less endogenous p21 than wild-type p53 (Fig.
1A, p21 panel) suggests the luciferase assay does
not faithfully reveal the transcriptional activity of p53 and various
mutants in inducing the endogenous p21 gene. This is not surprising
since a transiently transfected promoter is more accessible to an
activator than the same endogenous promoter. To ensure an equivalent
expression level of wild-type p53 and various p53 mutants, we also
performed Western blot analysis and found that their expression was
comparable (Fig. 1D). Furthermore, we determined the level
of endogenous p21 induced by transiently expressed wild-type p53 and
various mutants (Fig. 1D, p21 panel). We found
that p21 was highly induced by wild-type p53, but little, if any, by
p53(R249S), p53(AD1
), p53(AD2
),
p53(AD1
AD2
), and p53(
PRD). Although p21
was slightly induced by p53(
BD), the level of its induction was
significantly less by p53(
BD) than by wild-type p53. This is
consistent with the data observed in the stable cell lines (Fig.
1A).
Wild-type p53 and Various p53 Mutants with an Intact DNA Binding
Domain Have Similar Sequence-specific DNA Binding Activity--
Except
the tumor-derived mutant p53(R249S), all of the mutants used in this
study have an intact DNA binding domain. Because the DNA binding
ability of p53 is necessary for, and correlated with, p53
transcriptional activity (10, 16, 38), it is possible that some p53
mutants have an altered DNA binding ability, which leads to decreased
induction of endogenous p21. To test this, we used ChIP-PCR to
investigate the ability of wild-type p53 and various mutants to bind
the p53-responsive elements in the p21 promoter. Because two
p53-responsive elements (RE1 and RE2) exist in the p21 promoter (Fig.
2A), we designed two pairs of
primers to amplify them separately. We found that anti-p53 antibodies (pAb1801 and pAb421) were able to pull down both RE1 and RE2 in the p21
promoter when wild-type p53 was expressed. However, neither RE1 nor RE2
was captured by a control antibody (Fig. 2, B and D), suggesting that wild-type p53 specifically binds both
RE1 and RE2. We also found that p53(AD1
),
p53(AD2
), and p53(AD1
AD2
) had
an equivalent ability to bind RE1 and RE2 compared with wild-type p53
(Fig. 2, B-E). These results are not surprising since
mutations in either AD1 or AD2 do not affect the specific DNA binding
ability of p53. Furthermore, both p53(
BD) and p53(
PRD) had an
equivalent ability to bind both RE1 and RE2 as wild-type p53 (Fig. 2,
B-E), although their ability to induce p21 was
significantly diminished. In addition, we also used anti-HA antibody to
capture p53(
BD)-DNA complex in the ChIP assay, and similar results
were obtained (data not shown). As expected, p53(R249S), which had a
mutation in the p53 DNA binding domain, was incapable of binding RE1 or
RE2 (Fig. 2, B-E).

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Fig. 2.
Wild-type p53 and various mutants with an
intact DNA binding domain have similar sequence-specific DNA binding
activity. A, schematic representation of the p21
promoter, the location of the p53-responsive elements (p53RE1 and
p53RE2), the TATA box, and the location of various primers used for
ChIP assay. B and D, wild-type p53 and various
mutants with an intact DNA binding domain bound to p53RE1 (B) and p53RE2 (D) with an
equivalent affinity. ChIP assay was performed as described under
"Experimental Procedures." p53-DNA complexes were captured with
anti-p53 antibodies (pAb421 and pAb1801). pAb419 antibody was used as a
control. C and E, the intensity of PCR fragments
in B and D was quantitated. The fold increase
induced by wild-type p53 was set as 100%. The relative binding ability
of p53 mutants was derived from the fold increase induced by mutant p53
divided by that induced by wild-type p53.
|
|
Histones Are Acetylated on the Region When Bound by p53--
When
transcription factors bind to specific sequences within a promoter,
they often recruit a co-activator with histone acetyltransferase activity to the promoter, leading to acetylation of the N-terminal tails of histones H3 and H4. Acetylation can disrupt the interaction of
core histones with DNA, thereby increasing the accessibility of the
promoter to the basal transcriptional machinery and facilitating the
initiation of transcription (3, 4). Because the ability of p53 mutants
to bind both RE1 and RE2 in the p21 promoter was not compromised, we
decided to determine whether wild-type p53 and various mutants can
equivalently increase the acetylation of histones on the regions
containing RE1 and RE2. As shown in Fig.
3, A and C,
acetylation of histones H3 and H4 on both RE1 and RE2 was increased
significantly when wild-type p53 was expressed, suggesting that
wild-type p53 can recruit histone acetyltransferases to RE1 and RE2.
However, the ability of p53(AD1
), p53(AD2
),
or p53(AD1
AD2
) to increase acetylation of
histones H3 and H4 on both RE1 and RE2 was severely compromised
compared with wild-type p53 (Fig. 3, A-D). This indicates
that these mutants probably recruit less histone acetyltransferase
activity to RE1 and RE2 than wild-type p53 despite showing equivalent
DNA binding ability as wild-type p53. This is further supported by our
finding that the ability of p53(
BD) or p53(
PRD) to increase
acetylation of histones H3 and H4 on both RE1 and RE2 was also
impaired, albeit to a lesser extent than that of
p53(AD1
), p53(AD2
), or
p53(AD1
AD2
) (Fig. 3,
A-D). Nevertheless, once p53 binds to its
responsive element, histones bound on the region containing the
responsive element become accessible for acetylation. Interestingly, we
noticed that histone H3 was always acetylated to a higher extent than histone H4 regardless of whether wild-type p53 or various mutants were
induced (Fig. 3, B and D, compare histones
H3 and H4 panels).

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Fig. 3.
Histones are acetylated on the region when
bound by p53. A and C, the histones H3- or
H4-DNA complexes bound to the region containing the p53 RE1
(A) and p53 RE2 (C) were captured by
anti-acetylated histone H3 or anti-acetylated histone H4. B
and D, the fold increase of acetylated histones bound to the
region containing p53 RE1 (B) and p53 RE2 (D).
The fold increase was the product of the amount of PCR products in the
presence of p53 divided by that in the absence of
p53.
|
|
The Level of Histone Acetylation on the Proximal p21 Promoter
Correlates with the Degree of Induction of Endogenous p21 by
p53--
The exposure of the proximal promoter, including the TATA
box, to the basal transcriptional machinery is a prerequisite for transcriptional initiation (2, 5). One of the critical processes of
chromatin structural disruption is acetylation of histones on the
proximal promoter containing the TATA box. Because some p53 mutants,
such as p53(AD1
AD2
), were deficient in
inducing endogenous p21 but still showed some, although compromised,
activity to increase the acetylation of histones on RE1 and RE2 (Fig.
3), we examined whether these p53 mutants are capable of increasing
acetylation of histones on the region proximal to the TATA box. As
shown in Fig. 4, A-B, acetylation of both histones H3 and H4 on the proximal p21 promoter was
significantly increased when wild-type p53 was induced. Acetylation of
histone H3 was also increased upon induction of p53(
BD) or p53(
PRD) (Fig. 4, A-B), although to a lesser extent than
that after induction of wild-type p53. However, little, if any,
increase in histone H3 or H4 acetylation was found when
p53(AD1
), p53(AD2
), or
p53(AD1
AD2
) was induced (Fig. 4,
A-B). Thus, upon p53 expression, the extent of histone
acetylation on the proximal p21 promoter correlates with the degree of
induction of the endogenous p21 gene.

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Fig. 4.
The level of acetylated histones
(A) and the fold increase of acetylated histones
(B) bound to the proximal p21 promoter after
p53 expression. The experiments were performed as in Fig. 3.
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|
p53 Induction of Endogenous p21 Is Impaired by Down-expression of
p300/CBP--
p300 and CBP, two histone
acetyltransferase-containing co-activators, have been found to
physiologically interact with p53 and increase the ability of p53 to
induce endogenous p21 or activate promoters of several p53 target genes
(17, 19, 20). A recent study also showed that the amount of p300/CBP
binding to the p21 promoter correlates with the level of acetylation of
histones H3 and H4 (22), indicating that the recruitment of p300/CBP to
the p21 promoter by p53 is probably responsible for histone acetylation
and subsequent activation of p21. To address what roles p300/CBP play
in the p53-dependent induction of p21, we used siRNAs
targeting the p300/CBP mRNAs. We found that p300/CBP were
specifically knocked down upon transfection of plasmids expressing p300/CBP siRNAs (Fig. 5, lanes
1 and 4) but not a control plasmid (lanes 2 and 5). Interestingly, we found that the ability of
wild-type p53 to induce p21 was much less in cells lacking p300/CBP
than in control cells (Fig. 5, compare lane 1 with
lanes 2-3). Thus, our data are consistent with, and extend,
the previous finding that the dominant-negative CBP decreases the
induction of endogenous p21 by p53 (21).

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Fig. 5.
Down-regulation of p300/CBP by siRNA impairs
the p53-dependent induction of p21. H1299 cells were
transfected with a mixture of 0.5 µg each of pBabe-U6-p300-1,
pBabe-U6-p300-2, pBabe-U6-CBP-1, and pBabe-U6-CBP-2 alone or together
with 0.1 µg of pcDNA3-HAp53. H1299 cells were also transfected
with 2 µg of control pBabe-U6 or pcDNA3 alone or together with
0.1 µg of pcDNA3-HAp53. The cell extracts were collected on the
second day after transfection. The level of p300, CBP, p53, and p21 was
detected by anti-p300, anti-CBP, anti-p53, and anti-p21, respectively.
The level of actin was also detected to ensure an equal loading.
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The Interaction of p300/CBP with Various p53 Mutants Is
Compromised--
Our study showed that down-regulation of p300/CBP
impairs the induction of endogenous p21 by wild-type p53 (Fig. 5).
Additionally, we showed that p53 mutants, which are compromised in
inducing p21, are also compromised in increasing the acetylation of
histones on the p21 promoter (Figs. 3-4). These findings prompt us to
determine whether the compromised ability of various p53 mutants to
increase the acetylation of histones on the p21 promoter and to induce p21 is due to their decreased interaction with p300/CBP. To test this,
we performed immunoprecipitation with anti-HA or anti-p53 antibodies to
capture p53-p300/CBP complexes and Western blot analysis with anti-CBP
and anti-p300 antibodies to detect the amount of p300/CBP in the p53
immunocomplexes (Fig. 6). We found that
wild-type p53 and p300/CBP interacted in vivo (Fig. 6,
lane 1), consistent with the previous observation (17, 20,
21, 23). In addition, we found that p53(
BD) and p53(
PRD) were capable of interacting with p300/CBP (Fig. 6A). However, the
amount of p300/CBP interacting with these two mutants was much less
than that interacting with wild-type p53 (Fig. 6, A,
C, and D), although the amount of wild-type p53
and various p53 mutants in the immunocomplexes was comparable (Fig.
6B). In contrast, p53(AD1
),
p53(AD2
), and p53(AD1
AD2
)
showed almost no interaction with p300/CBP (Fig. 6, A,
C, and D). This suggests that mutations in AD1
and AD2 and deletions of PRD and BD decrease the interaction between
p53 and p300/CBP, although such mutations and deletions do not
interfere with the sequence-specific DNA binding ability of p53. We
would like to note that the amount of p300/CBP, which interacts with
wild-type p53 and various mutants, is nearly proportional to the extent of histone acetylation on the proximal p21 promoter (compare Fig. 4B with Fig. 6, C-D).

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Fig. 6.
Wild-type p53 and various mutants
differentially interact with p300/CBP. A, cell extracts
were prepared from ~2 × 108 cells that were induced
to express wild-type p53 or various mutants and immunoprecipitated with
anti-HA (lanes 1 and 3-6) or anti-p53 antibodies
(pAb1801 and pAb421) (lane 2). 1% of the eluted
immunocomplexes was used for Western blot analysis to determine the
level of p53 captured by immunoprecipitation, and 90% was used to
measure the level of CBP and p300. B, the relative level of
wild-type p53 and various mutants captured by immunoprecipitation. The
amount of wild-type p53 was set as 100%. The relative level of mutant
p53 is the product of the amount of mutant p53 divided by that of
wild-type p53. C, the relative level of p300 that interacts
with p53. The relative level of p300 that interacts with mutant p53 is
the product of the amount of p300 associated with mutant p53 divided by
that associated with wild-type p53. D, the relative level of
CBP that interacts with p53. The calculation was performed as in
C.
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DISCUSSION |
The transcriptional activity of p53 is regulated by various
functional domains in p53 (10, 16, 28, 29, 35, 39). Because most
tumor-derived p53 mutants have a mutation in the DNA binding domain
that inevitably leads to p53 dysfunction (10, 40, 41), many studies
have focused on whether various domains in p53 regulate the
sequence-specific DNA binding activity of the core DNA binding domain.
Indeed, several different experimental approaches have shown that the
extreme C-terminal basic domain regulates the activity of the core DNA
binding domain in vitro. For example, the core DNA binding
activity is increased when the basic domain is deleted,
phosphorylated, acetylated, or bound by a peptide or an anti-p53
antibody (10, 19). These data lead to a hypothesis that p53 exists in
two states, latent and active (42). However, a recent NMR study
indicated that both latent and active forms of p53 are identical in
conformation (43), suggesting that the basic domain may not have any
effect on the DNA binding domain. In this study, we directly tested the
DNA binding activity in vivo and showed that the core DNA
binding activity is not affected by the basic domain (Fig. 2). In
addition, we found that p53(
PRD), p53(AD1
),
p53(AD2
), and p53(AD1
AD2
),
all of which contain an intact core DNA binding domain, have an
unaltered DNA binding ability (Fig. 2). Because these p53 mutants are
compromised in the transcriptional activity, especially in inducing
endogenous p21, the question is posed of what is responsible for
their deficiency?
Disruption of the dense chromatin structure by chromatin remodeling
complexes is a prerequisite for transcriptional activation (1, 2, 5).
Histone acetyltransferase complexes, which are well defined
chromatin remodeling complexes, acetylate the tail of histones and make
the promoter accessible to the basal transcriptional machinery (3, 6).
Recently, several studies demonstrate that p53 physically interacts
with p300/CBP both in vitro and in vivo, and
co-expression of p300/CBP can potentiate p53 to activate the p21
promoter (17, 20, 21, 23). A dominant-negative CBP mutant is able to
impair the p53-dependent induction of endogenous p21 (21).
In addition, a double-point mutation in the activation domain 1 or the
activation domain 2, which abolishes the transcriptional activity of
p53, also abolishes the interaction of p53 with CBP in vitro
(21, 23). Therefore, we have analyzed histone acetylation on the p21
promoter upon expression of p53 in vivo. We found that when
p53 binds to the responsive elements in the p21 promoter, histone
acetylation on the region proximal to the p21 promoter is nearly
proportional to the extent of p21 induction by wild-type p53 and
various mutants. Furthermore, we found that the extent of histone
acetylation on the proximal p21 promoter is also proportional to the
amount of p300/CBP that interacts with wild-type p53 and various
mutants. Additionally, we found that down-regulation of p300/CBP can
lead to impairment of p53-dependent induction of endogenous
p21. Thus, our data extend the previous observations and show for the
first time that both activation domains in p53 are necessary for
interacting with p300/CBP in vivo, histone acetylation on
the proximal p21 promoter, and subsequently, the promoter
accessibility. Furthermore, we found that although the basic domain and
the proline-rich domain are not essential for the interaction between
p53 and p300/CBP, both domains do influence the extent of this
interaction in vivo and, subsequently, histone acetylation
and the promoter accessibility.
We also mention that histone acetylation on the region surrounding the
p53-responsive elements in the p21 promoter is increased by
p53(AD1
AD2
) (Fig. 3), but the mutant is
almost completely deficient in inducing p21 (Fig. 1A) and in
interacting with p300/CBP (Fig. 6, A, C, and
D). These data suggest that once this mutant binds to the p53-responsive elements, it alters the chromatin structure of these
local regions, which concurrently allows for access of some histone
acetyltransferase-containing proteins, and subsequently, the
acetylation of histones in these regions. However, these histone acetyltransferase-containing proteins are not enough to spread the
acetylation to the region proximal to the p21 promoter. Because the
proximal promoter is the region to which the basal transcriptional machinery binds, lack of chromatin remodeling on, and subsequently, lack of accessibility of, the proximal p21 promoter are probably responsible for the deficiency of
p53(AD1
AD2
) to induce endogenous p21.