Research Article

Chromatin Immunoprecipitationon-Chip Reveals Stress-Dependent

p53 Occupancy in Primary Normal Cells but Not in
Established Cell Lines
1

Helena Shaked, Idit Shiff, Miriam Kott-Gutkowski, Zahava Siegfried,

3
1
Ygal Haupt, and Itamar Simon

1
Department of Molecular Biology, 2Microarray Service Laboratory, The Core Research Facility, and 3Lautenberg Center for
General and Tumor Immunology, Hebrew University Medical School, Jerusalem, Israel

Abstract
The p53 tumor suppressor protein is a transcription factor
that plays a key role in the cellular response to stress and
cancer prevention. Upon activation, p53 regulates a large
variety of genes causing cell cycle arrest, apoptosis, or
senescence. We have developed a p53-focused array, which
allows us to investigate, simultaneously, p53 interactions
with most of its known target sequences using the chromatin
immunoprecipitation (ChIP)-on-chip methodology. Applying
this technique to multiple cell types under various growth
conditions revealed a profound difference in p53 activity
between primary cells and established cell lines. We found
that, in peripheral blood mononuclear cells, p53 exists in a
form that binds only a small subset of its target regions. Upon
exposure to genotoxic stress, the extent of targets bound
by p53 significantly increased. By contrast, in established
cell lines, p53 binds to essentially all of its targets irrespective
of stress and cellular fate (apoptosis or arrest). Analysis of
gene expression in these established lines revealed little
correlation between DNA binding and the induction of gene
expression. Our results suggest that nonactivated p53 has
limited binding activity, whereas upon activation it binds
to essentially all its targets. Additional triggers are most
likely required to activate the transcriptional program of p53.
[Cancer Res 2008;68(23):96717]

Introduction
The p53 protein plays a key role in the cellular response to stress
and cancer prevention, and its function is impaired in most human
cancers. The p53 protein is activated upon many types of stress,
including DNA damage, oncogene activation, and hypoxia. Its
activation leads to a variety of cellular events, among them, cell
cycle arrest, DNA repair, apoptosis, or senescence, depending on
the cell-specific and tissue-specific context and the type and extent
of the stress. The p53 protein exerts its function, in part, as a
transcription factor, which controls the expression of its target
genes through direct binding to response elements. The increas-

ingly growing list of p53-regulated genes includes those involved in

a variety of biological activities, such as apoptosis, growth arrest,
senescence, DNA repair, metabolism, cell adhesion, and differentiation (1).
The recognition of the importance of p53 in tumor development
has made it an obvious target for cancer therapy (2, 3). The
ultimate goal of these strategies is to reactivate p53 to specifically
promote apoptosis of cancer cells with minimal effect on neighboring healthy cells. While p53-induced apoptosis will eliminate
cancer cells, induction of cell cycle arrest will protect these cells
from the effects of chemotherapeutic agents. A clearer understanding of how induction of p53 leads specifically to apoptosis
and not to other cellular responses is crucial for taking advantage
of p53 in cancer therapy.
It is widely believed that p53 activation is needed for its
interaction with the DNA and that it binds and regulates different
sets of target sequences as a result of various types of stress
conditions (46). p53 differential regulation is suggested to be
mediated, at least on some targets, by cofactors, such as the ASPP
proteins (7), p63 and p73 (8), and p53 modifications, such as
phosphorylation (9), acetylation (1012), ubiquitylation (13), and
methylation (14). These results are incompatible with recent
observations that p53 occupies at least some of its target
promoters both before and after stress induction (1518).
To clarify this dispute, we decided to explore p53 association
with its target sequences on a large scale. To this end, we generated
a p53-focused array, which includes all the known paired-end ditag
(PET)-p53 target sites (19) and previously validated p53 target
promoters. Our results indicate that, in primary normal cells, p53
interacts with most of its target sequences only upon activation.
However, in established cell lines, the same DNA regions are bound
irrespective of stress conditions. In the latter, the binding to DNA
does not correlate with gene expression. Our results challenge the
p53-selective binding view and highlight the importance of
additional factors that may regulate p53 action or contribute to
the choice of cellular fate in parallel with p53.

Materials and Methods

Note: Supplementary data for this article are available at Cancer Research Online
(http://cancerres.aacrjournals.org/).
H. Shaked and I. Shiff contributed equally to this work.
Requests for reprints: Itamar Simon, Department of Molecular Biology, Hebrew
University, Jerusalem 91120, Israel. Phone: 972-2-6758544; Fax: 972-2-6758992; E-mail:
itamarsi@ekmd.huji.ac.il.
I2008 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-08-0865

www.aacrjournals.org

Array design. The p53-focused array includes 540 p53-PET sites and 62
additional p53 target regions (Supplementary Table S1), 846 randomly
chosen human promoter regions, and two types of yeast sequences, which
can be used for spike in controls (Supplementary Table S2). We PCR
amplified (average size, 800 bp) and printed (Microgrid II Compact,
Biorobotics) all these regions on GAPS IIcoated slides (Corning). The array
positions of the p53 targets and the controls were chosen randomly.
The eight yeast intergenic region spots were printed five times on each grid.
The entire array was printed four times on each slide.

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Cell growth and treatments. Primary foreskin fibroblasts transfected
with the h-TERT gene (FF-T), HCT116, HCT116 (p53 / , a generous gift from
Prof. B. Vogelstein, Johns Hopkins University), U2OS, U2OS p53 small
interfering RNA (siRNA), and LacZ siRNA cells (a generous gift from
Prof. M. Oren, Weizmann Institute) were grown in McCoys5A or DMEM
supplemented with 10% FCS to subconfluence. Buffy coats from healthy
blood donors were acquired through the blood bank. Peripheral blood
mononuclear cells (PBMC) were isolated by Ficoll gradient centrifugation
using Lymphocyte Separation Medium (MP Biomedicals). Cells were washed
in PBS, and isolated cells were counted and tested for viability by trypan blue
exclusion. Cells were cultured in RPMI 1640 with 10% FCS in the presence of
phytohemagglutinin (PHA; 5 Ag/mL); 12 h later, interleukin-2 (IL-2; 3 ng/mL)
was added, and 48 h later, cells were irradiated.
Chromatin immunoprecipitationon-chip. Chromatin immunoprecipitation (ChIP)-on-chip analysis was performed essentially, as previously described (20), using 10 Ag anti-p53 antibody DO1 (Santa Cruz).
For most experiments, 5 to 10  107 cells were used. In experiments
with untreated PBMCs, 8-fold more cells were used to compensate for the
lower amounts of p53 protein in these cells. Under these conditions,
the amount of p53 in the untreated PBMC samples was in excess to the
amount detected in other nontreated cells (Supplementary Fig. S4). In
the PBMC ChIP-on-chip experiments, immunoprecipitation and input DNA
from several donors were pooled to avoid donor-specific effects. The array
was scanned and analyzed with GenePix Pro software, and the fluorescence
intensity in both channels was obtained for each spot. As the array is spotted
four times, median Cy3 and Cy5 intensities were calculated for each spot.
The two channels were normalized according to the median intensity of the
random human promoter spots using LOWESS normalization, and the Cy5/
Cy3 ratio of each spot was calculated. The experiment was performed in
either triplicate or duplicate (Supplementary Table 3), and the average
binding ratio for each spot was calculated. The significance of the
enrichment observed in each spot was determined by calculating the
deviation of each ratio from the mean of the random promoters control
spots (Z score). Only f1% of the random promoters obtained Z of >2.5;
thus, this cutoff is equivalent to an FDR of 0.01. See Supplementary Table S3
for processed data (raw data was deposited in ArrayExpress, accession
A-MEXP 1036). For gene-specific validation, the ChIP assay was performed as
described above and the nonamplified immunoprecipitation and input
fractions were subjected to 36 cycles of semiquantitative PCR.
Reverse transcriptionPCR. RNA was extracted from HCT116 or
HCT116 (p53 / ) cells treated as described in the text, using TriPure
reagent (Roche). Semiquantitative PCR was performed on cDNA (in two
concentrations) in the presence of 32P-dCTP using appropriate primer pairs.
The number of PCR cycles for each gene was chosen to keep the linearity,
varying from 20 to 28 cycles. The products were run on a 6% acrylamide gel
and quantified from the phosphoimager using TINA software. The average
intensity and the SD were calculated between the repeats and normalized to
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression. For
this analysis, we chose genes bound by p53 either within 1 kb upstream of
the TSS or in the first intron, within 5% of the gene.
Fluorescence-activated cell sorting and Western analysis. Cells were
fixed with ethanol, stained with propidium iodine, and subjected to
fluorescence-activated cell sorting (FACS) analysis for DNA content. For
Western analysis, the cellular lysates were separated on a denaturing
polyacrylamide gel, with equal protein amounts loaded on the gel for each
sample, then transferred to a nitrocellulose membrane and incubated with
mouse anti-p53 (DO-I; Santa Cruz), goat antih-actin (I-19; Santa Cruz),
rabit anti-p53 (FL-393; Santa Cruz), or mouse anti-GAPDH (GAPDH-71.1;
Sigma) antibodies and horseradish peroxidaseconjugated secondary
antibodies. The signal was visualized via enhanced chemiluminescence
reaction and exposure to film.
Bioinformatics. The p53 binding site score was calculated with the
PoSSum software (21) using the PSSM of the two p53 half sites from (19).
The score of two half sites <14 bp apart was added, and the maximum
score for each region was used for further analysis. For the correlation of
p53 binding enrichment and its binding site (Fig. 1C), we used a sliding
window of 20.

Cancer Res 2008; 68: (23). December 1, 2008

Figure 1. ChIP-on-chip analysis using the p53-focused array. A, scatter plot

of the results of a representative ChIP-on-chip experiment performed on
HCT116 cells treated with 5-FU for 6 h. p53-PET sites (triangles ) and known p53
sites (rectangles ) are shown in black, whereas the random human promoter
regions are shown in gray. In a control ChIP-on-chip experiment using HCT116
(p53 / ) cells, no enrichment for p53 targets was observed (Supplementary
Fig. S1). B, p53-PET sites were divided into six groups according to the PET
cluster size (19), and the average enrichment (Z score) for each group is shown.
C, all p53-PET sites were ranked according to their p53-binding site scores,
and the p53-binding enrichment for each site is reported (see Materials and
Methods).

Results
p53-focused array. To monitor p53 binding to its target sites
under multiple conditions, we developed a p53-focused microarray
containing most of the experimentally validated p53 binding sites
in the human genome. Our array contains all the genomic locations identified by Wei and colleagues (19) as putative p53-binding
sites and 62 additional genomic loci previously identified to be
associated with p53 (see Supplementary Table S1), among them are

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promoters of genes involved in cell cycle control, DNA repair, and

apoptosis. We also included, on the array, a variety of negative
controls for normalization and spike in controls (see Materials and
Methods for details).
The p53-focused array is ideal for validating the p53-binding
sites reported by the ChIP-PET method. To this end, we used the
same cells, HCT116 cells expressing wt p53, under the same
experimental conditions used by Wei and colleagues We performed
ChIP using anti-p53 antibody (DO-1) on HCT116 cells treated for
6 hours with 5-fluorouracil (5-FU) and hybridized the enriched
DNA to the p53-focused array. Even with a fairly stringent cutoff
(FDR < 1%, see Materials and Methods), we detected binding of
p53 to 453 of 540 (83%) p53-PET sites (Fig. 1A), confirming that the
list of 540 p53-PET sites is highly enriched for regions occupied by
p53 in these cells under these conditions.
Binding sites may differ in their affinity to p53 and, thus, in the
amount of time they are occupied by p53. Such differences are
captured by the ChIP-PET method as the number of different
sequences mapped to the same overlapping region (cluster size)
and by the ChIP-on-chip method as the enrichment measured for
each region. Comparison of the promoter occupancy measured
by both methods (Fig. 1B) indicates that, to a certain extent,
quantitative information can be derived from our ChIP-on-chip
data. Interestingly, we also found a certain correlation between the
observed enrichment of a site and the strength of the p53
consensus binding site it contains (Fig. 1C), suggesting that
differences in enrichment indeed reflect differences in p53-binding
affinity to the DNA. Overall, these results are highly consistent with
those published by Wei and colleagues (19).
p53 interaction with chromatin of primary cells is enhanced
upon stress. p53 is activated in a variety of situations, including

genotoxic and oncogenic stresses (6). To study p53-DNA interactions in a nonactivated state, we decided to use primary cells
exposed to minimal perturbations. To this end, we isolated PBMCs,
and used the ChIP-on-chip methodology for the identification
of p53 interactions to most of its known targets using the p53
focused array. Cell cycle proliferation was induced in PBMCs
by treatment with PHA and IL-2. Cells were then exposed to
g-irradiation (5 Gy) and harvested at different time points after
exposure. As expected, p53 levels were increased in response to
irradiation (Fig. 2A), and a significant increase in apoptosis was
observed (Fig. 2B). p53-DNA interactions were monitored both
before and 4 hours after irradiation by ChIP followed by
hybridization to the p53-focused array (ChIP-on-chip). We found
that in the nonactivated primary cells, p53 is bound to a small
set of target sequences, including many of the well-studied
p53 targets (such as CDKN1A, DDB2, and GADD45). On the other
hand, after irradiation, a dramatic increase in the number of
genomic sites occupied by p53 was observed from 62 sites before to
175 sites after irradiation (Fig. 2C). Semiquantitative PCR confirms
the array resultsregions that were bound only after irradiation
(such as PET367 and PET494) showed significant increase in p53
occupancy whereas regions prebound by p53 (such as PET32 and
PET180) did not (Fig. 2D). Taken together our results show that
p53-DNA interactions are increased upon g-irradiation in PBMCs.
p53-DNA interactions in established cell lines. In contrast to
normal cells, in which p53 is inactive before exposure to genotoxic
stress, in established cell lines, p53 may be activated by oncogenic
or culturing stress even under normal growth conditions (22).
Thus, we applied our comprehensive ChIP-on-chip methodology
to determine p53 occupancy in a variety of established cell lines.
Normal foreskin fibroblasts immortalized with the h-TERT gene

Figure 2. p53-DNA interactions in primary blood cells. A, Western blot analysis of PBMCs with p53 and h-actin antibodies at the indicated time points after g-irradiation
(5 Gy). B, FACS analysis of PBMCs with no treatment or irradiated with g (5 Gy). The cells were analyzed 24 h after the treatment. Percentages of cells in sub
G1 and S phases. C, scatter plot of the results (only p53-PET and known targets) of a representative ChIP-on-chip experiment performed on PBMCs before (gray ) and
after (black ) g-irradiation. The diagonal dashed line represents the cutoff used for the analysis (Z > 2.5). D, validation of p53 targets by semiquantitative PCR.
Immunoprecipitation-enriched DNA (IP ) and dilutions of input DNA (In ) were subjected to PCR with primers specific for the indicated p53-PET sites or for the promoter
region of EDN3 (a negative control). Note the distinct characteristics of the two types of targets.

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Figure 3. p53-DNA interaction in established cell lines. A, FACS analysis of FF-T, HCT116, and U2OS cells untreated or treated with genotoxic stresses
(12 Gy g-irradiation, 375 Amol/L 5-FU, and 10 Gy g-irradiation, respectively). The cells were analyzed 24 to 48 h after the treatment. Percentages of cells in sub-G1 and
S phases. B, Western blot analysis with p53 and h-actin antibodies on FF-T, HCT116, and U2OS cells at the indicated time points after treatments as in A. C,
for the indicated cell types, the fraction of p53 target loci bound by p53 in untreated cells of the total number of loci interacting with p53 in those cells. The difference
between PBMCs and the other cell lines is highly significant (P < 10 8, Fisher exact test). D, validation of p53 targets by semiquantitative PCR. immunoprecipitationenriched DNA (IP ) and dilutions of input DNA (In ) from HCT116 cells not treated or treated with 375 Amol/L 5-FU were subjected to PCR with primers specific
for the indicated p53-PET sites or for the promoter region of EDN3 (a negative control). Note that, in all regions tested, p53 was found on the DNA, even in not treated
cells. Similar results were obtained in HCT116 cells exposed to other types of stress (Supplementary Fig. S2).

(FF-T) and tumor-derived lines HCT116 and U2OS were exposed to

different genotoxic stress (12 Gy, 375 Amol/L 5-FU, and 10 Gy,
respectively) to activate p53. These treatments stabilize p53 and
cause either apoptosis (in HCT116 cells) or cell cycle arrest (in the
FF-T and U2OS cells; Fig. 3A and B). ChIP-on-chip analysis of
p53-DNA interactions performed before and 5 hours (FF-T and
U2OS) or 6 hours (HCT116) after the stress revealed that, in
contrast to the PBMCs, in all three cell lines, p53 is bound to most
of its target locations before the genotoxic stress (Fig. 3C). For
example, 418 of 453 sites (92%) occupied by p53 in HCT116 cells
treated with 5-FU were also detected in untreated HCT116 cells.
Moreover, most of the remaining 35 regions show marginal
enrichment of p53 binding also in the nontreated cells. Semiquantitative PCR analysis of the ChIP samples further confirmed
the conclusion that, in established cell lines, p53 occupies almost
all its targets both before and after activation (Fig. 3D). These
results are in line with previous experiments using limited specific
promoter analysis in established cell lines (1518).
To determine whether the extensive binding seen in untreated,
transformed, or immortalized cells can be attributed only to the
high p53 levels in these cells, compared with PBMCs, we used U2OS
cells expressing p53 siRNA to lower the levels of p53. Western blot
analysis shows that p53 levels in these cells are similar to levels in
PBMCs (Fig. 4A). Performing ChIP-on-chip on untreated U2OS p53
siRNA cells revealed that p53 was bound to 71% of the targets
bound in untreated U2OS cells (Fig. 4B), suggesting that low levels
of p53 are sufficient to support extensive DNA binding.
Similar p53 binding profiles lead to different cellular
outcomes in cancer cell lines. To examine the effect of different

Cancer Res 2008; 68: (23). December 1, 2008

stress treatments on p53 occupancy, we exposed HCT116 and

U2OS cells to various genotoxic agents, leading to either apoptosis
(5-FU in HCT116 and 15 J/cm2 UV irradiation in U2OS) or cell cycle
arrest (0.17 Amol/L doxorubicin and 1.5 Amol/L HU in HCT116
cells and 10 Gy g-irradiation in U2OS cells). In both cell lines,
p53-binding profiles were essentially the same under all tested
conditions (Fig. 5A and B) and p53 was bound to the promoters of
apoptotic genes (such as BAX, NOXA and TNFRSF10B), cell cycle
regulation genes (such as CDKN1A and SNK), and DNA repair
genes (such as DDB2, PCNA, and RRM2B) under all conditions.
These results suggest that cell fate is not determined exclusively
by p53 binding to the DNA.
To examine the effect of cumulative DNA damage on p53-binding
profile, we exposed HCT116 cells to doxorubicin (0.17 Amol/L) for
longer times (24 hours) and performed ChIP-on-chip experiments.
p53 binding profile after 24 hours of treatment was essentially
identical to that observed after 6 hours of treatment (Fig. 5C).
Binding of p53 to promoters is insufficient for expression
regulation. Our observation that, in established cell lines, p53
constitutively binds its target sequences raised the question as
to whether these are transcriptionally active. To address this
question directly, we measured the mRNA levels of selected
p53 targets, all of which are constitutively associated with p53.
We performed reverse transcriptionPCR (RT-PCR) on RNA isolated from HCT116 and HCT116 (p53 / ) cells grown in the
absence or presence of different genotoxic stresses (Fig. 6). The
results reveal that, although p53 is associated with the promoters
of all of these genes, their pattern of expression seems to be
distinct. The expression of some of these genes is affected by

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p53-DNA Interaction

These results support the notion that in established cell lines

differential gene regulation by p53 is not determined solely at the
level of p53-DNA interaction with subsets of its target promoters.

Discussion

Figure 4. The effect of p53 cellular levels on p53-DNA interaction. A,

Western blot analysis with p53 and GAPDH antibodies on untreated PBMCs,
U2OS, U2OS LacZ siRNA, and U2OS p53 siRNA cells. B, Venn diagram
depicting the overlap between p53 targets in untreated U2OS and U2OS p53
siRNA cells. The number in parentheses under each cell type represents the
number of p53 targets bound by p53 in that experiment. ChIP-on-chip on
U2OS LacZ siRNA cells gave almost identical results to U2OS cells
(Supplementary Table S3).

p53 even in untreated cells (CDKN1A, RPS27L, and APBB2 were

induced by p53 and S100A2 was repressed); others show p53
dependency upon exposure to stress (Fig. 6, second panel), whereas a
third group did not show any dependency on p53 in their expression
patterns (at least at the time point examined; Fig. 6, third panel).

To study the effect of genotoxic stress on p53-DNA interactions,

we investigated p53 binding to most of its known binding sites in
a variety of cell types. There has been an ongoing debate in the
p53 field as to whether stress, which induces posttranslational
modifications of p53, promotes p53 binding to its DNA targets.
Initially, it was thought that stress induces binding of p53 to the
DNA. However, subsequent studies did not provide evidence for the
existence of p53 in a nonDNA-binding state in unstressed cells
(18). Our findings help to resolve this apparent discrepancy. On one
hand, in unstressed PBMCs, we show p53 binding to only a subset
of its target regions. Importantly, exposure of the PBMCs to
irradiation significantly increased the number of target regions
bound by p53. On the other hand, in multiple established cell lines
(FF-T, U20S, and HCT116), the binding of p53 to DNA occurred,
irrespective of stress stimulation. Although the levels of p53 protein
in untreated PBMCs are quite low, the observed low p53 occupancy
is not due to the limitation in the sensitivity of the ChIP-on-chip
technique, because we compensated for p53 protein levels by using
8-fold more chromatin extract in each untreated PBMC reaction
(Supplementary Fig. S4).
p53 in the established cell lines differs from p53 in untreated
PBMCs in two respects: (a) p53 levels, which are lower in PBMCs
and (b) p53 activation. In established cell lines, such as HCT116
and U2OS, which were derived from tumors, the cells are under
constant oncogenic stress (22). The FF-T cells, although not
oncogenic, have been grown in culture for an extended period of
time, which has been shown to activate p53 (2325). Furthermore,
these cells were immortalized by the introduction of the h-TERT
gene, a process that was shown to affect the p53 pathway (26).
In contrast to these established cell lines, the PBMCs are not

Figure 5. Stress-independent p53

occupancy in cancer cells. A, heat map
representation of ChIP-on-chip results.
The Z scores of all the p53-PET sites are
shown for the indicated experiments. The
different stresses lead to activation of p53
and to distinct cellular responses. 5-FU and
UV lead to apoptosis, whereas Dox, HU,
and g-irradiation cause cell cycle arrest
(Supplementary Fig. S3). B, correlation
matrix representing the Pearson correlation
values between ChIP-on-chip results
obtained in the various growth condition in
HCT116 and U2OS cells. Four hundred
seventy-one targets that showed binding in
at least one condition tested were included
in this analysis. Note that, in all cases, the
correlation was >0.8 between cell types
and 0.9 within cell types, indicating that
there are almost no differences in p53
occupancy in the various conditions.
C, scatter plot representing the binding
intensity (Z score) of p53 to all spots
represented on the array after short (6 h)
or long (24 h) exposure of HCT116 cells to
doxorubicin (0.17 Amol/L).

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Figure 6. Expression analysis. Semiquantitative RT-PCR analysis of selected genes in HCT116 or HCT116
(p53 / ) cells exposed to various growth conditions for 6 h. The expression values in the bar graphs represent the
enrichment over the expression in the untreated HCT116 cells, after averaging two repeats and normalization to
GAPDH . Error bars represent 1 SD. The genes were grouped according to their expression patterns (see text).

transformed and were kept in culture only for a few days, a stress
that was insufficient to induce p53 and to promote its binding to
target DNA. Thus, in these primary cells, p53 exists in its native and
low activity state. To identify which of these two differences is the
main reason for the limited binding activity of p53 in PBMCs,
we used the U2OS p53 siRNA cells, in which activated p53 is
maintained at low levels. Our finding of extensive p53 binding in
these cells (Fig. 4) reveals that low levels of activated p53 are
sufficient for its DNA-binding activity. Taken together, these results
support the p53 latency model, which suggests that p53 activation
is crucial to increase its DNA-binding activity.
In the established cell lines, p53 is found on most of its target
sequences before its activation (Fig. 3) and remains bound
regardless of cell fate (Fig. 5), suggesting that p53 interaction with
the DNA is insufficient for carrying out its function. Indeed,
analysis of the RNA levels (in HCT116 cells) of several genes, all
bound by p53, reveals divergent results. Some of the genes are
induced in a p53-dependent manner already before exposure to
genotoxic stress, whereas others are either induced only after p53
activation or are unaffected by p53 binding (Fig. 6). Our results
raise the question of what determines p53 ability to regulate its
target genes? One possibility is that the quantity of p53 bound to its
targets determines gene regulation. Although genotoxic stress
caused an increase in p53 levels in the cells, we did not observe

Cancer Res 2008; 68: (23). December 1, 2008

significant differences in p53 occupancy before and after stress.

This may be due to limitations in the quantitative abilities of our
methods. Indeed, an increase in p53 promoter occupancy in response to genotoxic stress has been reported by others (15, 16, 18).
Another possibility is that modification of p53 determines p53
activity (27). For instance, acetylation of p53 was shown to increase
its activity (28, 29), and specific acetylations were associated with
subsets of p53 targets (1012). A third possibility is that genotoxic
stress induces various p53 cofactors, which can contribute to
p53 specificity. Indeed, recent experiments have shown that p53
activation of certain genes is affected by cofactors, such as the
ASPP proteins (7), p63 and p73 (8), Mdm2 (30), and Hzf (31).
We have found that the global pattern of p53 occupancy to its
genomic response elements does not change significantly among
different cell lines treated with distinct stimuli, which, although
activate p53, lead to different cellular responses (Fig. 5). These
results suggest that the above-mentioned modifications and cofactors do not affect p53 binding and probably contribute only to
differential gene activation. Whereas this conclusion is in
agreement with some previous works (1013, 3234), it contradicts
other studies that provide several examples in which p53 association with apoptotic genes is affected by cofactors or modifications (79, 31, 35). Although it is possible that p53 binding to
promoter regions of selected genes is regulated by additional

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factors, we would like to point out that some of these studies were
carried out by overexpressing or knocking-out key p53 cofactors
or modifiers that could influence p53-DNA interaction. Our results
suggest that, under physiologic levels of these proteins, p53 binds
all of its target sequences, and this pattern does not change upon
extended exposure to genotoxic stress (Fig. 5C). However, we
cannot rule out that such changes may occur at a later time
point as has been recently shown for U20S cells treated with
etoposide (31).
In conclusion, our results in the context of previous studies
suggest that p53-mediated induction of target genes cannot be
explained by a simple model. Naive p53 seems to have a very
limited activity. Many types of stress signals, including extended
culturing of the cells, are sufficient for the activation of p53 to
achieve its full DNA-binding capacity. However, this interaction
is insufficient for the induction of gene expression. p53 transactivation function requires additional triggers, such as cofactors

References
1. Vousden KH, Lane DP. p53 in health and disease. Nat
Rev Mol Cell Biol 2007;8:27583.
2. Haupt S, Haupt Y. Manipulation of the tumor
suppressor p53 for potentiating cancer therapy. Semin
Cancer Biol 2004;14:24452.
3. Haupt S, Haupt Y. Importance of p53 for cancer onset
and therapy. Anticancer Drugs 2006;17:72532.
4. Meek DW. Mechanisms of switching on p53: a
role for covalent modification? Oncogene 1999;18:
766675.
5. Oren M. Decision making by p53: life, death and
cancer. Cell Death Differ 2003;10:43142.
6. Vousden KH. Outcomes of p53 activation-spoilt for
choice. J Cell Sci 2006;119:501520.
7. Samuels-Lev Y, OConnor DJ, Bergamaschi D, et al.
ASPP proteins specifically stimulate the apoptotic
function of p53. Mol Cell 2001;8:78194.
8. Flores ER, Tsai KY, Crowley D, et al. p63 and p73 are
required for p53-dependent apoptosis in response to
DNA damage. Nature 2002;416:5604.
9. Oda K, Arakawa H, Tanaka T, et al. p53AIP1, a
potential mediator of p53-dependent apoptosis, and its
regulation by Ser-46-phosphorylated p53. Cell 2000;102:
84962.
10. Knights CD, Catania J, Di Giovanni S, et al. Distinct
p53 acetylation cassettes differentially influence geneexpression patterns and cell fate. J Cell Biol 2006;173:
53344.
11. Sykes SM, Mellert HS, Holbert MA, et al. Acetylation
of the p53 DNA-binding domain regulates apoptosis
induction. Mol Cell 2006;24:84151.
12. Tang Y, Luo J, Zhang W, Gu W. Tip60-dependent
acetylation of p53 modulates the decision between cellcycle arrest and apoptosis. Mol Cell 2006;24:82739.
13. Le Cam L, Linares LK, Paul C, et al. E4F1 is an
atypical ubiquitin ligase that modulates p53 effector
functions independently of degradation. Cell 2006;127:
77588.

www.aacrjournals.org

or modifications that may also contribute to its specificity. Further

research is needed to decipher the identity and role of these factors
in p53 activation of target genes.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments
Received 3/6/2008; revised 9/3/2008; accepted 9/23/2008.
Grant support: FP6 funding of the European Commission contract 503576 and
Israel Cancer Association through the Ber-Lehmsdorf Memorial Fund in memory of
the late Prof. Natan Trainin. This publication reflects only the authors views. The
European Commission is not liable for any use that may be made of the information
herein.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Yoav Smith and Shlomit Farkash-Amar for assistance in data analysis
and Gali Hager-Price, Nathalie Friedman, and Ariella Simon for assistance in printing
the arrays.

14. Huang J, Perez-Burgos L, Placek BJ, et al. Repression

of p53 activity by Smyd2-mediated methylation. Nature
2006;444:62932.
15. Ceribelli M, Alcalay M, Vigano MA, Mantovani R.
Repression of new p53 targets revealed by ChIP on chip
experiments. Cell Cycle 2006;5:110210.
16. Espinosa JM, Verdun RE, Emerson BM. p53 functions
through stress- and promoter-specific recruitment of
transcription initiation components before and after
DNA damage. Mol Cell 2003;12:101527.
17. Imbriano C, Gurtner A, Cocchiarella F, et al. Direct
p53 transcriptional repression: in vivo analysis of
CCAAT-containing G2/M promoters. Mol Cell Biol
2005;25:373751.
18. Kaeser MD, Iggo RD. Chromatin immunoprecipitation analysis fails to support the latency model for
regulation of p53 DNA binding activity in vivo . Proc Natl
Acad Sci U S A 2002;99:95100.
19. Wei CL, Wu Q, Vega VB, et al. A global map of p53
transcription-factor binding sites in the human genome.
Cell 2006;124:20719.
20. Lee TI, Johnstone SE, Young RA. Chromatin immunoprecipitation and microarray-based analysis of protein location. Nat Protocol 2006;1:72948.
21. Beckstette M, Homann R, Giegerich R, Kurtz S. Fast
index based algorithms and software for matching
position specific scoring matrices. BMC Bioinformatics
2006;7:389.
22. Horn HF, Vousden KH. Coping with stress: multiple
ways to activate p53. Oncogene 2007;26:130616.
23. Brummelkamp TR, Fabius AW, Mullenders J, et al. An
shRNA barcode screen provides insight into cancer cell
vulnerability to MDM2 inhibitors. Nat Chem Biol 2006;2:
2026.
24. Sedelnikova OA, Horikawa I, Zimonjic DB, Popescu
NC, Bonner WM, Barrett JC. Senescing human cells and
ageing mice accumulate DNA lesions with unrepairable
double-strand breaks. Nat Cell Biol 2004;6:16870.
25. Mendrysa SM, Perry ME. The p53 tumor suppressor
protein does not regulate expression of its own

9677

inhibitor, MDM2, except under conditions of stress.

Mol Cell Biol 2000;20:202330.
26. Noble JR, Zhong ZH, Neumann AA, Melki JR, Clark SJ,
Reddel RR. Alterations in the p16(INK4a) and p53 tumor
suppressor genes of hTERT-immortalized human fibroblasts. Oncogene 2004;23:311621.
27. Espinosa JM, Emerson BM. Transcriptional regulation by p53 through intrinsic DNA/chromatin binding
and site-directed cofactor recruitment. Mol Cell 2001;8:
5769.
28. Barlev NA, Liu L, Chehab NH, et al. Acetylation of
p53 activates transcription through recruitment of
coactivators/histone acetyltransferases. Mol Cell 2001;
8:124354.
29. Luo J, Li M, Tang Y, Laszkowska M, Roeder RG, Gu W.
Acetylation of p53 augments its site-specific DNA
binding both in vitro and in vivo . Proc Natl Acad Sci
U S A 2004;101:225964.
30. White DE, Talbott KE, Arva NC, Bargonetti J. Mouse
double minute 2 associates with chromatin in the
presence of p53 and is released to facilitate activation of
transcription. Cancer Res 2006;66:346370.
31. Das S, Raj L, Zhao B, et al. Hzf Determines cell
survival upon genotoxic stress by modulating p53
transactivation. Cell 2007;130:62437.
32. Budhram-Mahadeo VS, Bowen S, Lee S, et al. Brn-3b
enhances the pro-apoptotic effects of p53 but not its
induction of cell cycle arrest by cooperating in transactivation of bax expression. Nucleic Acids Res 2006;34:
664052.
33. Homer C, Knight DA, Hananeia L, et al. Y-box factor
YB1 controls p53 apoptotic function. Oncogene 2005;24:
831425.
34. Schumm K, Rocha S, Caamano J, Perkins ND.
Regulation of p53 tumour suppressor target gene
expression by the p52 NF-nB subunit. EMBO J 2006;25:
482032.
35. Wei G, Li AG, Liu X. Insights into selective activation
of p53 DNA binding by c-Abl. J Biol Chem 2005;280:
122718.

Cancer Res 2008; 68: (23). December 1, 2008