Supplemental Data
Results
Identification of two classes of genes bound by p53 in the absence of activating
stimuli
Of the identified 2432 common p53-bound fragments, only 309 were present in
the library from mock-treated cells (Figure S3). Although these 309 peaks were much
weaker, with an average area of 19, compared to 118 in nutlin-treated samples, they were
present in all four libraries, strongly suggesting that they are high confidence p53-bound
sites. 143 of these were located within 10kbp from TSS (Figure S3). Interestingly, out of
these 143 we found 48 peaks (corresponding to 30 protein-coding genes) that were
significantly increased (>3 fold) to fairly large areas (>50) upon nutlin3a treatment (Class
I, Figure S3). Notably, nearly all these peaks (96%) contained consensus p53 motif as
revealed by p53Scan program. In line with these data, the expression of nearby genes
upon p53 activation was increased in 80% of cases (Figure S4A). It appears that the
products of these genes are involved mainly in survival function of p531, i.e., regulation
of cell cycle and DNA repair (CDKN1A, DDB2) and inhibition of apoptosis (NOTCH,
MDM2, MDM4). Since p53 was bound to the promoters of this set of genes even before
its activation, they probably represent a basal p53’s transcriptional program. Indeed, p53
depletion by siRNA reduced the expression level of these genes, confirming their
regulation by p53 in the absence of stress (Figure S4B).
On the other hand, 70 relatively weak peaks (Figure S3, class II), (corresponding
to 51 protein-coding genes) did not contain p53 consensus motif, in sharp contrast to
nutlin-induced peaks with the same area, 66% of which contained p53 consensus (data
not shown). In spite of this and the absence of significant increase of p53 occupancy upon
nutlin3a, the expression of nearby genes was changed upon nutlin3a in 45% of cases,
suggesting that the binding of p53 to these sites plays a functional role (Figure S4C). The
basal level of expression of these genes is also partially controlled by p53, as it changed
upon p53 depletion (Figure S4C).
Thus, we identified two types of p53 binding to chromatin in non-treated cells that
play a functional role. Canonical class I peaks carrying p53 consensus motif are increased
proportionally to the p53 level, linked to the basal transcriptional activity of p53, and are
probably reflecting mainly the survival function of p53. Identification of noncanonical
peaks without p53 consensus, whose size is relatively low and does not depend on p53
activation, suggest the existence of additional mechanisms of transcriptional regulation
by p53, since occupation of these sites correlates with changes in gene expression upon
p53 activation (Table S11). Transcriptional regulation by p53 through its binding to
noncanonical DNA motifs has been previously demonstrated, although the mechanisms
remain to be fully elucidated2. The absence of induction of p53 binding to these sites
proportionally to the increase of p53 level suggests an alternative binding mode of p53
or/and particular chromatin structure at these sites which affects p53 interaction with
DNA3, 4.
Identification of novel p53 target genes
Next, we addressed the question, how the p53 binding to promoters correlates
with the differential gene expression. We selected p53 peaks located within 10 kb from
the TSS of an adjacent gene, which yielded 913 peaks out of 2432 (Figure 1E). Only 143
(16%) of these were present in mock-treated cells, but were significantly weaker (Figure
2F, right). We identified 734 genes in which the p53-enriched peaks in all three libraries
were located within a region encompassing -10kb of the TSS to +10kb.
Comparison with the list of known direct p53 targets5, 6 revealed that 75% (117
out of 157 genes) are bound by p53 in our experiment upon any of treatments, while 39%
(61 out of 157 genes) are detected upon all three treatments. Taking into account the
diversity of cell lines and conditions used to compile the list of p53 targets, we suggest
that at least these 61 known targets are strongly bound by p53 independently of the cell
type and mechanism of p53 activation. 75% of these 61 genes had p53 peaks after all
treatments within 10 kbp from the TSS (46 genes with 58 peaks out of 61 genes) (Table
S5, Figure 1F). The remaining 855 peaks representing 688 genes not previously described
to be regulated by p53, are likely to be novel direct p53 target genes.
Using Affymetrix microarrays, we compared the pattern of p53-bound genes with
p53-induced gene expression changes upon nutlin3a. We identified 4849 differentially
expressed genes, 1993 induced and 2856 repressed (FDR 5%) (Figure 2A). Among these,
we found 85 known direct p53 targets, including CDKN1A, BBC3, PMAIP1 and others.
Next, we analysed the peaks and genes affected upon nutlin3a treatment only.
Analysis of p53-enriched sites irrespective to the distance from the TSS and area of the
peak (non-prefiltered) showed that 78% (67 out of 85) of promoters of the known direct
p53 targets which changed expression upon nutlin3a are p53-bound (Table S7). In
addition, 39 known p53 target genes were bound by p53 without significant change in
their expression (Table S8).
On the other hand, 22% of known p53 targets were differentially expressed upon
nutlin3a treatment without detectable p53 binding (Table S9). Notably, the proportion of
repressed genes is much higher in this category, i.e. 44% of non-bound differentially
expressed genes were repressed, whereas among p53-bound genes 12% were repressed.
This suggests that p53-mediated transcriptional repression of some target genes might be
indirect, and/or the binding mode of p53 is different on repressed promoters (i.e.,
transient occupancy, binding via other TFs or far away from TSS), making it more
difficult to detect.
Of the 688 p53-bound genes, which we identified as potential novel p53 targets
by ChIP-seq, 41% (280 genes) were differently expressed upon nultin3a (Figure 2A).
4484 genes that changed their expression but were not p53-bound most likely represent
the secondary effects of p53. Alternatively, p53 might regulate some of them via binding
outside of our defined promoter region (-/+ 10 kb from the TSS).
We detected the p53 binding to the promoters of 408 genes which did not change
their expression (Figure 2A). Thus, our data suggest that the binding of p53 per se is not
sufficient to regulate gene expression.
In summary, we identified 280 novel genes whose promoters p53 binds and which
change their expression upon nutlin3a treatment. These genes are likely to be novel
functional p53 target genes (Table S10).
Clinical relevance of novel p53 target genes
To address the biological relevance of the newly identified p53 target genes in a
clinical setting, we used gene expression data obtained in 251 breast cancer patient
samples (58 p53-mutant and 193 p53-wild type tumors, as detected by sequencing7-9. By
applying unsupervised hierarchical clustering to analyze the expression of p53 targets, we
identified two clusters which correlate with the p53 status (Supplemental Figures 7A and
B). We found that p53-activated target genes showed higher expression in wild type p53
tumor samples, whereas those we found to be repressed by p53 were expressed at higher
levels in p53-mutant tumors. Thus, the expression of the genes we have identified is p53dependent in a clinical setting without external p53 modulation.
Notably, higher expression of p53-activated genes and lower expression of p53repressed genes were associated with a lower grade and less metastatic tumors
(Supplemental Figures 7A and B). Further, using Kaplan-Meier curves we showed that
the lower expression of p53-activated target genes and higher expression of repressed
genes in cluster C2 was significantly associated with poor survival (P=0.008 and
P=0.047) (Supplemental Figure 7C), suggesting that the newly identified p53 target genes
might be helpful in the clinical setting as prognostic markers.
Moreover, we found that the direct p53 target genes AURKA and ATAD2 were
among the most significantly differentially expressed genes between these two clusters
(Supplemental Figure 7D), suggesting their high importance in p53-mediated prevention
of tumor progression in patients.
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