The pro-longevity gene FoxO3 is a direct target of the p53 tumor suppressor
VM Renault1, PU Thekkat1, KL Hoang1, JL White1, CA Brady2,3, D Kenzelmann Broz2,
OS Venturelli1, TM Johnson2,3, PR Oskoui1, Z Xuan4, EE Santo5, MQ Zhang4,6, H Vogel7,
LD Attardi1,2,3and A Brunet1,3
1Department of Genetics, Stanford University, Stanford, CA, USA;2Department of Radiation Oncology, Stanford University,
Stanford, CA, USA;3Cancer Biology Graduate Program, Stanford University, Stanford, CA, USA;4Department of Molecular and
Cell Biology, Center for Systems Biology, University of Texas at Dallas, Richardson, TX, USA;5Department of Human Genetics,
Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands;6MOE Key Laboratory of Bioinformatics &
Bioinformatics Division, TNLIS, Tsinghua University, Beijing, China and7Department of Pathology, Stanford University,
Stanford, CA, USA
FoxO transcription factors have a conserved role in
longevity, and act as tissue-specific tumor suppressors
in mammals. Several nodes of interaction have been
identified between FoxO transcription factors and p53, a
major tumor suppressor in humans and mice. However,
the extent and importance of the functional interaction
between FoxO and p53 have not been fully explored.
Here, we show that p53 regulates the expression of
FoxO3, one of the four mammalian FoxO genes, in
response to DNA damaging agents in both mouse
embryonic fibroblasts and thymocytes. We find that p53
transactivates FoxO3 in cells by binding to a site in the
second intron of the FoxO3 gene, a genomic region
recently found to be associated with extreme longevity in
humans. While FoxO3 is not necessary for p53-dependent
cell cycle arrest, FoxO3 appears to modulate p53-
dependent apoptosis. We also find that FoxO3 loss does
not interact with p53 loss for tumor development in vivo,
although the tumor spectrum of p53-deficient mice appears
to be affected by FoxO3 loss. Our findings indicate that
FoxO3 is a p53 target gene, and suggest that FoxO3 and
p53 are part of a regulatory transcriptional network that
may have an important role during aging and cancer.
Oncogene advance online publication, 21 March 2011;
Keywords: FoxO transcription factors; p53; tumor
suppression; cancer; aging; longevity
Aging and cancer are intimately linked. Many cancers
have a striking age-dependent onset. Interventions that
extend lifespan, such as dietary restriction, decrease the
incidence of tumors (Hursting et al., 2003; Masoro,
2005). The connection between aging and cancer raises
the possibility that genes that extend lifespan may also
be part of a molecular network that suppresses
tumorigenesis. An example for such genes is provided
by FoxO transcription factors, which have a pivotal role
at the interface between longevity and tumor suppres-
sion (Greer and Brunet, 2005). In invertebrates, FoxO
factors are necessary to extend lifespan downstream of
the insulin pathway (Kenyon, 2005). In mammals, the
four FoxO family members (FoxO1, FoxO3, FoxO4
and FoxO6) also function downstream of the insulin-
signaling pathway (Greer and Brunet, 2005). Single-
nucleotide polymorphisms in the FoxO3 gene have
recently been found to be associated with extreme
longevity in humans, suggesting a conserved function
for FoxO3 in longevity (Willcox et al., 2008; Anselmi
et al., 2009; Flachsbart et al., 2009; Pawlikowska et al.,
2009). Interestingly, the FoxO family has also been
found to act as a lineage-specific tumor suppressor in
mammals (Paik et al., 2007). Combined somatic deletion
of FoxO1, FoxO3 and FoxO4 in mice leads to the
development of tumors, particularly thymic lymphomas
and hemangiomas (Paik et al., 2007). FoxO3?/?mice can
also develop cancer, but at a lesser frequency and later
in life than FoxO1/FoxO3/FoxO4 compound mutant
mice (Paik et al., 2007). In humans, FoxO3 inactivation
is correlated with poor prognosis of breast cancers (Hu
et al., 2004). Conversely, ectopic expression of FoxO3 in
human cells is sufficient to delay tumor development in
xenograft models (Hu et al., 2004; Seoane et al., 2004).
Thus, FoxO3 may be an important part of a regulatory
network that controls both aging and cancer.
FoxO3 is a potent transcriptional activator that
triggers the expression of a program of genes involved
in cell cycle arrest, DNA repair, hypoxia response and
apoptosis (Brunet et al., 1999; Medema et al., 2000;
Tran et al., 2002; Seoane et al., 2004; You et al., 2006a;
Bakker et al., 2007; Tothova et al., 2007; Paik et al.,
2009; Renault et al., 2009). FoxO3 transcriptional
activity is inhibited in response to insulin and growth
factors through phosphorylation-dependent nuclear
export (Brunet et al., 1999, 2001, 2002). Although the
regulation of FoxO3 activity by post-translational
Received 20 December 2010; accepted 18 January 2011
Correspondence: Dr A Brunet, Department of Genetics, Stanford
University, 300 Pasteur Drive, Alway M336, Stanford, CA 94305,
Oncogene (2011) 1–15
& 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11
modifications, such as phosphorylation, has been well
studied (Van Der Heide et al., 2004; Calnan and Brunet,
2008), the molecular mechanisms that regulate the
expression of the FoxO3 gene remain mostly unclear.
Given the connection between aging and cancer, it is
interesting to note that there are a number of parallels
between FoxO3 and the tumor suppressor protein p53.
Like FoxO3, p53 induces cell cycle arrest, apoptosis and
DNA repair (Vousden and Lu, 2002). Several FoxO3
target genes, such as Gadd45, Wip1, p21Cip1, Puma and
Sestrin1/PA26, are also regulated by p53 (Kastan et al.,
1992; el-Deiry et al., 1993; Fiscella et al., 1997; Velasco-
Miguel et al., 1999; Nakano and Vousden, 2001; Yu
et al., 2001). FoxO3 and p53 are extensively modified in
response to stress stimuli, through phosphorylation
and acetylation (Calnan and Brunet, 2008; Vousden
and Prives, 2009), and both p53 and FoxO3 bind to and
are deacetylated by the Sirt1 deacetylase (Luo et al.,
2001; Vaziri et al., 2001; Brunet et al., 2004; Motta et al.,
2004). These extensive similarities between FoxO3 and
p53 suggest that both transcription factors may be part
of a common regulatory complex.
A number of direct and indirect links between FoxO3
and p53 have already been uncovered. First, FoxO3
directly binds to p53, at least in the context of
overexpression (Nemoto et al., 2004; Wang et al.,
2008). Second, FoxO3 leads to stabilization of the p53
protein and activation of p53-dependent apoptosis
(You et al., 2006b). FoxO3 also upregulates p19ARF, a
positive upstream regulator of p53 (Bouchard et al.,
2007). Conversely, p53 has been reported to inhibit
FoxO3 function indirectly by upregulating serum- and
glucocorticoid-induced protein kinase (SGK), thereby
resulting in the phosphorylation of FoxO3 and its
sequestration in the cytoplasm (You et al., 2004). In
addition, p53 has been found to inhibit FoxO3
transcriptional activity under conditions of oxidative
stress (Miyaguchi et al., 2009) and to induce FoxO3
degradation (Fu et al., 2009). Whether FoxO3 and p53
intersect in other ways, for example by regulating each
other’s transcription, remains largely unknown.
p53 is a potent tumor suppressor in humans, as
underscored by the fact that nearly all human tumors
have mutations or deletions in the p53 gene itself or in
the p53 pathway (Vogelstein et al., 2000). Mutations in
p53 have been linked to poor prognosis in a variety of
human cancers, including lung (Quinlan et al., 1992),
breast (Deng et al., 1994) and gastric cancers (Scott
et al., 1991), as well as lymphomas (Gaidano et al., 1991;
Lo Coco et al., 1993). Consistent with the prevalence of
p53 loss in human tumors, p53?/?mice are highly prone
to cancer early in life (Donehower et al., 1992; Harvey
et al., 1993a, 1993b; Jacks et al., 1994). p53þ/?mice also
develop tumors with high frequency (Donehower et al.,
1992; Harvey et al., 1993a, 1993b; Jacks et al., 1994).
The connection between FoxO3 and p53 in cells raises
the possibility that FoxO3 functionally interacts with
p53 for tumor suppression. A dominant-negative form
Myc-driven tumorigenesis by blocking p53-dependent
apoptosis (Bouchard et al., 2007). However, the genetic
interaction between FoxO3 and p53 loss in cancer
progression in the absence of oncogenic stimulation
has never been tested.
Here, we explore the connections between FoxO3, a
ubiquitously expressed FoxO family member, and p53 in
cells and in mice. We find that p53 acts as a direct
upstream transcriptional activator of the FoxO3 gene in
response to DNA damage in mouse embryonic fibro-
blasts (MEFs) and in lymphocytes. We show that p53
regulates the transcription of the FoxO3 gene by binding
to a site in the second intron of the FoxO3 gene.
Although FoxO3 is not necessary for p53-dependent
cell cycle arrest, FoxO3 appears to have a role in
p53-dependent apoptosis. While FoxO3 loss does not
synergize with p53 loss for tumor development in vivo,
tumor spectrum in p53-deficient mice appears to be
affected by the loss of one or both FoxO3 alleles. These
results reveal a regulatory mechanism linking FoxO3
and p53, two critical molecules involved in the control of
longevity and tumor suppression.
DNA damage and nutlin treatment increase FoxO3
protein levels in a p53-dependent manner in fibroblasts
To test whether p53 regulates FoxO3 expression in
mammalian cells, we compared FoxO3 protein levels in
p53þ/þand p53?/?primary MEFs in the absence or
presence of doxorubicin, a DNA damaging agent that
activates endogenous p53. We found that doxorubicin
treatment increased FoxO3 protein expression in p53þ/þ
MEFs, but not in p53?/?MEFs (Figure 1a). Changes in
FoxO3 protein levels were similar to those of p21Cip1, a
well-known target of p53 (Figure 1a). To activate p53
in a more specific manner, we used nutlin, a chemical
compound that inhibits binding of p53 to Mdm2, a
0 4 8 4 8
4 8 4 8
expression that is p53-dependent in MEFs. (a) Western blot of
protein extracts from p53þ/þand p53?/?MEFs incubated in the
absence (?) or presence (þ) of doxorubicin (Dox, 0.2mg/ml) for 8h,
using antibodies to FoxO3, p21Cip1(a well-known target of p53), p53
and Mek1 (loading control). (b) Western blot of protein extracts from
p53þ/þand p53?/?MEFs incubated with nutlin (10mM), a p53
activator, or doxorubicin (Dox, 0.2mg/ml) for 0, 4 and 8h, using
antibodies to FoxO3, p53 and b-actin (loading control). Western
blots are representative of at least two independent experiments,
conducted on independent cultures of MEFs.
Doxorubicin and nutlin elicit an increase in FoxO3 protein
FoxO3 is a direct target of p53
VM Renault et al
ubiquitin ligase critical for p53 degradation (Vassilev
et al., 2004). Similar to what we observed for
doxorubicin, nutlin treatment increased FoxO3 protein
in p53þ/þMEFs, but not in p53?/?MEFs (Figure 1b).
Together, these results indicate that p53 is necessary
for FoxO3 protein accumulation in MEFs in response to
DNA damage and nutlin.
p53 is necessary for FoxO3 mRNA upregulation
in response to DNA damage or nutlin treatment
To determine whether the p53-dependent accumulation
of FoxO3 protein is due to transcriptional or post-
transcriptional changes, we compared FoxO3 mRNA
levels in p53þ/þand p53?/?MEFs in response to nutlin
or doxorubicin (Figure 2a). We found that nutlin or
doxorubicin led to an upregulation of FoxO3 mRNA
that was significantly attenuated in p53?/?MEFs
(Figure 2a), similar to two known p53 targets, p21Cip1
and Mdm2 (Figures 2b and c). We noted that FoxO3
mRNA expression at basal levels is lower in p53?/?
MEFs than in p53þ/þMEFs, whereas FoxO3 protein
expression is similar in MEFs of both genotypes
(see Figure 1), suggesting that there are additional levels
of regulation of the FoxO3 protein by p53. In contrast
to FoxO3, other FoxO family members (FoxO1, FoxO4
and FoxO6) did not show a p53-dependent increased
mRNA expression in response to nutlin and doxorubi-
cin (Figures 2d–f). FoxO6 mRNA was induced in
response to doxorubicin, but in a p53-independent
manner (Figure 2f), raising the possibility that other
members of the p53 family (for example, p73) may be
responsible for FoxO6 regulation in response to DNA
damage. Collectively, these observations indicate that
p53 is necessary for the upregulation of FoxO3 mRNA
in MEFs in response to signals that activate p53.
p53 is necessary for FoxO3 mRNA upregulation in
response to DNA damage in lymphocytes
We next asked whether the induction of FoxO3 mRNA
by p53 was also observed in other cell types. We found
that FoxO3 mRNA was upregulated in response to
FoxO3 mRNA /Gapdh mRNA
p21Cip1 mRNA /Gapdh mRNA
Mdm2 mRNA /Gapdh mRNA
FoxO1 mRNA /Gapdh mRNA
FoxO4 mRNA /Gapdh mRNA
FoxO6 mRNA /Gapdh mRNA
*** *** ***
analysis of FoxO3 (a), p21Cip1(b), Mdm2 (c), FoxO1 (d), FoxO4 (e), and FoxO6 (f) mRNA levels in p53þ/þand p53?/?MEFs in
response to 4 and 8h of treatment with nutlin (10mM) or doxorubicin (Dox, 0.2mg/ml). Mean þ/? s.e.m. of two independent
experiments conducted in triplicate. *Po0.05, **Po0.01, ***Po0.001 between p53þ/þand p53?/?MEFs at a given time point,
two-way analysis of variance with Bonferroni post-test.
p53 is necessary for FoxO3 mRNA upregulation in response to doxorubicin or nutlin in MEFs. Real-time quantitative PCR
FoxO3 is a direct target of p53
VM Renault et al
g-irradiation in mouse thymocytes, but that this
upregulation was no longer observed in p53?/?thymo-
cytes (Figure 3a). The changes in FoxO3 mRNA levels
in thymocytes were similar to the changes observed for
two well-known targets of p53, p21Cip1(Figure 3b)
and Mdm2 (Figure 3c). The expression of other FoxO
family members (FoxO1, FoxO4 and FoxO6) was not
strongly upregulated in response to g-irradiation in a
p53-dependent manner (Figures 3d–f), although FoxO1
mRNA was slightly affected by p53 (Figure 3d). These
findings indicate that the p53-dependent regulation of
FoxO3 mRNA by DNA damaging agents is relatively
specific tothis FoxOisoform,
in multiple cell types.
and is observed
p53 transcriptional activity is necessary and sufficient
to regulate FoxO3 mRNA in fibroblasts
To determine whether FoxO3 mRNA upregulation in
response to doxorubicin is mediated by p53’s ability to
act as a transcriptional activator, we examined FoxO3
mRNA levels in knock-in MEFs expressing a transcrip-
tionally impaired mutant of p53 under the control of the
endogenous p53 promoter (Figure 4a). This mutant
p53 is transcriptionally deficient because leucine 25 and
tryptophan 26, two key residues for p53 transcriptional
activity, are replaced by glutamine and serine, respec-
tively (Johnson et al., 2005). In addition, the transcrip-
tionally impaired p53 allele and a control wild-type
(WT) allele of p53 were rendered inducible by the
presence of a transcriptional/translational STOP cas-
sette flanked by LoxP sites upstream of the p53 coding
region (p53LSL?WTand p53LSL?25,26) (Figure 4a) (Johnson
et al., 2005). In p53LSL?WTand p53LSL?25,26knock-in
MEFs, introduction of Cre recombinase allows expres-
sion of the WT or transcriptionally impaired p53 alleles
(Johnson et al., 2005). Northern blot experiments
revealed that the expression of FoxO3 mRNA was
higher in p53LSL?WTMEFs in the presence of Cre (that
is, expressing WT p53) than in p53LSL?WTMEFs in the
absence of Cre (that is, not expressing p53), similar to
p21Cip1mRNA, a classical p53 target. These results
confirm that p53 is necessary for expression of the
FoxO3 mRNA (Figure 4b). In the presence of Cre,
doxorubicin elicited a significant increase in FoxO3 and
p21Cip1mRNA in p53LSL?WTMEFs, but not in p53LSL?25,26
MEFs (Figure 4b). There was a slight upregulation of
FoxO3 and p21Cip1mRNA in p53LSL?25,26MEFs in the
presence of Cre and doxorubicin (Figure 4b), consistent
with the fact that the p5325,26mutant is not completely
Together, these data indicate that p53 transcriptional
/ Gapdh mRNA
analysis of FoxO3 (a), p21Cip1(b), Mdm2 (c), FoxO1 (d), FoxO4 (e), and FoxO6 (f) mRNA levels in p53þ/þand p53?/?thymocytes, 3h
after g-irradiation (g-IR, 10Gy). Meanþ/? s.e.m. of two independent experiments conducted in triplicate on samples from 3–5 mice
per genotype. *Po0.05, ***Po0.001 between p53þ/þand p53?/?thymocytes at a given time point, two-way analysis of variance with
p53 is necessary for FoxO3 mRNA upregulation in response to g-irradiation in thymocytes. Real-time quantitative PCR
FoxO3 is a direct target of p53
VM Renault et al
activity is necessary for the DNA damage-dependent
increase in FoxO3 mRNA.
To test whether p53 transcriptional activation is
sufficient to induce FoxO3 mRNA, we used MEFs with
a knock-in mutation of p53 fused to the transactivation
domainof the herpes virus
(p53LSL?VP16, Figure 4a), thus rendering p53 maximally
active (Johnson et al., 2005, 2008). We observed that
similar to p21Cip1mRNA, FoxO3 mRNA was potently
induced in Cre-treated p53LSL?VP16MEFs, even in the
absence of doxorubicin (Figure 4b). These findings
indicate that p53 transactivation activity is sufficient
for FoxO3 mRNA upregulation. Taken together,
these results indicate that FoxO3 gene expression is
transcriptionally regulated by p53.
p53 directly binds and transactivates regulatory regions
in the FoxO3 gene
To determine whether FoxO3 is a direct target gene of
p53, we searched the 5-kb upstream regulatory region,
as well as all introns of the mouse FoxO3 gene, for the
presence of putative p53 binding sites, based on the
known p53 consensus binding site (el-Deiry et al., 1992).
This analysis identified four potential p53 binding
elements: three (p53-1, p53-2 and p53-3) in the FoxO3
promoter, and one (p53-4) in the second intron of the
FoxO3 gene (Figure 5a). p53-4 has a near-perfect match
to the p53 consensus binding site and only one base pair
of spacing between the two half-sites (Figure 5a), which
is a characteristic of optimal p53 binding sites (el-Deiry
et al., 1992). In addition, although the region containing
p53-4 is not perfectly conserved in human FoxO3, there
is also an optimal p53 binding site in the second intron
of the human FoxO3 gene, suggesting that the presence
of p53 binding site in the second intron may be a
conserved feature of the FoxO3 gene. Interestingly, the
second intron of the human FoxO3 gene contains single-
nucleotide polymorphisms associated with extreme
human longevity (Willcox et al., 2008; Anselmi et al.,
2009; Flachsbart et al., 2009).
To assess whether p53 binds to these p53 binding
sites in MEFs, we performed chromatin immunopreci-
pitation (ChIP) assays with antibodies to p53 in
chromatin extracts from MEFs that had been treated
with doxorubicin. The recruitment of p53 to the
regulatory regions of the FoxO3 gene was assessed by
quantitative PCR using primers surrounding the p53-1
or p53-4 binding sites (Figure 5b). We found that
endogenous p53 occupied the p53-4 binding site in the
intron of the FoxO3 gene in doxorubicin-treated MEFs
(Figure 5b). In contrast, p53 was not bound to the p53-
1, p53-2, or p53-3 binding sites in the FoxO3 promoter
(Figure 5b and data not shown). Endogenous p53 was
bound to the p53-4 binding site in the intron of the
FoxO3 gene even in the absence of doxorubicin, but the
recruitment of p53 to that site was slightly increased in
(Figure 5c). p53 occupancy at the p53 binding site in
FoxO3 second intron was similar to that in the p21Cip1
promoter, although p53 recruitment to the p21Cip1
promoter in response to short treatment with doxor-
ubicin was more robust (Figure 5c). Taken together,
these results indicate that p53 directly binds to a site
within the second intron of the mouse FoxO3 gene
p53 is necessary for FoxO3 transactivation
To determine whether p53 can transactivate the FoxO3
gene by binding to the p53-4 binding site, we generated
reporter constructs in which luciferase expression is
driven by an SV40 minimal promoter fused to a 500-bp
region surrounding p53-1, p53-2, p53-3 or p53-4, and
analyzed the luciferase activity of these reporter
constructs in p53þ/þand p53?/?MEFs (Figure 5d and
data not shown). The region surrounding the p53-4 site
in the FoxO3 second intron induced transcriptional
DNA binding domainAD1
FoxO3 mRNA upregulation. (a) Schematic of the p53 knock-in
alleles used. p53LSL?WT: inducible allele encoding a form of wild-
type p53. p53LSL?25,26: inducible allele encoding a transcriptionally
impaired mutant of p53 in which leucine 25 is replaced by a
glutamine, and tryptophan 26 is replaced by a serine. p53LSL?VP16:
inducible allele encoding a mutant of p53 in which the transactiva-
tion domains (AD1 and AD2) are replaced by the transactivation
domain of VP16. AD: activation domain; Olig.; oligomerization
domain. The star indicates the location of the 25,26 mutations.
(b) Northern blot analysis of MEFs in which the endogenous allele
of p53 has been replaced by an allele encoding inducible forms of
WT p53 (p53LSL?WT), a transcription-deficient mutant (p53LSL?25,26),
or a mutant of p53 in which the transactivation domains of p53
were replaced by that of VP16 (p53LSL?VP16). The addition of an
adenovirus containing Cre recombinase (Ad-Cre) allows the
deletion of the Lox-STOP-Lox (LSL) cassette upstream of each
allele and allows the expression of each p53 variant. Cells were
exposed to 8h of doxorubicin (Dox, 0.2mg/ml). Northern blots
were analyzed with a probe to FoxO3, p21Cip1(a known target of
p53) and GAPDH (loading control).
p53 transcriptional activity is necessary and sufficient for
FoxO3 is a direct target of p53
VM Renault et al
activation of the luciferase reporter gene in p53þ/þ
MEFs, but not in p53?/?MEFs (Figure 5d). In contrast,
the transcriptional activity of the p53-1, p53-2 and p53-3
regions was low, regardless of the presence of p53
(Figure 5d, data not shown). Note that the region
surrounding the p53-4 binding site induced transcrip-
tional activation of the luciferase reporter gene in a p53-
dependent manner even in the absence of doxorubicin,
perhaps because transfection itself triggered a stress
to the cells or because basal FoxO3 mRNA levels are
also regulated by p53 (see Figure 2a). Together, these
findings indicate that the region surrounding p53-4 in
the second intron of the FoxO3 gene can be transacti-
vated by p53.
To examine whether the transcriptional activity of the
region containing p53-4 was indeed due to this p53
binding site, we created a luciferase reporter construct
with a mutated version of p53-4 (p53-4m) that can no
+/+ -/- +/+ -/- +/+ -/-
p53 IgG p53 IgG p53 IgG p53
(p53-1, p53-2, p53-3 and p53-4) in the promoter and second intron of the mouse FoxO3 gene. R: G or A; W: T or A; Y: C or T; E:
exon; I: intron. Also depicted are the consensus for p53 binding sites, the p53 binding site in the p21Cip1promoter, and the mutant of
critical bases in p53-4 (p53-4m). (b) ChIP on MEFs treated with doxorubicin (Dox, 0.2mg/ml) for 16–20h, using antibodies to p53
(colored bars) or control IgG (white bars). The chromatin bound to p53 or to the control IgG was analyzed by quantitative PCR with
primers surrounding a region that did not contain p53 binding sites (?), the distal p53 binding site in the FoxO3 promoter (p53-1), the
p53-4 binding site in the FoxO3 intron 2 (p53-4), and the p53 binding site in the p21Cip1promoter (p53 (p21Cip1)). The fold enrichment
over the IgG control is represented. Mean þ/? s.e.m. of three independent experiments. **Po0.01, one-way analysis of variance.
(c) ChIP on p53þ/þand p53?/?MEFs in the absence or presence of doxorubicin (Dox, 0.2mg/ml) for 6h, using antibodies to p53 or
control IgG. The chromatin bound to p53 or to the control IgG was analyzed by quantitative PCR with primers surrounding a region
that did not contain p53 binding sites (?), the distal p53 binding site in the FoxO3 promoter (p53-1), the p53-4 binding site in the
FoxO3 intron 2 (p53-4) and the p53 binding site in the p21Cip1promoter (p53 (p21Cip1)). The fold enrichment over the IgG control is
represented. Mean þ/? s.d. from triplicates of one experiment. (d) Normalized activity of luciferase reporter constructs driven by
500bp surrounding the p53 binding sites p53-1 or p53-4 in p53þ/þ(black) and p53?/?(white) MEFs. Mean þ/? s.e.m. of four
independent experiments conducted in triplicate. *Po0.05 between p53-4 and control in p53þ/þMEFs, **Po0.01 between p53þ/þ
and p53?/?MEFs for p53-4, one-way ANOVA. (e) Normalized activity of luciferase reporter constructs driven by the region
surrounding the p53-4 binding site or by the region surrounding the p53-4 binding site in which the p53 binding site was mutated (p53-
4m) in p53þ/þ(black) and p53?/?(white) MEFs. Mean þ/? s.e.m. of three independent experiments conducted in triplicate.
**Po0.01 between p53-4m and p53-4 in p53þ/þMEFs, one-way ANOVA.
p53 is recruited to a binding site in the second intron of the FoxO3 gene. (a) Location and sequence of the p53 binding sites
FoxO3 is a direct target of p53
VM Renault et al
longer be bound by p53 because it is missing the critical
bases necessary for p53 binding (Figure 5a). Mutating
the p53-4 site abolished the transcriptional activity of
the luciferase reporter gene (Figure 5e). These experi-
ments indicate that p53 binding to p53-4 in FoxO3
second intron is pivotal for the regulation of the FoxO3
gene by p53.
p53-induced FoxO3 does not have a role in cell cycle
arrest in MEFs
p53 is necessary for cell cycle arrest in response to
double-strand breaks in MEFs (Kastan et al., 1992).
FoxO3 can also promote cell cycle arrest when over-
expressed in cells (Medema et al., 2000), although
FoxO3 is not necessary for cell cycle arrest in response
to DNA damage in MEFs (Castrillon et al., 2003). To
test whether the upregulation of FoxO3 by p53 mediates
part of the cell cycle arrest controlled by p53, we isolated
MEFs from FoxO3þ/þp53þ/þ(þ/þ), FoxO3?/?and
p53?/?mice and assessed Bromodeoxyuridine (BrdU)
incorporation in these cells in response to doxorubicin
(Figure 6a) and nutlin (Figure 6b). We found that
FoxO3?/?MEFs underwent cell cycle arrest to the same
extent as þ/þ MEFs in response to doxorubicin
(Figure 6a) and nutlin (Figure 6b). In contrast, p53?/?
MEFs were partially resistant to cell cycle arrest caused
by doxorubicin (Figure 6a) and completely resistant to
cell cycle arrest induced by nutlin (Figure 6b). These
results indicate that unlike p53, FoxO3 is not necessary
for DNA damage- and p53-dependent cell cycle arrest in
MEFs or that there is compensation by another factor,
perhaps another FoxO isoform (see below). Figure 6c
shows that FoxO3 was dispensable for cell cycle arrest
induced by long-term treatment by chronic oxidative
stress (H2O2) and by hydroxyurea, which both lead to
p53-dependent cellular senescence (Marusyk et al., 2007)
(TMJ and LDA, unpublished). However, FoxO3?/?
MEFs displayed significant cell cycle arrest compared
with þ/þ MEFs in basal long-term culture conditions
(Figure 6c), suggesting that FoxO3 loss may itself induce
cell cycle arrest over several cellular passages. Consistent
with the absence of role for FoxO3 in p53-dependent cell
cycle arrest, the expression of p27Kip1, a well-known
target of FoxO3 involved in cell cycle arrest (Kops et al.,
2002), did not change in response to nutlin or
doxorubicin in p53þ/þMEFs, and was even upregulated
in p53?/?MEFs (Figure 6d). These observations indicate
that the p53-dependent upregulation of FoxO3 mRNA
and protein is not accompanied by an increase in FoxO3
transcriptional activity toward p27Kip1, which is likely
due to the fact that the FoxO3 protein is exported to the
cytoplasm in response to DNA damage in different cell
types (AB, data not shown) (You et al., 2004).
The role of FoxO3 in p53-dependent cell cycle arrest may
be masked by compensation by other FoxO family
FoxO3 loss has been found to be compensated by other
FoxO family members (Bouchard et al., 2007; Paik
et al., 2007). Even though other FoxO family members
are not regulated by p53 to the same extent as FoxO3 in
MEFs and thymocytes (see Figures 2 and 3), we
determined whether interfering with more than one
FoxO family member had a more pronounced impact
on cell cycle arrest than interfering with FoxO3 alone.
We found that nutlin-induced cell cycle arrest was
attenuated in MEFs infected with lentiviruses expressing
a short hairpin RNA directed to several FoxO family
members (‘pan FoxO’) (Hribal et al., 2003) (Figures 6e
and f), although nutlin still caused some cell cycle arrest
in these cells. These findings suggest that the FoxO
family partially contributes to p53-dependent cell cycle
arrest and that the role of FoxO3 in p53-dependent
cell cycle arrest may be masked by compensation by
other FoxO isoforms.
p53-induced FoxO3 appears to have some role
FoxO has been shown to be important for p53-
dependent apoptosis in the context of Myc oncogene-
transformed cells (Bouchard et al., 2007). We asked
whether FoxO3 had a role in p53-dependent apoptosis
in MEFs. MEFs do not usually respond to p53
activation by undergoing apoptosis, unless they have
the adenovirus E1A protein (Lowe et al., 1993). We
found that nutlin treatment combined with serum
starvation slightly enhanced the percent of cells under-
going apoptosis in E1A-transformed wild-type MEFs
(Figure 7a). FoxO3?/?E1A-transformed MEFs ap-
peared to be slightly impaired in their ability to undergo
apoptosis in response to nutlin and serum starvation,
although this effect was more modest than that observed
in p53?/?E1A-transformed MEFs (Figure 7a). We also
found that Bim, a well-known FoxO3 target involved
in apoptosis (Dijkers et al., 2000), was upregulated in
response to g-irradiation in thymocytes and that this
upregulation was dependent on p53 (Figure 7b). Taken
together, these results are consistent with the notion that
FoxO3 contributes, at least in part, to p53-mediated
FoxO3 loss does not cooperate with p53 loss for tumor
suppression in mice, but appears to have an impact on
Our study and published findings indicate that FoxO3
and p53 interact in many different ways in cells.
Although the relevance of the interaction between
FoxO3 and p53 in vivo has been tested in a mouse
lymphoma model (Bouchard et al., 2007), it has not
been assessed in the absence of oncogenic stimulation.
Loss of one or both p53 alleles in mice results in
predisposition to cancer and death at an early age
(Donehower et al., 1992; Jacks et al., 1994). In contrast,
FoxO3?/?mice develop tumors only at a very low
frequency, and tumor development in these mice
manifests late in life (Paik et al., 2007), probably due
to the redundancy of FoxO family members. To test
whether FoxO3 and p53 interacted in vivo for overall
survival and tumor survival, we generated a cohort
FoxO3 is a direct target of p53
VM Renault et al
of compound FoxO3/p53 mutant mice in a mixed
FVB/N-129Sv/J background and monitored overall
survival in these mice (Figure 8). Loss of one or both
alleles of FoxO3 did not accelerate the mortality rate of
p53?/?mice in this genetic background (Figure 8a).
Similarly, loss of one or both alleles of FoxO3 did not
significantly affect the survival rate of p53þ/?mice
(Figure 8b), indicating that FoxO3 and p53 loss do not
cooperate to diminish overall survival in vivo. This result
further suggests that a model lacking an activated
oncogene may not be sufficient to reveal FoxO3
interaction with p53 for tumor suppression. Alterna-
tively, the absence of synergy between FoxO3 and p53
loss for tumor development could be due to the fact that
both molecules are the in same genetic pathway.
Histopathological analysis on a subset of FoxO3/p53
8 48 0 48 4 8
FoxO3 loss. (a) Percent BrdU-positive cells in FoxO3þ/þp53þ/þ(þ/þ), FoxO3?/?and p53?/?MEFs in the presence or absence of
doxorubicin (Dox, 0.2mg/ml) for 24h. Mean þ/? s.e.m. of three independent experiments, two of which were conducted
with independent MEF cultures from distinct animals. ***Po0.001 between þ/þ and p53?/?MEFs for the same treatment; ns: non
significant between þ/þ and FoxO3?/?MEFs for the same treatment, two-way analysis of variance with Bonferroni post-test.
(b) Percent BrdU-positive cells in FoxO3þ/þp53þ/þ(þ/þ), FoxO3?/?and p53?/?MEFs in the presence or absence of nutlin (10mM)
for 24–36h. Mean þ/? s.e.m. of three independent experiments. *Po0.05, ***Po0.001 between þ/þ and p53?/?MEFs for the same
treatment; ns: nonsignificant between þ/þ and FoxO3?/?MEFs for the same treatment, two-way ANOVA with Bonferroni post-test.
(c) Percent BrdU-positive cells in FoxO3þ/þp53þ/þ(þ/þ), FoxO3?/?and p53?/?MEFs in the presence or absence of chronic
treatment of H2O2or hydroxyurea. Mean þ/? s.e.m. of two independent experiments with different lines of MEFs. **Po0.01,
***Po0.001 between þ/þ and FoxO3?/?or p53?/?MEFs for the same treatment; ns: non significant between þ/þ and FoxO3?/?
MEFs for the same treatment, two-way ANOVA with Bonferroni post-test. (d) Real-time quantitative PCR analysis of p27Kip1mRNA
levels in p53þ/þand p53?/?MEFs in response to 4 and 8h of treatment with nutlin (10mM) or doxorubicin (Dox, 0.2mg/ml). Meanþ/?
s.e.m. of two independent experiments conducted in triplicate. ***Po0.001 between p53þ/þand p53?/?MEFs at a given time point,
two-way ANOVA with Bonferroni post-test. (e) Percent BrdU-positive cells in MEFs infected with control lentiviruses (pSicoR (PSR))
or with lentiviruses expressing a short hairpin RNA to FoxO family members (PSR ‘pan FoxO’) in the presence or absence of
nutlin (Nutlin, 10mM) for 24h. The data are expressed as fold decrease, respective to the value obtained in the absence of nutlin.
Meanþ/? s.e.m. of two independent experiments. **Po0.01, between PSR and PSR ‘pan FoxO’-infected MEFs in the presence of
nutlin, two-way ANOVA with Bonferroni post-test. (f) Western blot of protein extracts from MEFs infected with control lentiviruses
(PSR) or with lentiviruses expressing a short hairpin RNA to FoxO family members (PSR ‘pan FoxO’), using antibodies to FoxO1,
FoxO3, FoxO4, FoxO6 and b-actin.
FoxO3 is not necessary for p53-dependent cell cycle arrest in MEFs, but other FoxO family members may compensate for
FoxO3 is a direct target of p53
VM Renault et al
compound mutant mice revealed that the loss of FoxO3
in the context of p53?/?mice resulted in the appearance
of tumors, such as adenocarcinomas and angiolipomas
(Figures 8c and d). As these tumor types are rarely seen
in p53?/?mice in the same genetic background (Sharp-
less et al., 2002), this result is consistent with the notion
that FoxO3 loss may impact the tumor spectrum of
p53-deficient mice. Collectively, these findings suggest
that FoxO3 and p53 are part of a common transcrip-
tional network that may affect cellular and organismal
responses that are important to counter aging and
Our results indicate that p53 transactivates the expres-
sion of the FoxO3 gene by binding to a site located in
the second intron of the FoxO3 gene. The regulation of
FoxO3 gene expression is likely an important mechan-
ism for the generation of a pool of FoxO3 molecules
that could be made active or inactive by post-transla-
tional modifications. Our data indicate that DNA
damage upregulates the expression of FoxO3 mRNA.
FoxO3 gene expression has been shown to be upregu-
lated in response to a number of other environmental
stimuli, including nutrient deprivation, growth factor
deprivation and hypoxia (Furuyama et al., 2002; Imae
et al., 2003; Bakker et al., 2007; Essaghir et al., 2009),
raising the possibility that p53 might transduce the
expression of FoxO3 in response to some of these
stimuli. The induction of FoxO3 mRNA by hypoxia
in MEFs is dependent on hypoxia inducible factor 1-a
(Bakker et al., 2007), although whether hypoxia
inducible factor 1 directly binds to FoxO3 regulatory
regions is not known. In addition, E2F1, a transcription
factor involved in cell cycle progression and apoptosis,
has been shown to upregulate FoxO3 mRNA in human
neuroblastoma cell lines by binding to two conserved
sites in the promoter of the human FoxO3 gene (Nowak
et al., 2007). These observations raise the possibility that
p53, hypoxia inducible factor 1 and E2F1 may all
interact in controlling the expression of the FoxO3 gene.
The regulation of FoxO3 gene expression by p53 may
be specific to a subset of tissues or cell types. FoxO3 and
p53 are both expressed relatively ubiquitously, but may
function more prominently in some tissues/cells versus
others. A cell-type specific regulation has indeed been
observed for the FoxO3 gene. For example, FoxO3
mRNA is upregulated by E2F1 in neuroblastoma cell
lines and in U2OS cells, but not in HeLa cells, human
diploid foreskin fibroblasts or PC12 cells (Nowak et al.,
2007). Similarly, FoxO3 mRNA expression is down-
regulated by growth factors in human AG01518
fibroblasts, but not in BJ-hTert fibroblasts (Essaghir
et al., 2009). Whether the regulation of FoxO3 by p53,
which we have identified in MEFs and thymocytes, is
observed in all cell types will be interesting to test. While
our study was being completed, another study reported
that in normal adult mouse liver, p53 could bind a
response element 4kb upstream of the FoxO3 transcrip-
tional start site and transactivate FoxO3 mRNA
expression (Kurinna et al., 2010). During hepatic
regeneration, the binding of p53 to the binding site
upstream of the FoxO3 gene was disrupted, leading to a
decrease in FoxO3 mRNA expression. Consistent with
our findings, p53-dependent upregulation of FoxO3 was
also observed in MEFs and in mouse hepatoma cells
overexpressing p53 (Kurinna et al., 2010). Interestingly,
another member of the p53 family, p73, also binds to the
same regulatory region in the promoter of the FoxO3
gene (Kurinna et al., 2010). Together with our study,
these findings indicate that the p53 family of transcrip-
tion factors regulates FoxO3 in a number of different
cell types, by binding to at least two different binding
sites in the FoxO3 gene, one 4kb upstream of FoxO3
transcriptional start site and one in the second intron of
the FoxO3 gene (our study). It is possible that p53
occupancy at different sites is dependent on cell type or
specific environmental conditions.
p53 and FoxO3 interact at many levels: (1) p53 and
FoxO3 proteins physically interact (Nemoto et al., 2004;
Wang et al., 2008); (2) p53 and FoxO3 share common
target genes (Zhao et al., 2000; Tran et al., 2002;
Vousden and Lu, 2002; Jacobs et al., 2007; Riley et al.,
Percent cleaved caspase 3
Bim mRNA /Gapdh
(a) Percent cleaved caspase 3-positive cells in E1A-transformed
MEFs (FoxO3þ/þp53þ/þ(þ/þ), FoxO3?/?and p53?/?) in the
absence of treatment (?) or in response to nutlin (N), serum
starvation (S), and nutlinþserum starvation (NþS). Mean þ/?
s.e.m. of three independent experiments. (b) Real-time quantitative
PCR analysis of Bim mRNA levels in p53þ/þ
thymocytes, 3h after g-irradiation (g-IR, 10Gy). Mean þ/?
s.e.m. of two independent experiments conducted in triplicate on
samples from 3–5 mice per genotype. *Po0.05 between p53þ/þand
p53?/?thymocytes at a given time point, two-way analysis of
variance with Bonferroni post-test.
FoxO3 hasa role in p53-dependentapoptosis.
FoxO3 is a direct target of p53
VM Renault et al
2008); (3) FoxO3 stabilizes p53 protein (You et al.,
2006b); (4) FoxO3 indirectly activates p53 by upregulat-
ing p19ARF, which inhibits the p53 ubiquitin ligase
Mdm2 (Bouchard et al., 2007); (5) p53 indirectly inhibits
FoxO3 activity by inducing SGK (You et al., 2004), or
more directly, by inhibiting FoxO3 transcriptional
activity (Miyaguchi et al., 2009) and by inducing FoxO3
degradation through Mdm2 (Fu et al., 2009); and
(6) FoxO3 is a p53 target gene (this study and (Kurinna
et al., 2010)). Thus, p53 and FoxO3 likely form a
regulatory network that elicits appropriate cellular
responses in response to stress stimuli. Negative and
positive feedback loops within this transcriptional
circuit could be beneficial for triggering a finely tuned
response to cellular stresses. The observation that p53
upregulates FoxO3 mRNA but also indirectly inhibits
FoxO3 protein activity (You et al., 2004; Fu et al., 2009;
Miyaguchi et al., 2009) suggests that co-incident signals
may be needed to activate the FoxO3 molecules
generated by p53-dependent transcription. Careful
analysis of the kinetics of FoxO3 mRNA upregulation
and FoxO3 protein activation in response to co-
occurring signals will be required to tease apart the
molecular links between p53 and FoxO3.
The presence of a binding site for p53 in the second
intron of the FoxO3 gene is a feature that is conserved in
the human genome. Mining the genome-wide ChIP data
available for p53 (Wei et al., 2006) revealed that p53
binds to FoxO3 second intron in the human genome in
human HCT116 cells. These observations suggest that
the binding of p53 in the second intron of the FoxO3
gene may be crucial for the regulation of FoxO3 gene
FoxO3-/- p53-/- (n=21)
FoxO3+/- p53-/- (n=29)
FoxO3+/+ p53-/- (n=8)
FoxO3-/- p53+/- (n=21)
FoxO3+/- p53+/- (n=62)
FoxO3+/+ p53+/- (n=21)
200 4000 600
(a) Percent survival of mice with different alleles of FoxO3 in the p53?/?background as a function of time. Kaplan–Meier survival
curves with the number of mice indicated for each genotype. P¼0.10, logrank test. (b) Percent survival of mice with different alleles of
FoxO3 in the p53þ/?background as a function of time. Kaplan–Meier survival curves with the number of mice indicated for
each genotype. P¼0.13, logrank test. (c) Tumor types and glomerulonephritis in mice with different alleles of FoxO3 in the p53þ/?and
p53?/?background. The number of mice is indicated for each genotype. (d) Examples of sarcomas and carcinomas in compound
FoxO3/p53 mutant mice. Main panels: ?50. Insets: ?630. (i) Subcutaneous fibrosarcoma in a FoxO3þ/?p53?/?mouse; (ii)
osteosarcoma in the leg of a FoxO3?/?p53?/?mouse; (iii) colon carcinoma in a FoxO3þ/?p53?/?mouse; (iv) uterine carcinoma in a
FoxO3þ/?p53þ/?mouse; (v) breast carcinoma in a FoxO3þ/?p53þ/?mouse; (vi) muscle carcinoma in the arm of a FoxO3þ/?p53þ/?
FoxO3 loss does not affect survival in mice that are lacking one or both p53 alleles, but may alter tumor spectrum.
FoxO3 is a direct target of p53
VM Renault et al
expression in mammals. Binding sites in introns have
been reported previously for a number of p53 target
genes, including TRAIL (Takimoto and El-Deiry, 2000),
GADD45 (Kastan et al., 1992; Smith et al., 1994) and
Mdm2 (Juven et al., 1993; Wu et al., 1993). Thus, p53
Particularly notable are the recent findings that the
second intron of the human FoxO3 gene contains single-
nucleotide polymorphisms associated with extreme
longevityin human centenarians
German and Italian descent (Willcox et al., 2008;
Anselmi et al., 2009; Flachsbart et al., 2009). Although
the causative single-nucleotide polymorphism associated
with longevity in the FoxO3 gene has not been located
yet, these observations raise the intriguing possibility
that sequence variations in the second intron of the
FoxO3 gene may be important for human longevity,
perhaps by leading to subtle differences in transcription
factor binding in this region, ultimately affecting FoxO3
Given that FoxO3 and p53 share so many common
functions, it is surprising to note that these two
molecules have been reported to have antagonistic roles
on organismal lifespan. Indeed, FoxO factors extend
lifespan in invertebrates (Henderson and Johnson, 2001;
Giannakou et al., 2004; Hwangbo et al., 2004), and
mutants of the insulin/IGF-1 receptor—which lead to
FoxO activation—also display an extended lifespan
in mice (Bluher et al., 2003; Holzenberger et al., 2003).
In contrast, p53 activity appears to promote aging in
worms and flies (Bauer et al., 2005; Arum and Johnson,
2007). Interestingly, ectopic expression of p53 decreased
lifespan in male flies, but increased lifespan in female
flies (Shen and Tower, 2010). In a FoxO-null back-
ground, p53 no longer shortened the lifespan of males,
but still extended the lifespan of females, suggesting
that, in males and females, FoxO has a differential role
downstream of p53 (Shen and Tower, 2010). Increased
p53 activity has also been shown to elicit premature
aging in mice (Tyner et al., 2002; Maier et al., 2004),
although wildtype p53 overexpression can actually
prolong mouse lifespan in the context of p19Arfover-
expression (Matheu et al., 2007). The molecular bases
for the differences between FoxO3 and p53 in regulating
lifespan are not known, but understanding of the
connections between these two molecules should give
crucial insights into the mechanisms that regulate
Materials and methods
A bacterial artificial chromosome containing the full-length
mouse FoxO3 gene was purchased from BACPAC Resource
Center (Oakland, CA, USA). The 500-bp regions surrounding
the four putative p53 binding sites in the FoxO3 regulatory
region were amplified by PCR, and were subcloned into the
pGL3 vector with the minimal SV40 promoter (SV40-pGL3).
The mutated p53-4 binding site was generated by site-directed
mutagenesis (Quikchange, Agilent, Santa Clara, CA, USA)
using the following primers: forward 50-GGAGGGTCCTGG
GGACCCTCC-30. The mutated fragment was entirely se-
quenced and subcloned into SV40-pGL3. The E1A plasmid
was described previously (Lowe and Ruley, 1993). The ‘Pan
FoxO’ short hairpin RNA construct was generated by
subcloning the following primers (Hribal et al., 2003) into
the pSicoR lentiviral expression vector between the HpaI and
XhoI sites (Ventura et al., 2004): forward 50-TGGATAAGG
TCCTTTTTTC-30and reverse 50-TCGAGAAAAAAGGAT
Antibodies to human FoxO1, FoxO4, FoxO4, and mouse
FoxO6 were generated by immunizing rabbits with a fusion
protein between glutathione S-transferase and each FoxO
family member, and purified by affinity (Quality Controlled
Biochemicals, Hopkinton, MA, USA). The FoxO3 antibody
(Greer et al., 2007; Renault et al., 2009) and the FoxO6
antibody were used previously (de la Torre-Ubieta et al.,
2010). The antibody to Mek1 was described previously
(Lenormand et al., 1993). Antibodies to p53 were obtained
from Oncogene Science (Cambridge, MA, USA) (Ab1), from
Novocastra/Leica Microsystems (Bannockburn, IL, USA)
(CM5), and from Santa Cruz Biotechnology (Santa Cruz,
CA, USA) (DO-1, SC-126X; pAb 1801, S-98X). Antibodies
to p21Cip1and b-actin were purchased from Santa Cruz
Biotechnology and Novus Biologicals (Littleton, CO, USA),
respectively. Antibodies to cleaved caspase 3 and to BrdU
were purchased from Cell Signaling Technology (Danvers,
MA, USA) and from AbD Serotec (Raleigh, NC, USA),
Thymocytes were extracted from 6- to 10-week-old mice of
as described (Ihrie et al., 2003). The following genotypes
were compared: p53þ/þversus littermate p53?/?or p53LSL?WT
mice, which result in complete p53 deficiency. Briefly, mice
were euthanized and the thymi of the mice were removed
and placed in phosphate-buffered saline (PBS) on ice. Each
thymus was passed through a 40-mm nylon cell strainer (BD
Biosciences, San Jose, CA, USA) and divided into two plates
(one treated and one untreated). Thymocytes were then
g-irradiated with 10Gy using a
was isolated 3h later using Trizol (Life Technologies,
Carlsbad, CA, USA).
137Cs irradiator, and RNA
In all, 1mg of total RNA was reverse transcribed with random
hexamers using Superscript II reverse transcriptase (Invitrogen)
or Moloney murine leukemia virus reverse transcriptase
(Invitrogen), according to the manufacturer’s protocol. Real-
time PCR was performed on a BIO-RAD (Hercules, CA, USA)
iCycler using iQ SYBR green (BIO-RAD) with the following
forward and reverse primers:
FoxO3 forward: 50-AGTGGATGGTGCGCTGTGT-30
FoxO3 reverse: 50-CTGTGCAGGGACAGGTTGT-30
Mdm2 forward: 50-AGCGCAAAACGACACTTACA-30
Mdm2 reverse: 50-ACACAATGTGCTGCTGCTTC-30
FoxO3 is a direct target of p53
VM Renault et al
FoxO1 forward: 50-ACGAGTGGATGGTGAAGAGC-30
FoxO1 reverse: 50-TGCTGTGAAGGGACAGATTG-30
FoxO4 forward: 50-GGTGCCCTACTTCAAGGACA-30
FoxO4 reverse: 50-GGTTCAGCATCCACCAAGAG-30
FoxO6 forward: 50-TGCCCTACTTCAAGGATAAAGG-30
FoxO6 reverse: 50-CAGCTGCTTCTTGCTCG-30
Bim forward: 50-TCCTGTGCAATCCGTATCTCC-30
Bim reverse: 50-CGCAAGCTTCCATACGACAGT-30
Gapdh forward: 50-TGTGTCCGTCGTGGATCTGA-30
Gapdh reverse: 50-TTGCTGTTGAAGTCGCAGGAG-30
The experiments were conducted in triplicate and the results
were expressed as 2?(gene-of-interest number of cycles?b-actin number of cycles).
MEFs were isolated at embryonic day 13.5 from mice of
different genotypes (p53LSL?WT, p53LSL?25,26and p53LSL?VP16).
MEFs were infected by adenovirus expressing the Cre
recombinase (University of Iowa) at a multiplicity of infection
of 100 for 24h, as described (Johnson et al., 2005). RNA was
isolated using the Trizol protocol. For northern blot experi-
ments, 15mg of RNA was resolved on a denaturing agarose gel
and transferred onto a nylon membrane. Prehybridization and
hybridization were performed using ExpressHyb hybridization
solution (Clontech, Mountain View, CA, USA). Probes were
prepared using a Prime-It II random primer labeling kit
(Agilent, Santa Clara, CA, USA). A KpnI-NotI fragment of
the mouse FoxO3 complementary DNA was used as a probe.
The probe for p21Cip1was described (Attardi et al., 2000).
Cells were lysed in lysis buffer (Tris–HCl pH 8.0 (50mM),
NaCl (100mM), ethylene glycol tetraacetic acid (2mM), NaF
(10mM), b-glycerophosphate (40mM), Triton X-100 (0.4%),
aprotinin (10mg/ml), phenylmethylsulfonyl fluoride (1mM). In
all, 50mg of protein extract was resolved by SDS–polyacryla-
mide gel electrophoresis and transferred to nitrocellulose
membranes. The membranes were incubated with primary
antibodies, and the primary antibodies were visualized using
horseradish peroxidase-conjugated anti-mouse or anti-rabbit
secondary antibodies and enhanced chemiluminescence (GE
Healthcare, Piscataway, NJ, USA).
ChIP with p53 antibodies
MEFs were seeded in 15-cm dishes at a density of 105–4?105
cells per ml. Twelve to twenty-four hours after plating, cells
were stimulated with doxorubicin (0.2mg/ml) for 18–20h
(Figure 4b) or 6h (Figure 4c). Cells were cross-linked with
formaldehyde (1%) for 10min and incubated with glycine
(0.125 M) for 5min. Cells were washed with PBS and lysed in
the Farnham lysis buffer (5mM PIPES, 85mM KCl, 0.5%
NP-40, protease inhibitor cocktail (#11697498001, Roche
USA, Indianapolis, IN, USA). Nuclear extracts were collected
by centrifuging at 2000r.p.m. for 5min. The cell nuclei
were re-suspended in RIPA buffer (1?PBS, 1% NP-40, 0.5%
Na-deoxycholate, 0.1% SDS, supplemented with Roche
5–8 times, for 30s each time, with a Sonics VirCell 130
sonicator (Sonics & Materials, Newtown, CT, USA) equipped
with a stepped microtip. The chromatin was flash-frozen
in liquid nitrogen and an aliquot was used to
sonication was effective. Antibodies to p53 (Figure 5b: DO-1
and pAb 1801 from Santa Cruz, 2.5mg each; Figure 5c: CM5
reaction) or IgGcontrol
(Life Technologies or Sigma Aldrich, St Louis, MO, USA)
were coupled to rabbit secondary antibody-coated Dynal
magnetic beads (Life Technologies) in PBSþ5mg/ml BSA
overnight at 41C. Chromatin extracts were pre-cleared on
beads and then incubated with the beads coupled to the
p53 antibody overnight at 41C. The beads were washed twice
in 1ml low-salt ChIP buffer (0.1% SDS, 1% Triton X-100,
2mM EDTA, 20mM Tris–HCl, pH 8.1, 150mM NaCl), three
times in 1ml high-salt ChIP buffer (0.1% SDS, 1% Triton
X-100, 2mM EDTA, 20mM Tris–HCl, pH 8.1, 500mM
NaCl), four times with 1ml LiCl ChIP buffer (0.25M LiCl,
1% IGEPAL CA630, 1% deoxycholic acid (sodium salt), 1mM
EDTA, 10mM Tris, pH 8.1), and once or twice in 1ml TE
buffer. The chromatin complex was eluted in 200–300ml of IP
elution buffer (1% SDS, 0.1 M NaHCO3) at 651C overnight.
alcohol and by PCR purification columns (Qiagen, Valencia,
CA, USA). Quantitative PCR was performed in triplicate
using SYBR green (BIO-RAD) and a 7900HT Fast Real-
Time PCR Machine (Life Technologies) or a BIO-RAD
iCycler on 2.5ml of eluted DNA using the following sets of
Mouse negative control forward: 50-GGGGGATAATGAT
Mouse negative control reverse: 50-GCGTGGACAGAGAT
p53-1 forward: 50-CCTAATGCCACAGCAGAACTCATC-30
p53-1 reverse: 50-TGGGAATGGAACTCAGTCAGTGC-30
p53-4 forward: 50-GGGTGGGGGATTCTTTTCACTC-30
p53-4 reverse: 50-CGAGGTAAGCCAGCACATACAAAT
p53 (p21Cip1) forward: 50-GAGACCAGCAGCAAAATCG-30
p53 (p21Cip1) reverse: 50-CAGCCCCACCTCTTCAATTC-30
For each primer set, a standard curve was established using
a 5- to 10-fold dilution series.
(Life Technologies) and antibiotics (50U/ml penicillin, 50mg/
ml streptomycin, 2mM glutamine). For luciferase assays,
MEFs were seeded at 35000 cells per well in 24-well plates.
In all, 125ng of vector encoding the luciferase reporter under
different promoters was transfected together with 62.5ng of a
vector expressing Renilla luciferase to control for variation in
transfection efficiency. Two days after transfection, cells
were lysed and firefly luciferase and Renilla luciferase were
measured according to the Promega protocol (Promega,
Madison, WI, USA).
Lentiviral infection of MEFs
Lentiviruses were produced by transfection of 293T cells with
the pSicoR lentiviral vectors and the helper plasmids 8.2 and
VSVg. MEFs were plated at 105cells per ml in 10-cm plates,
and 0.45-mm-filtered 293T supernatant was applied 24h later
and the infection was repeated 2–3 times in the presence
of polybrene (8mg/ml). Puromycin (5mg/ml) was added
to select MEFs that were infected with the virus. On day
5 post-infection, MEFs were used for BrdU or western
BrdU incorporation assays
FoxO3þ/þp53þ/þ, p53?/?or FoxO3?/?MEFs were seeded in
cover chambers at 300000 cells per ml. Cells were stimulated
with doxorubicin (0.2mg/ml) or nutlin (10mM) for 24h, unless
FoxO3 is a direct target of p53
VM Renault et al
otherwise noted. BrdU was added for the last 16h, and the
cells were fixed in 4% formaldehyde for 10min and
permeabilized in 0.4 % Triton for 30min. Coverslips were
incubated with 2 N HCl for 30min, and washed extensively
with PBS. Non-specific antibody-binding sites were blocked by
incubation with PBS containing 10% goat serum and 7.5%
BSA. Coverslips were then incubated with primary antibodies
(rat anti-BrdU, 1:500) for 2h, and washed five times with PBS.
Cells were then incubated for 1h with a secondary antibody
(goat anti-rat Alexa 488, 1:400). Coverslips were mounted in
Vectashield (Vector Laboratories, Burlingame, CA, USA)
containing 4-6-diamidino-2-phenyl indole and examined under
epifluorescent illumination using a Zeiss (Thornwood, NY,
USA) microscope digital camera with AxioVision 4 software.
For quantification, at least 250–400 cells per coverslip were
counted in a blinded manner. The ratio of BrdU-positive
nuclei over the total number of nuclei was calculated.
Apoptosis assays in E1A-transformed MEFs
Phoenix cells were plated at 200000 cells per ml in 10-cm plates
and transfected with 10mg of the E1A-encoding plasmid with
the calcium phosphate method. Phoenix cell supernatant was
filtered through 0.45-mm filters onto MEFs (70000 cells per ml
in 10-cm plates) in the presence of polybrene (8mg/ml) and the
infection was repeated once. Infected MEFs were selected
24h after the last infection with selection media containing
250mg/ml hygromycin. E1A-transformed MEFs were seeded
on coverslips at 200000 cells per well.
24h later, MEFs were stimulated with nutlin (10mM) for 8h
in the presence or absence of serum in the medium. Cells were
fixed in 4% formaldehyde for 10min and permeabilized in
0.4% Triton for 30min. Non-specific antibody binding sites
were blocked by incubation with PBS containing 10% goat
serum and 7.5% BSA. Coverslips were then incubated with
primary antibodies (rat anti-cleaved caspase 3, 1:500) for 2h,
and washed five times with PBS. Cells were then incubated for
1h with a secondary antibody (goat anti-rat Alexa 555, 1:400).
Coverslips were mounted
4-6-diamidino-2-phenyl indole and examined under epifluor-
escent illumination using a Zeiss microscope digital camera
with AxioVision 4 software. For quantification, at least
250–400 cells per coverslip were counted in a blinded manner.
The proportion of cleaved caspase-3-positive nuclei over the
total number of nuclei was calculated.
Mouse crosses and survival curves
Aging cohorts were produced by three different mating
strategies: (i) FoxO3?/?p53þ/?(male) and FoxO3þ/?p53?/?
(female), (ii) FoxO3þ/?p53þ/?
FoxO3?/?p53?/?, 29 FoxO3þ/?p53?/?and 8 FoxO3þ/þp53?/?
62 FoxO3þ/?p53þ/?and 21 FoxO3þ/þp53þ/?mice were
generated. The cohorts were on a mixed FVB/N and 129Sv/J
background. Animals were genotyped by PCR and allowed to
age at a maximum of five mice per cage with standard chow
and water ad libitum in a standard light–day cycle. Mice were
monitored once to twice a week, and were killed by CO2
asphyxiation and scored as a death in survival analysis when
moribund or if external tumors exceeded 1cm in diameter.
Survival analysis was performed using Kaplan–Meier curves
with the logrank test.
All tissues, except skin and bone marrow, were fixed in 10%
formaldehyde for 24 to 48h. Bones from the head, legs and rib
cage were fixed in Bouin’s fixative for 1 week before being
decalcified for 48h. Tissues were embedded in paraffin,
sectioned in 5-mm sections, dewaxed and stained with
Department, Stanford Medical School. Tumors were identified
in a blinded manner.
Conflict of interest
The authors declare no conflict of interest.
We thank Dr Ron DePinho for his generous gift of the FoxO3?/?
mice. We thank Julien Sage for critical discussion, reading of the
for taking the pictures for Figure 8d. We thank Jamie Brett
for reading the manuscript. We thank Pauline Chu (Compara-
tive Medicine Department, Stanford Medical School) for her
help in processing the histopathology samples. We thank Dan
Calnan for participating in earlier aspects of this work. This
work was supported by NIH R01 AG026648 and a McCormick
Award for Women in Science (AB). ZX, EES and MQZ were
supported partly by NIH R01 HG001696. VMR received
support from the Dean’s Fellowship at Stanford University.
Author contributions: VMR designed, performed and analyzed
the lifespan and tumor spectrum of compound FoxO3/p53
mice (Figure 8). PUT completed Figure 1b, Figure 5b,
Figure 6b, Figure 6e, Figure 7a and helped with Figure 2
and Figure 6d. KLH completed Figure 5d, Figure 5e,
Figure 6a, Figure 6b and Figure 6c. JLW completed Figure 2,
Figure 6d, and helped with Figure 3 and Figure 7b. CAB
completed Figure 3 and Figure 7b. DKB completed Figure 5c.
OSV initiated this project as her Honors Thesis and completed
Figure 1a. TMJ completed Figure 4b. PRO completed
Figure 6f. ZX and EES identified p53 binding sites in FoxO3
regulatory regions (Figure 5a) under the supervision of MQZ.
HV performed the tumor identification (Figure 8b). LDA
supervised CAB, DKB and TMJ, and provided ideas. AB
supervised VMR, PUT, KLH, JLW, OSV and PRO. The
manuscript was written by AB, VMR and OSV, with input
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