1 Institute for Amphibian Biology, Hiroshima University Graduate School of
Science, Kagamiyama 1-3-1, Higashi-Hiroshima 739-8526, Japan
2 Department of Molecular Pathology, Graduate School of Medicine, University of
Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Author for correspondence (e-mail:
asuzuki{at}hiroshima-u.ac.jp)
Accepted 13 May 2003
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SUMMARY |
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Key words: Axis formation, TGFß, p53, Xenopus, Embryogenesis
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INTRODUCTION |
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Homeobox genes are not only induced by TGFß ligands but also play a
pivotal role in the regional specification of cell fates during development.
The even-skipped-like homeobox gene Xhox3, which is responsive to
both activin and BMPs, is expressed in ventral and posterior mesoderm during
gastrulation and functions as a ventro-posteriorizing factor
(Ruiz i Altaba and Melton,
1989a; Ruiz i Altaba and
Melton, 1989b
; Ruiz i Altaba
et al., 1991
; Dale et al.,
1992
; Jones et al.,
1992
). Mix.1 was initially isolated as an immediate early
response gene to activin, and its expression was detected in endoderm and
mesoderm (Rosa, 1989
).
Moreover, Mix.1 has been proposed to function in the BMP pathway as BMP4
induces the expression of Mix.1 and requires functional Mix.1 to
cause ventro-posteriorization of embryos
(Mead et al., 1996
).
The p53 gene is a tumor suppressor gene that is most frequently
mutated or inactivated in a wide range of human tumors
(Levine, 1997;
Prives and Hall, 1999
;
Vogelstein et al., 2000
). p53
protein functions as a sequence-specific transcription factor and its tumor
suppressor function is attributed to its ability to regulate gene expression.
Several p53 target genes mediating p53-induced responses have been reported,
which include the cell-cycle inhibitor p21/WAF as well as the growth
and differentiation factor inhibitors IGFBP3 and Dkk1
(El-Deiry et al., 1993
;
Buckbinder et al., 1995
;
Wang et al., 2000
). The
transcriptional regulation of genes involved in growth factor signaling
suggests that p53 has a role in cell differentiation processes. In fact, it
has been shown that overexpression of dominant-negative forms of human p53 or
the p53 negative regulator dm-2 in Xenopus embryos affects terminal
differentiation of neural and mesodermal tissues
(Wallingford et al., 1997
).
However, p53 appears to be largely dispensable for normal development during
mouse embryogenesis (Donehower et al.,
1992
), although a small proportion of p53 null mice develop
defects in neural tube closure (Armstrong
et al., 1995
; Sah et al.,
1995
). Therefore, the precise function of p53 during vertebrate
development and the mechanisms by which p53 regulates cellular differentiation
remain largely unknown.
In this paper, we describe a novel embryonic function for the transcription factor p53. We demonstrate that p53 functionally and physically interacts with the intracellular signaling of the TGFß pathway to regulate the expression of homeobox genes Mix.1/2 and Xhox3 directly in Xenopus embryos. Furthermore, we show that in vivo function of p53 is required for the development of mesoderm.
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MATERIALS AND METHODS |
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Expression library screening and RT-PCR analysis
Capped RNA was synthesized from a Xenopus laevis gastrula library
(Weinstein et al., 1998;
Suzuki and Hemmati-Brivanlou,
2000
), and injected in combination with noggin mRNA (200 pg) in
the animal pole of two-cell embryos. Animal caps were isolated from blastulae
and subjected to RT-PCR analysis at neurula stages as described
(Wilson and Hemmati-Brivanlou,
1995
) except that PCR cycles were increased by two or three more
cycles to allow the detection of amplified products by ethidium bromide
staining. Primers used in the RT-PCR were described previously
(Suzuki and Hemmati-Brivanlou,
2000
). Other primer sequences are as follows: xp53,
5'-GGG TTC ACT GTA AGA TAT GG-3' and 5'-GGC TGG AGG GCA CTA
TTA CC-3'; Sox17, 5'-CAG AGC AGA TCA CAT CCA ACC
G-3' and 5'-GGA AAG GAC AGA AGA AAT GGG C-3';
Mix.1, 5'-AAT GTC TCA AGG CAG AGG TT-3' and 5'-AGA
TAC AGG TAT CTG AGG GC-3'. Nucleotide sequence of a positive single
clone (pDH105-xp53) was determined and deposited with GenBank (Accession
Number AY221266).
Whole-mount in situ hybridization
Whole-mount in situ hybridization was carried out as described previously
(Suzuki et al., 1997a). For
bleaching of wild-type embryos, the hybridized embryos were treated with
bleaching solution (0.5xSSC with 1% hydrogen peroxide and 5% formamide)
under a fluorescent light.
Plasmids
xp53RD, xp53:GR, xp53Nmut:GR, Myc-tagged xp53 and Myc-tagged
xp53
RD were made by a PCR-based strategy. The PCR fragments were cloned
into expression vectors pDH105 (a gift from R. Harland), pDH105-GRHA (a vector
constructed from the pSP64TGRHA vector)
(Tada et al., 1997
) or
Myc-pcDNA3 (Yagi et al.,
1999
). xp53Nmut:GR was designed to have conservative mutations,
refractory to translational inhibition by xp53-MO, downstream of the
initiation methionine. xp53 (R255T), Mix.2 (Smad mut), Mix.2 (FAST mut), Mix.2
(p53 mut) reporter mutants were made by a PCR-based method
(Sawano and Miyawaki, 2000
).
p53 (X3), a p53 reporter gene was constructed by cloning annealed double
strand oligonucleotides containing the p53-binding sites found in the human
PA26 gene (Velasco-Miguel et al.,
1999
) into the Otx minimal promoter vector pGL3-HpOtxE
(-139
+180) (Kiyama et al.,
1998
). pXeX-RL was made by cloning a PstI/XbaI
fragment from pRL-CMV (Promega) into pXeX
(Johnson and Krieg, 1994
)
downstream of the EF-1
promoter. A Mix.2 reporter
gene, pGL3-Mix.2 [-0.367], is a gift from M. Watanabe
(Chen et al., 1997
;
Watanabe and Whitman, 1999
;
Yeo et al., 1999
). For
FLAG-tagged human p53, pDH105-hp53 was constructed by cloning a
BamHI/XbaI fragment of pcDNA3flag-hp53 (a gift from Y. Taya)
into pDH105. Other plasmids used for mRNA synthesis are pSP64T-activinßB
(Thomsen et al., 1990
),
pSP64TBX-CA-ALK2 (Suzuki et al.,
1997b
), pDH105-Smad1, pDH105-Smad2
(Lagna et al., 1996
),
pSP64T-xE2F(1-88):GR (Suzuki and
Hemmati-Brivanlou, 2000
) and pSP64T-noggin
(Smith and Harland, 1992
). In
vitro translation of synthetic mRNA was carried out using Speed Read lysate
kit (Novagen) and SDS-PAGE was performed using standard methods.
Electromobility-shift assay (EMSA) and oligonucleotides for EMSA
Whole-cell extract was prepared from early gastrula embryos injected
animally with appropriate mRNA as described
(Germain et al., 2000). Binding
reactions were performed in 30 µl of buffer containing 1 µg Herring DNA,
3 mM DTT, 0.03% BSA, 20 mM HEPES, pH 7.6, 20% glycerol, 10 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.1% NP-40, protease inhibitor cocktail
(Roche), 5 µl extract, the appropriate 32P-labeled
double-stranded oligonucleotides and monoclonal anti-p53 antibody, Pab421
(Oncogene). It has been shown that Pab421 recognizes human p53 and facilitates
the binding of p53 to DNA in EMSA assay
(Hupp et al., 1992
). Thus, we
used human p53, instead of xp53, for EMSA assay in the presence of Pab421. For
supershift, anti-FLAG M2 monoclonal antibody (Sigma) was added to the binding
reactions before electrophoresis. In the case of competition experiments,
embryo extract were pre-incubated with competitor oligonucleotides before the
binding reaction.
Oligonucleotides
5'-CCA CAT CCC AGA CAA GTT CAC ACT TCA GAG CT-3'
(Mix.2-upstream)
5'-CTG AAG TGT GAA CTT GTC TGG GAT GTG GAG CT-3' (Mix.2-downstream)
5'-CCA CAT CCC ACA AAA CTG CAC ACT TCA GAG CT-3' (Mix.2 mut-upstream)
5'-CTG AAG TGT GCA GTT TTG TGG GAT GTG GAG CT-3' (Mix.2 mut-downstream)
Luciferase assay
Ten animal caps or four whole embryos injected with appropriate mRNA and
reporter plasmid were homogenized in 100 µl of 50 mM Tris-HCl, pH 7.4 and
centrifuged for 5 minutes at 4°C. Supernatant of lysate (10 µl) was
used to perform luminescence measurement using the Dual-Luciferase Reporter
Assay System (Promega) according to the manufacturer's protocol at half-scale.
As an internal control for luciferase assay, we used pXeX-RL, which contains
Renilla luciferase (RL) cDNA under the control of
EF-1 promoter.
Chromatin immunoprecipitation (ChIP)
Twenty animal caps were isolated at stage 9 from embryos injected with
appropriate mRNA, cultured until sibling embryos reached stage 11 and
crosslinked with 1% formaldehyde at room temperature for 20 minutes. After
rinse with ice-cold 0.5xMMR twice, animal caps were incubated in 100 mM
Tris-HCl, pH 9.0, 10 mM DTT for 30 minutes at 30°C and followed by steps
described by Shang et al. (Shang et al.,
2000). Primer sequences used in PCR are as follows: Mix.2
(upstream), 5'-GGT CTA TAG ATC TAT GGA GTG TGC C-3' and
Mix.2 (downstream), 5'-AGT GCT GCT CAG TTG ACT CAA TGA
C-3'; goosecoid (upstream), 5'-CGT TAA TGT CCC ATC ACG
CTC AAT G-3' and goosecoid (downstream), 5'-TGC AGA CTG
CAG TCC TCT CCC ATC T-3'. Nucleotide sequence of the PCR products was
confirmed by automated sequence.
Cell culture and immunoprecipitation
COS-7 cells were transiently transfected with the indicated plasmids using
FuGene6 transfection reagent (Roche) following the manufacturer's
instructions. Immunoprecipitation and immunoblotting were performed as
previously described (Yagi et al.,
1999).
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RESULTS |
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xp53 interacts functionally and physically with the TGFß pathway
for the regulation of homeobox gene expression
Our analysis and previous reports (see Fig. S1 at
http://dev.biologists.org/supplemental/)
(Vize, 1996) have shown that
BMPs and activin-like molecules are able to induce directly the expression of
Xhox3 and Mix.1 genes that are identified as potential
direct targets for p53 (Fig.
3C). Therefore, we analyzed if xp53 requires signals mediated by
TGFß ligands to activate Xhox3 and Mix.1 gene
expression in animal cap assays (Fig.
4A). In order to inhibit TGFß ligand-dependent signals, we
used a dominant-negative activin type II receptor (
ActR) that has been
shown to inhibit both activin and BMPs at the plasma membrane
(Hemmati-Brivanlou and Thomsen,
1995
; Wilson and
Hemmati-Brivanlou, 1995
;
Yamashita et al., 1995
;
Macias-Silva et al., 1998
). We
found that the expression of
ActR prior to activation of xp53:GR had
no effect on the ability of xp53:GR to activate Xhox3 and
Mix.1 genes (lane 5). In addition, in the presence of
ActR,
xp53:GR induced a reporter plasmid for the Mix.2 gene, the
transcriptional regulation of which is similar to that of Mix.1
(Fig. 4B). Thus, xp53 regulates
these homeobox genes either downstream of TGFß ligand-induced receptor
activation or independently of TGFß ligands. To distinguish these two
possibilities, we tested if endogenous xp53 is required for activin or
BMP-mediated induction of Xhox3 and Mix.1 gene expression.
We established that an antisense morpholino oligonucleotide designed around
the initiation methionine of xp53 (xp53-MO) is able to inhibit translation of
xp53 mRNA in vitro, while a control morpholino oligonucleotide containing five
mismatched sequence (5mis-MO) had no effect
(Fig. 4C). In addition, the
xp53-MO suppressed endogenous p53 activity as monitored by a p53 reporter gene
[p53 (X3)] (Fig. 4D). The
effect of p53-MO is specific because the inhibition of endogenous p53 activity
is restored by expression of xp53Nmut:GR, a transcript that is refractory to
the p53-MO inhibition because of conservative mutations in the p53-MO target
region (Fig. 4C). As shown in
Fig. 4E, injection of the
intracellular signal transducers Smad2 or Smad1 mRNA, which transmit activin
and BMP signals, respectively, induced the expression of both Xhox3
and Mix.1 genes, while co-injection of xp53-MO partially suppressed
these responses (lane 6). Furthermore, the inhibition of marker gene
expression is restored by p53Nmut:GR, indicating the effect of p53-MO is
specific (lanes 7 and 8). In summary, these results suggest that xp53
functions downstream of the receptor activation and may act together with a
transcriptional machinery involving Smads to regulate homeobox gene
expression.
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p53 binds directly to Mix.2 gene
We next examined if p53 binds to the putative p53-binding sites found in
the regulatory sequence of Mix.2 in vitro by using
electromobility-shift assays (EMSA). We identified binding complexes with a 26
bp labeled probe containing a p53-binding site from Mix.2 in cell
extracts from embryos injected with FLAG-tagged human p53 mRNA
(Fig. 6A, lane 2). The
formation of these complexes was diminished by addition of an excess amount of
the non-labeled probe, but not by probe bearing a mutation in the consensus
p53-binding site (lanes 3-6). Furthermore, addition of a monoclonal antibody
recognizing FLAG tag caused a large shift in the electrophoretic mobility of
the complexes (lane 7). To examine whether xp53 binds to this homeobox gene in
vivo, we performed a chromatin immunoprecipitation assay in which Myc-tagged
xp53 was expressed in embryos in the absence or presence of TGFß signals
and followed by precipitation of chromatin bound to xp53 with an anti-Myc
antibody (Fig. 6B). PCR
amplification using specific primer sets flanking p53-binding sites of
Mix.2 gene revealed the in vivo association of xp53 with the
proximity of these genes in response to activin and BMP signals (lanes 4 and
5), given that the size of the genomic DNA fragment produced by sonication is
300-1000 bp (not shown). In the absence of TGFß signals, no significant
binding of xp53 to Mix.2 gene was detected. This may be due to the
detection limit of this assay, because xp53 alone was able to activate the
target gene expression in the animal cap assay
(Fig. 2C). The
goosecoid gene was not precipitated with xp53 even in the presence of
TGFß signals, showing the specificity of xp53. Overall, these results, in
conjunction with the in vitro EMSA data described above, strongly suggest that
xp53 binds to p53-binding sites in the Mix.2 gene in vivo, and that
TGFß ligand stimulation can enhance the binding of p53 to its target
genes.
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DISCUSSION |
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Interplay between the TGFß and p53 pathways
Although we show that p53 overexpression induces a variety of genes
involved in the establishment of mesoderm and endoderm, we found that
goosecoid, an organizer-specific homeobox gene, is not induced by
xp53. As the goosecoid promoter contains an activin-responsive
element that has been shown to bind a transcription complex involving Smads
(Watabe et al., 1995;
Labbé et al., 1998
),
this result indicates that not all genes that respond to the Smad-mediated
activin pathway are regulated by p53. The extent to which p53 interacts with
the TGFß pathway as well as the selection of the TGFß pathways to be
connected to p53 could be a context dependent and may involve other factors
that are differentially expressed in different cell or tissue types. For
example, p21/WAF1, a p53 target gene, is known to be directly
regulated by TGFß signaling in several types of cells, but at least in
the cultured mouse B-cell hybridoma cells, inactivation of endogenous p53 does
not affect the TGFß ligand-mediated induction of p21 gene
expression (Yamato et al.,
2001
). However, using bioinformatic and microarray approaches for
the human genome, Wang et al. have found that the majority of
TGFß1-induced genes they characterized contain p53-binding sites
(Wang et al., 2001
). Based on
our analysis, we propose that the interplay between TGFß and p53 pathways
at the level of transcription is crucial for mesoderm formation in
Xenopus embryos. However, the interplay may be limited to genes
involved in early development. It will be interesting to address if the
interplay is also subject to downstream genes important for other aspects of
p53 function such as apoptosis and cell cycle arrest in physiological contexts
including early mammalian embryogenesis and primary cell culture from tumor
tissues.
In addition to the importance of the p53-binding site in the Mix.2 promoter, we observed that both Smad and FAST-1-binding sites also are important for p53-mediated Mix.2 expression (Fig. 5D). The mutual requirement for p53 and Smad-binding sites for Mix.2 expression may result from the physical interaction between Smads and p53 (Fig. 5E). The identification of signals and mechanisms regulating the physical interactions in embryos may provide a clue to understanding the dynamics of interplay between p53 and TGFß signaling during embryogenesis.
Developmental functions for p53
Despite evidence that p53 appears to be largely dispensable for normal
development during mouse embryogenesis
(Donehower et al., 1992), we
have demonstrated that Xenopus p53 plays an important role in the
formation of mesoderm. This result is consistent with the previous
observation, by Wallingford et al.
(Wallingford et al., 1997
),
that the blockade of p53 activity results in inhibition of terminal
differentiation of mesoderm and neural tissues. In addition, several lines of
evidence have already implied the developmental functions of p53 during early
mammalian development (Hall and Lane,
1997
). For example, it has been shown that the overexpression of
p53 in transgenic mice results in altered differentiation of the ureteric bud
without causing cell cycle arrest and apoptosis
(Godley et al., 1996
). Mice
homozygous for p53 are viable, but a significant proportion of
p53-/- mice die during embryogenesis due to a spectrum of
abnormalities including defects in neural tube closure and craniofacial
malformations (Armstrong et al.,
1995
; Sah et al.,
1995
). Mice embryos homozygous for mdm2, a negative
regulator of p53, die between implantation and days E6.5, but the phenotype is
rescued by the absence of p53, suggesting that the embryonic
lethality of the mdm2 null mutation is caused mainly by activation of
p53 (Jones et al., 1995
;
Montes de Oca Luna et al.,
1995
).
The fundamental question is why defects in mesoderm are observed in
Xenopus embryos, but not in the mouse, upon knockdown of p53? We
expect that the severity of the phenotype may depend on the extent of
redundancy among members of the p53 family (p53, p63 and p73) in a given
species. At least in Xenopus, major expression of p63 begins in the
ectoderm, not the mesoderm, at neurula stages, following the establishment of
early mesoderm formation (Lu et al.,
2001). This could explain our observation that p53 knockdown
affects mesoderm development in this species. In mammals, however, the
expression of p63 and p73 genes during gastrulation has not been examined and
the determination of the involvement of p63 and p73 in mesoderm formation
awaits further studies. We also do not rule out the possibility that mammalian
embryos contain a system that bypasses the loss of p53 function, rather than
the use of redundant functions among p53 family members. A detailed analysis
of the expression profile as well as the determination of functional
redundancy between p53 family members will be required to understand precisely
the developmental functions of this family during early vertebrate
embryogenesis.
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ACKNOWLEDGMENTS |
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Footnotes |
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* These authors contributed equally to this work
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