MOLECULAR AND CELLULAR BIOLOGY, Apr. 2002, p. 2220–2228
0270-7306/02/$04.00⫹0 DOI: 10.1128/MCB.22.7.2220–2228.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 22, No. 7
p53-Dependent S-Phase Damage Checkpoint and Pronuclear Cross
Talk in Mouse Zygotes with X-Irradiated Sperm
Tsutomu Shimura,1 Masao Inoue,2 Masataka Taga,1 Kazunori Shiraishi,1 Norio Uematsu,1
Norihide Takei,1 Zhi-Min Yuan,3 Takashi Shinohara,4 and Ohtsura Niwa1*
Department of Late Effect Studies, Radiation Biology Center,1 and Department of Medical Chemistry, Faculty of Medicine,4
Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, and Medical Research Institute, Kanazawa Medical University,
Uchinada-machi, Kahoku-gun, Ishikawa 920-02,2 Japan, and Department of Cancer Cell Biology,
Harvard School of Public Health, Boston, Massachusetts 021153
Received 4 September 2001/Returned for modification 5 October 2001/Accepted 20 December 2001
merge to enter M phase. This gives a unique opportunity to
analyze the mechanism of damage responses; damage can be
delivered through irradiated sperm while the responses can be
analyzed in damage-free female pronuclei. Indeed, our previous study clearly demonstrated that introduction of DNA damage by irradiated sperm induces genomic instability which destabilizes a maternally derived minisatellite allele in F1 mice (32).
In the present study, we have investigated the p53-dependent radioresponse in mouse zygotes fertilized by X-irradiated
sperm. The results definitely demonstrate p53-dependent pronuclear cross talk in zygotes. In addition, we have found a
novel function of p53 in the S-phase DNA damage checkpoint,
which suppresses the rate of pronuclear DNA synthesis in
mouse zygotes.
Ionizing radiation induces DNA double-strand breaks (DSB)
and inflicts a variety of damage responses which include induction of cell cycle checkpoint and apoptosis. The p53 pathway
plays a prominent role in the damage responses of higher
eukaryotes (26). The signal generated by DSB is transduced by
ATM and related sensor kinases to phosphorylate p53, which
activates various functions of the protein (5, 7, 20).
It has been well documented that embryonic development in
mouse is quite sensitive to radiation. A high level of p53 was
correlated to radiation induction of apoptosis without cell cycle
arrest in post-implantation stage mouse embryos (19, 23). This
p53-dependent apoptosis was shown to be the major mechanism to suppress radiation-induced teratogenesis in mice (33).
Earlier stages of mouse development, especially from fertilization to implantation, were shown to be highly radiosensitive
(14). However, the mechanisms of the high radiosensitivity
have been poorly elucidated. Mouse zygotes are known to
possess an extremely high level of p53 (21). This indicates that
p53 might play an important role in damage response of mouse
zygotes.
One difficulty in studying the damage response is that the
effect of damage itself and the effect of cellular responses are
hard to distinguish, because these two always come together in
irradiated cells. In a mammalian zygote, genomes of the incoming sperm and residing oocyte form two separate pronuclei. DNA synthesis occurs in two pronuclei, which thereafter
MATERIALS AND METHODS
Mice. ICR strains of mice 8 to 10 weeks old were used in the present experiment. The p53⫺/⫺ C57BL/6 ⫻ CBA mice were kindly provided by S. Aizawa
(42). The p21⫺/⫺ 129 mice were purchased from The Jackson Laboratory (Bar
Harbor, Maine). The p53⫺ allele was then introduced into ICR strains by repeated backcross. All mice were maintained in our animal facility on a 12 h of
light and 12 h of dark cycle starting at 7:00 a.m. under standard temperature
conditions (23 ⫾ 2°C).
X-irradiation. Partial irradiation was performed on the testicular area of male
mice, with a lead shield covering other parts of the body, at a dose rate of 1
Gy/min (250 keV; 15 mA with 1-mm-thick Al filter; Rigaku Radioflex X-ray
generator). Irradiation of isolated sperm and zygotes was carried out at room
temperature in microdrops of the medium submerged in liquid paraffin. Plasmid
DNA was irradiated in a buffer under the X-ray generator without the filter at a
dose rate of 30 Gy/min.
In vivo and in vitro fertilization. In vitro fertilization of mouse oocytes and
subsequent culture were carried out as described (30, 44). Virgin ICR female
mice were induced to superovulate with 5 U of pregnant mare’s serum
(Teikokuzoukiseiyaku Ltd., Tokyo, Japan), followed 46 to 48 h later by 5 U of
* Corresponding author. Mailing address: Department of Late Effect Studies, Radiation Biology Center, Kyoto University, Yoshida
Konoe, Sakyo-ku, Kyoto 606-8501, Japan. Phone: 81-75-753-7563. Fax:
81-75-753-7564. E-mail: [email protected].
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One difficulty in analyzing the damage response is that the effect of damage itself and that of cellular
response are hard to distinguish in irradiated cells. In mouse zygotes, damage can be introduced by irradiated
sperm, while damage response can be studied in the unirradiated maternal pronucleus. We have analyzed the
p53-dependent damage responses in irradiated-sperm mouse zygotes and found that a p53-responsive reporter
was efficiently activated in the female pronucleus. [3H]thymidine labeling experiments indicated that irradiated-sperm zygotes were devoid of G1/S arrest, but pronuclear DNA synthesis was suppressed equally in male
and female pronuclei. p53ⴚ/ⴚ zygotes lacked this suppression, which was corrected by microinjection of
glutathione S-transferase–p53 fusion protein. In contrast, p21ⴚ/ⴚ zygotes exhibited the same level of suppression upon fertilization by irradiated sperm. About a half of the 6-Gy-irradiated-sperm zygotes managed to
synthesize a full DNA content by prolonging S phase, while the other half failed to do so. Regardless of the DNA
content, all the zygotes cleaved to become two-cell-stage embryos. These results revealed the presence of p53dependent pronuclear cross talk and a novel function of p53 in the S-phase DNA damage checkpoint of mouse
zygotes.
VOL. 22, 2002
p53-DEPENDENT S-PHASE CHECKPOINT IN MOUSE ZYGOTES
RESULTS
Transactivation of a p53-responsive reporter in female pronuclei of zygotes with irradiated sperm. The p53-dependent
cross talk between two pronuclei was examined in zygotes
fertilized in vitro by unirradiated sperm (control zygotes) and
irradiated sperm (irradiated-sperm zygotes). A p53-responsive
reporter plasmid carrying the nucleus-localizing lacZ gene was
microinjected into female pronuclei at 8 h after fertilization.
These zygotes were cultured and then fixed at 20 h (zygote
stage) or 24 h (two-cell stage) after fertilization. There was no
staining for the lacZ activity in microinjected control zygotes
(data not shown). In contrast, a strong staining was observed in
both pronuclei when the control zygotes were irradiated with 3
Gy at 4 h after fertilization (Fig. 1a). This staining persisted in
two-cell-stage embryos (data not shown). A strong staining was
also observed in 6-Gy-irradiated-sperm zygotes (Fig. 1b). The
polar body which does not share cytoplasm with the zygote was
devoid of the staining (Fig. 1b). Activation of the reporter was
not observed in p53⫺/⫺ zygotes regardless of irradiation of
p53⫺ sperm (Fig. 1c).
Irradiation of the reporter plasmid with 3,000 Gy of X rays,
which was expected to introduce about one DSB per plasmid,
resulted in a weak but definite staining upon microinjection
into control zygotes (Fig. 1d). In addition, a weak staining was
also observed for the KpnI-linearized reporter (data not
shown). These results indicate that introduction of DNA damage, possibly DSB, by irradiated sperm triggered the p53-dependent pathway, which then activated the reporter in the
female pronucleus.
S-phase progression of irradiated-sperm zygotes. S-phase
progression in zygotes was examined by pulse-labeling with
[3H]TdR of control zygotes and 6-Gy-irradiated-sperm zygotes
fertilized in vitro and in vivo. Table 1 shows that pronuclear
DNA synthesis was first detected at 8 h after fertilization, both
in control and in irradiated-sperm zygotes in vitro. Control
zygotes gave the labeling index of 23% at 8 h after fertilization,
the value for irradiated sperm was 21%, and there was no
arrest at the G1/S border in irradiated-sperm zygotes, even
through activation of p53 was evident from Fig. 1b. In order to
avoid a possible artifact of in vitro fertilization, we also investigated in vivo-fertilized zygotes recovered from natural mating. Control and irradiated-sperm zygotes recovered this way
both entered S phase at 8 h after mating. The labeling index of
control zygotes was 44% at 8 h after in vivo fertilization, and
the value for irradiated sperm was 45% (Table 1). Direct
irradiation of these zygotes with 3 Gy of X rays at 4, 6, and 8 h
after mating was not effective in induction of G1/S arrest (data
not shown).
The labeling index increased to around 70% at 11 h and then
to 100% for both control and irradiated-sperm zygotes fertilized in vitro. The index dropped at 21 h. Delay in cessation of
DNA synthesis was noted in some of the irradiated-sperm
zygotes, and for these, uptake of [3H]TdR ceased at 22 h after
fertilization (Table 1).
A similar analysis was performed with control and 6-Gyirradiated-sperm zygotes fertilized in vivo. The duration of S
phase in control zygotes was from 8 to 18 h after fertilization in
vivo (Table 1). Some of the 6-Gy-irradiated-sperm zygotes
exhibited a delay in S phase, and on average the S phase of
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human chorionic gonadotropin (Teikokuzoukiseiyaku Ltd.). Female mice were
sacrificed at 15 to 16 h after human chorionic gonadotropin administration, and
the oocytes were released from the ampullar portion of the oviducts into the
medium submerged in equilibrated paraffin oil. Sperm was obtained from the
cauda epididymides of male ICR mice and suspended in medium submerged in
equilibrated paraffin oil. After capacitation by incubating for 2 h at 37°C under
5% CO2 in an air environment, sperm was added to the oocytes. Zygotes were
washed 6 h later and were transferred into fresh microdroplets of culture medium.
For in vivo fertilization, females of the proestrus stage were identified by
examination of vaginal smears at 5:00 p.m. These females were caged at 6:00 a.m.
the next morning with males for 1 h. Successful mating were identified by the
presence of a vaginal plug. Females were sacrificed at 6 h after mating, and the
zygotes were collected as described above. Zygotes were washed and transferred
into microdroplets of culture medium, the cumulus cells were removed by treatment with hyaluronidase (10 mg/ml; Sigma), and the zygotes were cultured
further for investigation.
Plasmid constructions, microinjection, analysis of lacZ expression, and protein purification. A p53-responsive lacZ reporter plasmid was constructed. The
HindIII fragment carrying 13 copies of the p53-responsive element (AGGCAA
GTCCAGGCAGGCC) and the basic promoter of polyomavirus was isolated
from PG13Py-lacZ plasmid (kindly provided by B. Vogelstein [22]). This was
inserted in the HindIII site of plasmid pENL (kindly provided by Y. Nabeshima).
The resulting plasmid, PG13-Py-NL-lacZ, carried the p53-responsive promoter
upstream of the nuclear localizing lacZ.
The plasmid described above was microinjected into the female pronuclei
of zygotes at 8 h postfertilization. Male and female pronuclei were distinguished by the size and the position with respect to the polar body. Expression of lacZ was examined at 20 and 24 h after fertilization by fixation with
1% glutaraldehyde and staining with 5-bromo-4-chloro-3-indolyl-␤-D-galactoside. The success rate of microinjection into female pronuclei was around
50%. Some zygotes degenerated after microinjection, possibly due to harsh
treatment, and some surviving zygotes did not stain. Therefore, we used at
least 30 zygotes for each microinjection experiment. When all the zygotes
were devoid of staining, as it was in the case of control zygotes and p53⫺/⫺
irradiated-sperm zygotes, then this was taken to represent a lack of response.
When we saw staining in about 50% of the 30 zygotes, this was taken as a
positive response. So, the data obtained here are qualitative rather than
quantitative.
A construct was made in which human p53 cDNA was fused downstream of
the glutathione S-transferase (GST) gene of pGEX plasmid (18). The plasmid
was introduced into Escherichia coli, and the culture was induced with 0.1 mM
isopropyl-␤-D-thiogalactopyranoside. The GST-p53 fusion protein was purified
with the standard procedure using glutathione agarose beads (39). The purified
protein was prepared in a buffer (17) at a concentration of 1 mg/ml and microinjected into the cytoplasm of the zygotes at 4 h after fertilization.
Analysis of pronuclear DNA synthesis. DNA synthesis and S-phase progression were monitored by pulse-labeling with 3H-labeled thymidine ([3H]TdR)
(28). Briefly, zygotes were pulse-labeled for 1 h with [3H]TdR (1 ␮Ci/ml) at 6 to
24 h after fertilization and chased with fresh medium containing 5 ⫻ 10⫺5 M
cold TdR for another 1 h. The total amount of DNA synthesized was monitored by continuous labeling with [3H]TdR (0.1 ␮Ci/ml) from 8 to 21 h after
fertilization for in vitro-fertilized zygotes and from 8 to 18 h for in vivofertilized zygotes. After completion of labeling and chasing, zygotes were
treated hypotonically in 0.9% sodium citrate and 3% fetal calf serum for 5
min at 37°C and fixed for 5 min in methanol-acetic acid-water (5:1:4, vol/vol/
vol). The fixed zygotes were placed onto glass slides and washed with methanol-acetic acid (3:1, vol/vol). Slides were then treated with 5% trichloroacetic acid solution for 1 h at 0°C and were washed with ethanol for 5 min at
room temperature. Slides were dipped in Kodak NTB2 emulsion and stored
at 4°C for 2 weeks. After development, autoradiographs were stained with
Giemsa stain, and the number of grains per nucleus was determined for male
and female pronuclei.
Measurement of DNA content. Zygotes were washed with phosphate-buffered
saline once and placed onto cover glasses. After fixation with 70% ethanol for 5
min, zygotes were stained with propidium iodide (PI) (0.1 mg/ml) in phosphatebuffered saline containing proteinase K (0.1 mg/ml) and RNase (1 mg/ml) at
37°C for 30 min. The relative intensity of PI fluorescence was measured by laser
scanning cytometer (LSC 101; Olympus, Tokyo, Japan). Care was taken to
compare the PI intensities of nuclei on the same slide so that the variation in the
staining was minimized.
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about a half of these embryos was longer by 2 h than that of
control zygotes. These results indicate that irradiation of sperm
did not trigger G1/S checkpoint in mouse zygotes but retarded
S phase in some.
Suppression of pronuclear DNA synthesis by irradiation to
sperm. The rate of DNA synthesized was analyzed by pulselabeling experiments. The numbers of grains were counted in
male and female pronuclei. The results indicated that the rate
of DNA synthesis was lower throughout S phase in both of the
pronuclei of 6-Gy-irradiated-sperm zygotes fertilized in vitro
(Fig. 2). A small but definite delay in S phase was evident for
irradiated-sperm zygotes.
The total amount of DNA synthesized in S phase was measured by continuous labeling of the zygotes. Zygotes were
incubated with [3H]TdR from 8 to 21 h after in vitro fertilization and from 8 to 18 h after in vivo fertilization. Control
zygotes had a large number of grains in both pronuclei, and
incorporation of [3H]TdR was also noted in the polar body
(Fig. 3a). The number of grains was suppressed drastically in
both male and female pronuclei of 6-Gy-irradiated-sperm zygotes (Fig. 3b). The grain numbers of both pronuclei were
TABLE 1. Duration of S phase in oocytes fertilized with irradiated
sperm in vitro and in vivo
Sperm irradiated with:
Fertilization
Time after
fertilization
(h)
0 Gy
6 Gy
No. of
oocytes
examined
No. of
labeled
oocytes (%)
No. of
oocytes
examined
No. of
labeled
oocytes (%)
In vitro
6
8
11
14
17
20
21
22
28
31
23
15
24
36
25
26
0 (0)
7 (23)
16 (70)
15 (100)
24 (100)
9 (25)
1 (4)
0 (0)
39
29
24
11
18
22
28
25
0 (0)
6 (21)
18 (75)
11 (100)
17 (95)
4 (18)
5 (18)
0 (0)
In vivo
6
8
16
17
18
19
20
15
18
8
8
24
28
0 (0)
8 (44)
8 (100)
3 (27)
2 (8)
0 (0)
12
31
12
15
26
39
10
0 (0)
14 (45)
11 (92)
9 (60)
13 (50)
14 (36)
2 (20)
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FIG. 1. Activation of p53-responsive reporter in irradiated-sperm zygotes. Construction of the p53-responsive reporter plasmid is described in
Materials and Methods. This reporter plasmid was microinjected into the female pronucleus at 8 h after fertilization. Zygotes were fixed and
stained with X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) at 20 h (zygote stage) or 24 h (two-cell stage) after fertilization. (a) Strong
staining with X-Gal was observed both in male pronuclei (么) and female pronuclei (乆) of control zygotes exposed to 3 Gy of X rays at 4 h after
fertilization. (b) This staining was also observed in 6-Gy-irradiated-sperm zygotes, but the polar body (arrowhead) was not stained. (c) There was
no staining in p53⫺/⫺ irradiated-sperm zygotes. (d) Weak staining was observed in control zygotes microinjected with 3,000-Gy-irradiated reporter
plasmid.
VOL. 22, 2002
p53-DEPENDENT S-PHASE CHECKPOINT IN MOUSE ZYGOTES
scored for each of the in vitro-fertilized oocytes, and the values
are plotted in Fig. 3c. The data indicated a strong reduction of
grain numbers in both pronuclei by irradiation of sperm. The
average of the grain counts is plotted in Fig. 3d, which demonstrates that the grain numbers in female and male pronuclei
of irradiated-sperm zygotes were suppressed to 35.4 and
35.1%, respectively, of those of the control zygotes. Similar
suppression was observed for irradiated-sperm zygotes recovered from natural mating, and the average grain counts decreased to 40.3% for female pronuclei and 44.1% for male
pronuclei (Fig. 3e and f). Suppression of DNA synthesis was
not specific for irradiated-sperm zygotes, and direct irradiation
of zygotes to 3 Gy of X rays at 8 h after fertilization was also
effective (data not shown).
p53-dependent suppression of pronuclear DNA synthesis.
Involvement of p53 in suppression of pronuclear DNA synthesis was tested. p53⫺/⫺ males were irradiated with 6 Gy of X
rays and mated immediately with p53⫺/⫺ females. Zygotes
were recovered at 6 h after mating and labeled with [3H]TdR
throughout S phase. The onset and the duration of S phase
were not affected by the p53-null state of the zygotes (data not
shown). Grain count analysis (Fig. 4) indicated that sperm
irradiation had no effect on DNA synthesis in p53⫺/⫺ zygotes.
However, the suppression was observed in p53 heterozygous
zygotes in which the p53-null allele was derived either from the
father or from the mother (data not shown). These results
demonstrate that the suppression of pronuclear DNA synthesis
in irradiated-sperm zygotes is p53 dependent. Direct irradiation of zygotes was also tested, and suppression of grain counts
was not observed in p53⫺/⫺ zygotes (data not shown).
In order to confirm the direct involvement of p53 in the
suppression, we microinjected GST-p53 fusion protein into the
cytoplasm of p53⫺/⫺ irradiated-sperm zygotes. Microinjection
of the fusion protein had no effect on pronuclear DNA synthesis of p53⫺/⫺ control zygotes (Fig. 4). In contrast, microinjection of the protein into p53⫺/⫺ irradiated-sperm zygotes
restored the suppression, and the average grain count in female and male pronuclei decreased to 48 and 38% of the
control values, respectively (Fig. 4).
Similar analysis was performed with p21⫺/⫺ zygotes. The
results show that pronuclear DNA synthesis in p21⫺/⫺ irradiated-sperm zygotes was suppressed as that in wild-type irradiated-sperm zygotes was (Fig. 4). Therefore, suppression of
pronuclear DNA synthesis was not dependent on p21.
Dose response of suppression of pronuclear DNA synthesis.
The dose response of suppression of pronuclear DNA synthesis was analyzed. Grain count analysis was done after continuous labeling of in vivo-fertilized zygotes from 8 to 18 h after
mating. Figure 5 shows the average grain count of female and
male pronuclei of p53⫹/⫹ and p53⫺/⫺ zygotes where the male
mice were exposed to 1, 2, 3, and 6 Gy of X rays and mated
with females immediately. In p53⫹/⫹ zygotes, X-irradiation of
sperm at doses higher than 1 Gy suppressed grain count in
both pronuclei and the grain count decreased dose dependently up to 3 Gy. In contrast, DNA synthesis was not suppressed by sperm irradiation in p53⫺/⫺ zygotes. Rather, the
grain count even increased in these zygotes when spermatozoa
were irradiated at doses of 1, 2, and 3 Gy. This increase in
p53⫺/⫺ zygotes was not due to the enhanced DNA synthesis,
because DNA content as assessed by the PI value was not
affected by sperm irradiation (data not shown). Therefore, the
increase was likely due to a decreased nucleotide pool size in
irradiated-sperm p53⫺/⫺ zygotes.
DNA content analyses of zygotes. [3H]TdR grain count can
be influenced by the size of the nucleotide pool in the cells.
Therefore, decreased grain count in irradiated-sperm zygotes
may not represent a real suppression of DNA synthesis. It may
be due to the increase in the nucleotide pool size and the
resulting decrease in the specific activity of [3H]TdR. In order
to rule out this possibility, we measured the PI values (in
arbitrary units) of the pronuclei. In Fig. 6a, the PI value of both
pronuclei was measured in control zygotes at 8 h after mating
(pre-S phase) and 18 h after mating (post-S phase). The PI
values of both pronuclei increased 2.1 times from pre-S phase
to post-S phase, demonstrating that the method was reliable in
estimating the relative DNA content. In contrast, the PI values
of irradiated-sperm zygotes increased only 1.5-fold in female
pronuclei and 1.4-fold in male pronuclei from 8 to 18 h after
mating (Fig. 6b). Thus, suppression of S phase was confirmed
by grain count analysis and by PI staining of the irradiatedsperm zygotes.
Similar analyses were made in p53⫺/⫺ zygotes. PI values of
both pronuclei in p53⫺/⫺ control zygotes increased about 2
times from 8 to 18 h after mating (Fig. 6c). PI values of both
pronuclei in p53⫺/⫺ irradiated-sperm zygotes also increased
about 2 times from 8 h to 18 h (Fig. 6d). These results are
consistent with those of labeling experiments (Fig. 4) and once
again demonstrate that the suppression of DNA synthesis is
p53 dependent.
M phase progression of irradiated-sperm zygotes. M phase
progression was examined for in vivo fertilized zygotes by scoring
two-cell-stage embryos (Fig. 7a). Control zygotes started to
cleave at 17 h and all of them completed the division by 22 h.
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FIG. 2. S-phase progression in zygotes. DNA synthesis was measured in pronuclei of zygotes by pulse-labeling with [3H]TdR for 1 h at
various time points after fertilization in vitro. The numbers of zygotes
examined are described in Table 1. Average grain counts of control
zygotes were plotted for male (—E—) and female (- - -E- - -) pronuclei.
Similarly, average grain counts of irradiated-sperm zygotes were plotted for male (—F—) and female (- - -F- - -) pronuclei. Error bars,
standard deviations.
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FIG. 3. Grain counts for total DNA synthesized in S phase. DNA synthesis was monitored by continuous labeling with [3H]TdR of zygotes
obtained from in vitro fertilization and in vivo natural mating. (a) A large number of grains was observed in control zygotes fertilized in vivo. (b)
Grain count was suppressed in irradiated-sperm zygotes fertilized in vivo, and this suppression was observed both in male pronuclei (么) and female
pronuclei (乆). The polar body is indicated by an arrowhead. Grain count was plotted for female pronuclei (abscissa) and male pronuclei (ordinate)
for in vitro-fertilized zygotes (c) and for in vivo-fertilized zygotes (e). The average of grain counts was plotted for in vitro-fertilized zygotes (d) and
for in vivo-fertilized zygotes (f). Average grain count was suppressed in irradiated-sperm zygotes (solid bars) compared with that of control zygotes
(open bars). Error bars, standard deviations.
M phase progression was delayed by about 2 h in irradiatedsperm zygotes. The cleavage was little delayed in irradiatedsperm p53⫺/⫺ zygotes.
DNA content in the nucleus was monitored in irradiatedsperm zygotes from 16 to 23 h after mating (data not shown).
At 20 h, the PI values were low in some of the irradiated-sperm
zygotes, but others showed values similar to those of the control. This suggested that some irradiated-sperm zygotes, especially those with late cleavage, may have managed to synthesize
a normal amount of DNA during the delay. We have con-
VOL. 22, 2002
p53-DEPENDENT S-PHASE CHECKPOINT IN MOUSE ZYGOTES
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firmed this possibility by measuring the PI values of two-cellstage embryos cleaved by 19 h (early cleavage) or those cleaved
at 21 h (late cleavage) after mating. PI values of irradiatedsperm two-cell-stage embryos of early cleavage were lower
than normal, but they were in the normal range for those of
late cleavage (Fig. 7b). This suggested that late-cleaving zygotes were those which spent more time in S phase to catch up
to the normal DNA content even under the lower rate of DNA
FIG. 5. Dose response of suppression of grain count in zygotes. (a)
Average grain count of pronuclei was plotted against dose at which
spermatozoa were irradiated. Each data point is the average of values
for more than 10 zygotes. In p53⫹/⫹ zygotes, grain counts of both
female pronuclei (- - -E- - -) and male pronuclei (—E—) decreased in a
dose-dependent manner up to 3 Gy of irradiation. In contrast, grain
counts of p53⫺/⫺ zygotes did not decrease in both female pronuclei
(- - -‚- - -) and male pronuclei (—‚—). Average grain counts even
increased in p53⫺/⫺ zygotes at doses of 1, 2, and 3 Gy. Error bars,
standard deviations.
synthesis. The PI values of the two-cell-stage irradiated-sperm
embryos with the p53⫺/⫺ genotype had a normal DNA content
(data not shown).
DISCUSSION
p53-dependent cross talk between male and female pronuclei in irradiated-sperm zygotes. The present study has shown
that irradiated sperm activated a p53-responsive reporter microinjected into the female pronucleus of the zygotes. Although DNA is packaged tightly by protamine in the sperm
head (4), this does not exempt DNA from being damaged by
ionizing radiation (2). The number of DSB induced per unit
dose of X rays was calculated to be around 30 in somatic cells
(27), and a similar number might be expected in the sperm
head. High-dose X-irradiation and KpnI digestion of the reporter also resulted in activation. These indicate that DSB
might be responsible for induction of the p53-dependent pronuclear cross talk observed in this study.
The p53-dependent pronuclear cross talk occurs across the
cytoplasm. This is in contrast to the nuclear limited activation
of the RAD9-dependent damage checkpoint in Saccharomyces
cerevisiae (9). Activation of p53 by radiation involves phosphorylation at the serine 15 and serine 20 residues (31). Phosphorylation of the serine 15 site is carried out by ATM, while
that of the serine 20 site is carried out by Chk2 (5, 7, 20). It is
easily envisaged that DNA damage is detected during decondensation of the sperm head and activates these protein kinases. Phosphorylated p53 would then be distributed in the
male pronucleus in cis and in the female pronucleus in trans
during pronuclear formation.
It is interesting that the activation was also observed in both
p53⫹/⫺ and p53⫺/⫹ irradiated-sperm zygotes, and the latter
indicates that the p53 gene product is made from a paternally
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FIG. 4. Grain count analysis of p53⫺/⫺ and p21⫺/⫺ zygotes. The average grain count was plotted for both pronuclei of control and 6-Gyirradiated-sperm wild-type (n ⫽ 18 and 36), p53⫺/⫺ (n ⫽ 12 and 18), p53⫺/⫺–GST-p53 (n ⫽ 20 and 10), and p21⫺/⫺ (n ⫽ 11 and 12) zygotes. Grain
count was not suppressed in p53⫺/⫺ zygotes by sperm irradiation. Average grain count was not suppressed in p53⫺/⫺ control zygotes by
microinjection of GST-p53 fusion protein. In contrast, grain count was clearly suppressed by microinjection of GST-p53 fusion protein in p53⫺/⫺
irradiated-sperm zygotes. In p21⫺/⫺ irradiated-sperm zygotes, grain count was suppressed as much as that in wild-type irradiated-sperm zygotes.
Error bars, standard deviations.
2226
SHIMURA ET AL.
MOL. CELL. BIOL.
inherited allele. One-cell-stage transcription of the p53 gene is
consistent with the previous report (21).
Lack of G1/S arrest in irradiated-sperm zygotes. One of the
major functions of the p53-dependent damage response is to
arrest the cells at the G1/S border. In our experiment, however,
the onset of DNA synthesis was not affected by 6-Gy X-irradiation of sperm, even though activation of p53 was evident by
the reporter experiment. p53 is known to transactivate p21,
which inhibits cyclin D-CDK4/6 complexes to arrest the cells at
the G1/S border (31). It was reported that embryonal stem cells
and embryonal carcinoma cells lack the G1/S checkpoint even
though p53 is highly activated after X-irradiation (37, 40). In a
recent study on ␥-irradiated F9 embryonal carcinoma cells, p53
was shown to activate transcription of the p21 gene, but a rapid
degradation blocked accumulation of p21 protein, leading to
elimination of the G1/S checkpoint (29). A similar mechanism
may also be responsible for the lack of G1/S checkpoint in
mouse zygotes.
p53 dependence of S-phase damage checkpoint in mouse
zygotes. Suppression of DNA synthesis is attained by depletion
of the pyrimidine nucleotide pool by treatment of the cells with
N-(phosphonacetyl)-L-aspartate, and this suppression was reported to be p53 dependent (1). In this study we observed
suppression of DNA synthesis in both pronuclei of irradiatedsperm mouse zygotes. The suppression was dependent on the
presence of functional p53. Microinjection of the GST-p53
fusion protein restored the suppression in irradiated-sperm
p53⫺/⫺ zygotes. The same treatment did not affect DNA synthesis in control zygotes, showing that the fusion protein by
itself had no ability to suppress DNA synthesis in the absence
of radiation damage. Thus, the present report is the first to
implicate p53 in the S-phase DNA damage checkpoint of
mouse zygotes.
It is well known that p21 is transactivated by p53 and arrests
somatic cells at the G1/S and G2/M border (6, 10, 11). Lack of
G1/S arrest in zygotes may be due to the failure of accumulation of p21, as discussed in the previous section, or p21 may
have a different function in mouse zygotes. In our study, suppression of DNA synthesis was observed in p21⫺/⫺ irradiatedsperm zygotes, indicating that p21 is not involved in the Sphase damage checkpoint. Indeed, S-phase block by hydroxyurea
results in accumulation of p53 but not accumulation of p21
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FIG. 6. DNA content analyses of zygotes. DNA content was estimated by PI staining of zygotes at 8 h (pre-S-phase stage [open bars]) and 18 h
(post-S-phase stage [striped bars]) after mating. PI value was measured in male pronuclei (么) and female pronuclei (乆). (a) The PI value of control
zygotes is plotted. The value of both pronuclei increased 2.1-fold from 8 h (n ⫽ 12) to 18 h (n ⫽ 13). (b) PI value of zygotes produced with sperm
irradiated with 6 Gy of X rays. The value increased only 1.5-fold in female pronuclei and 1.4-fold in male pronuclei from 8 h (n ⫽ 17) to 18 h (n
⫽ 15). (c) The same analysis was performed in a p53⫺/⫺ background. The PI value of p53⫺/⫺ control zygotes was plotted. The value increased
2.1-fold in female pronuclei and 1.9-fold in male pronuclei from 8 h (n ⫽ 11) to 18 h (n ⫽ 11). (d) PI value of p53⫺/⫺ zygotes produced with sperm
irradiated with 6 Gy of X rays. The value increased 2.1-fold in female pronuclei and 1.9-fold in male pronuclei from 8 h (n ⫽ 13) to 18 h (n ⫽
13). Error bars, standard deviations.
VOL. 22, 2002
p53-DEPENDENT S-PHASE CHECKPOINT IN MOUSE ZYGOTES
2227
(15). Detailed analysis of a series of mutant p53 proteins is in
progress to elucidate the domain required for the S-phase
damage checkpoint.
The S-phase damage checkpoint operates by two mechanisms:
suppression of replication origin firing and suppression of replication fork movement (35). Many of the works on the S-phase
DNA damage checkpoint come from the study of S. cerevisiae
(34). Suppression of origin firing in hydroxyurea-treated budding yeast was shown to be dependent on Mec1 and Rad53
(36). Involvement of Rad53 in the S-phase damage checkpoint
was also reported (38). In higher eukaryotes, the S-phase
checkpoint and suppression of replication origin are dependent on the gene product ATM, a mammalian homologue of
Mec1, and this has explained the long-known radioresistant
DNA synthesis in AT cells (24). The p53 gene product was
reported to have no relevance in the ATM-dependent S-phase
damage response in many of the cell types studied (25, 43).
Sperm irradiation resulted in suppression of DNA synthesis
throughout S phase. This suggested that replication fork movement was likely to be the target of p53-dependent suppression
of DNA replication in irradiated-sperm zygotes. Replication
machinery of eukaryotes is composed of DNA polymerase A
and primase. Replication protein A (RPA) is also required.
Mutation of any of these genes results in a defect of the
S-phase damage checkpoint (13). DNA polymerase, primase,
and RPA form foci in the nucleus during S phase. p53 is known
to bind to RPA and suppress its ability to associate with singlestranded DNA (12). It is possible that p53 might suppress
replication fork movement by binding to RPA. Indeed, colocalization of RPA and p53 was found in the S-phase nucleus of
Xenopus laevis embryos after UV and ␥-irradiation (41).
It has long been assumed that replication fork movement is
retarded in irradiated cells because the progression of the
replication machinery is hindered at the site of DNA damage.
Our present data demonstrated that DNA synthesis was suppressed not only in the damaged male pronucleus but also in
the female pronucleus which had no radiation damage. Therefore, suppression of DNA synthesis in mouse zygotes is truly a
checkpoint rather than a mechanical block of replication machinery at the damaged site.
The biological function of p53-dependent S-phase damage
checkpoint. In vivo fertilization gives better-synchronized zygotes than in vitro fertilization, and this is the reason for the
shorter S phase in the former zygotes (Table 1). Delay in S
phase can be better demonstrated for in vivo-fertilized zygotes,
and about half of the in vivo-fertilized irradiated-sperm zygotes
exhibited retardation of S phase by 2 h (Table 1). These delayed zygotes managed to synthesize the normal amount of
DNA, while those without delay had less than the normal DNA
content (Fig. 7b). Nevertheless, all zygotes went through M
phase (Fig. 7a).
Mouse zygotes irradiated prior to pronuclear formation
were reported to exhibit no G2/M arrest, while irradiation at
later stages led to cleavage block (16). This arrest has recently
been shown to correlate with the decreased activity of p34cdc2
(3). Irradiation of sperm is equivalent to irradiation of zygotes
prior to pronuclear formation. DNA damage at the pre-pronuclear stage, either from sperm irradiation or from direct
irradiation of zygotes, led to activation of the p53-dependent
S-phase damage checkpoint in mouse zygotes.
We propose that during the p53-dependent suppression of
DNA synthesis, DNA damage is modified in such a way that it
is no longer capable of activating the G2/M checkpoint. In
contrast to somatic cells, the irradiated-sperm zygotes which
fail to synthesis full DNA content still can proceed to G2 and
M (Fig. 7b). p53⫺/⫺ zygotes had no S-phase damage checkpoint, and they also did not show delay in M-phase entry,
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FIG. 7. Progression of M phase in zygotes. (a) Zygotes were recovered from in vivo fertilization and analyzed for cleavage. The frequency of
two-cell-stage embryos was scored from 16 to 23 h after mating. Cleavage started at 17 h and was completed at 22 h in p53⫹/⫹ control zygotes (—E—)
(n ⫽ 112) and in p53⫺/⫺ control zygotes (—‚—) (n ⫽ 28). In p53⫹/⫹ irradiated-sperm zygotes (- - -F- - -) (n ⫽ 186), cleavage was delayed for 2 h, while
the delay was marginal in p53⫺/⫺ irradiated-sperm zygotes (- - -Œ- - -) (n ⫽ 44). (b) The PI fluorescence value in two-cell-stage embryos was measured and
plotted for control embryos (open bars) and irradiated-sperm embryos (solid bars). DNA contents in irradiated-sperm early-cleaving embryos (cleaved
before 19 h [n ⫽ 14]) were less than those in control early-cleaving embryos (n ⫽ 14). In contrast, irradiated-sperm late-cleaving embryos (cleaved
at 21 h [n ⫽ 10]) had the same DNA content as control late-cleaving embryos (n ⫽ 14). Error bars, standard deviations.
2228
SHIMURA ET AL.
which is consistent with the requirement of p53 in the G2/M
checkpoint (8).
The biological function of the p53-dependent S-phase
checkpoint is likely to be one of protecting early-stage mouse
development. Our unpublished observation demonstrated that
all zygotes produced from 6-Gy-irradiated sperm progressed at
least to the eight-cell stage, and half of the fetuses were born
normally. This suggests the lack of p53-dependent apoptosis in
pre-implantation stage embryos. In contrast, 6-Gy-irradiatedsperm p53⫺/⫺ zygotes lacking S-phase suppression had a
higher frequency of micronucleus occurrence during subsequent cleavages and did not implant. Thus, the p53-dependent
S-phase damage checkpoint in mouse zygotes is yet another
function of p53 as a guardian of the mammalian genome.
REFERENCES
1. Agarwal, M. L., A. Agarwal, W. R. Taylor, O. Chernova, Y. Sharma, and
G. R. Stark. 1998. A p53-dependent S-phase checkpoint helps to protect cells
from DNA damage in response to starvation for pyrimidine nucleotides.
Proc. Natl. Acad. Sci. USA 95:14775–14780.
2. Ahmadi, A., and S. C. Ng. 1999. Fertilizing ability of DNA-damaged spermatozoa. J. Exp. Zool. 284:696–704.
3. Baatout, S., P. Jacquet, T. Jung, J. Hain, A. Michaux, J. Buset, C. Vandecasteele, L. de Saint-Georges, and L. Baugnet-Mahieu. 1999. Histone H1
kinase activity in one cell mouse embryos blocked in the G2 phase by
X-irradiation. Anticancer Res. 19:1093–1100.
4. Balhorn, R., B. L. Gledhill, and A. J. Wyrobek. 1977. Mouse sperm chromatin proteins: quantitative isolation and partial characterization. Biochemistry 16:4074–4080.
5. Banin, S., L. Moyal, S.-Y. Shieh, Y. Taya, C. W. Anderson, L. Chessa, N. I.
Smorodinsky, C. Prives, Y. Reiss, Y. Shiloh, and Y. Ziv. 1998. Enhanced
phosphorylation of p53 by ATM in response to DNA damage. Science
281:1674–1677.
6. Bunz, F., A. Dutriaux, C. Lengauer, T. Waldman, S. Zhou, J. P. Brown, J. M.
Sedivy, K. W. Kinzler, and B. Vogelstein. 1998. Requirement for p53 and p21
to sustain G2 arrest after DNA damage. Science 282:1497–1501.
7. Canman, C. E., D. S. Lim, K. A. Cimprich, Y. Taya, K. Tamai, K. Sakaguchi, E.
Appella, M. B. Kastan, and J. D. Siliciano. 1998. Activation of the ATM kinase
by ionizing radiation and phosphorylation of p53. Science 281:1677–1679.
8. Chan, T. A., P. M. Hwang, H. Hermeking, K. W. Kinzler, and B. Vogelstein.
2000. Cooperative effects of genes controlling the G2/M checkpoint. Genes
Dev. 14:1584–1588.
9. Demeter, J., S. E. Lee, J. E. Haber, and T. Stearns. 2000. The DNA damage
checkpoint signal in budding yeast is nuclear limited. Mol. Cell 6:487–492.
10. Deng, C., P. Zhang, J. W. Harper, S. J. Ellege, and P. Leder. 1995. Mice
lacking p21CIP1/WAF1 undergo normal development, but are defective in
G1 checkpoint control. Cell 82:675–684.
11. Dulic, V., W. K. Kaufmann, S. J. Wilson, T. D. Tlsty, E. Lees, J. W. Harper,
S. J. Ellege, and S. I. Reed. 1994. p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest. Cell 76:1013–1023.
12. Dutta, A., J. M. Ruppert, J. C. Aster, and E. Winchester. 1993. Inhibition of
DNA replication factor RPA by p53. Nature 365:79–82.
13. Foiani, M., A. Pellicioli, M. Lopes, C. Lucca, M. Ferrari, G. Liberi, M. M.
Falconi, and P. Plevani. 2000. DNA damage checkpoints and DNA replication controls in Saccharomyces cerevisiae. Mutat. Res. 451:187–196.
14. Goldstein, L. S., A. I. Spindle, and R. A. Pedersen. 1975. X-ray sensitivity of
the preimplantation mouse embryo in vitro. Radiat. Res. 62:276–287.
15. Gottifredi, V., S. Shieh, Y. Taya, and C. Prives. 2001. From the cover: p53
accumulates but is functionally impaired when DNA synthesis is blocked.
Proc. Natl. Acad. Sci. USA 30:1036–1041.
16. Grinfeld, S., and P. Jacquet. 1987. An unusual radiation-induced G2 arrest
in the zygote of the BALB/c mouse strain. Int. J. Radiat. Biol. 51:353–363.
17. Gross, S. D., D. P. Hoffman, P. L. Fisette, P. Baas, and R. A. Anderson. 1995.
A phosphatidylinositol 4,5-bisphosphate-sensitive casein kinase I alpha associates with synaptic vesicles and phosphorylates a subset of vesicle proteins. J. Cell Biol. 130:711–724.
18. Gu, J., D. Chen, J. Rosenblum, R. Rubin, and Z. M. Yuan. 2000. Identification of a sequence element from p53 that signals for Mdm2-targeted
degradation. Mol. Cell. Biol. 20:1243–1253.
19. Heyer, B. S., A. MacAuley, O. Behrendtsen, and Z. Werb. 2000. Hypersensitivity to DNA damage leads to increased apoptosis during early mouse
development. Genes Dev. 14:2072–2084.
20. Hirao, A., Y.-Y. Kong, S. Matsuoka, A. Wakeham, J. Ruland, H. Yoshida, D.
Liu, S. J. Elledge, and T. W. Mak. 2000. DNA damage-induced activation of
p53 by the checkpoint kinase Chk2. Science 287:1824–1827.
21. Jurisicova, A., K. E. Latham, R. F. Casper, and S. L. Varmuza. 1998.
Expression and regulation of genes associated with cell death during murine
preimplantation embryo development. Mol. Reprod. Dev. 51:243–253.
22. Kern, S. E., J. A. Pietenpol, S. Thiagalingam, A. Seymour, K. W. Kinzler, and
B. Vogelstein. 1992. Oncogenic forms of p53 inhibit p53-regulated gene
expression. Science 256:827–829.
23. Komarova, E. A., M. V. Chernov, R. Franks, K. Wang, G. Armin, C. R.
Zelnick, D. M. Chin, S. S. Bacus, G. R. Stark, and A. V. Gudkov. 1997.
Transgenic mice with p53-responsive lacZ: p53 activity varies dramatically
during normal development and determines radiation and drug sensitivity in
vivo. EMBO J. 16:1391–1400.
24. Larner, J. M., H. Lee, R. D. Little, P. A. Dijkwel, C. L. Schildkraut, and J. L.
Hamlin. 1999. Radiation down-regulates replication origin activity throughout the S phase in mammalian cells. Nucleic Acids Res. 27:803–809.
25. Lee, H., J. M. Larner, and J. L. Hamlin. 1997. A p53-independent damagesensing mechanism that functions as a checkpoint at the G1/S transition in
Chinese hamster ovary cells. Proc. Natl. Acad. Sci. USA 94:526–531.
26. Levine, A. J. 1997. p53, the cellular gatekeeper for growth and division. Cell
88:323–331.
27. Lobrich, M., P. K. Cooper, and B. Rydberg. 1996. Non-random distribution
of DNA double-strand breaks induced by particle irradiation. Int. J. Radiat.
Biol. 70:493–503.
28. Luthardt, F. W., and R. P. Donahue. 1973. Pronuclear DNA synthesis in
mouse eggs. Exp. Cell Res. 82:143–151.
29. Malashicheva, A. B., T. V. Kislaykova, N. D. Aksenov, K. A. Osipov, and V. A.
Pospelov. 2000. F9 embryonal carcinoma cells fail to stop at G1/S boundary
of the cell cycle after gamma-irradiation due to p21WAF/CIP degradation.
Oncogene 19:3858–3865.
30. Matsuda, Y., T. Yamada, and I. Tobari. 1985. Studies on chromosome
aberrations in the eggs of mice fertilized in vitro after irradiation. I. Chromosome aberrations induced in sperm after X-irradiation. Mutat. Res. 148:
113–117.
31. May, P., and E. May. 1999. Twenty years of p53 research: structural and
functional aspects of the p53 protein. Oncogene 18:7621–7636.
32. Niwa, O., and R. Kominami. 2001. Untargeted mutation of the maternally
derived mouse hypervariable minisatellite allele in F1 mice born to irradiated spermatozoa. Proc. Natl. Acad. Sci. USA 98:1705–1710.
33. Norimura, T., S. Nomoto, M. Katsuki, Y. Gondo, and S. Kondo. 1996.
p53-dependent apoptosis suppresses radiation-induced teratogenesis. Nat.
Med. 2:577–580.
34. Paulovich, A. G., and L. H. Hartwell. 1995. A checkpoint regulates the rate
of progression through S phase in S. cerevisiae in response to DNA damage.
Cell 8:841–847.
35. Rhind, N., and P. Russell. 2000. Checkpoints: it takes more than time to heal
some wounds. Curr. Biol. 10:908–911.
36. Santocanale, C., and J. F. X. Diffley. 1998. A Mec1-and Rad53-dependent
checkpoint controls late-firing origins of DNA replication. Nature 395:615–618.
37. Schmidt-Kastner, P. K., K. Jardine, M. Cormier, and M. W. McBurney.
1998. Absence of p53-dependent cell cycle regulation in pluripotent mouse
cell lines. Oncogene 16:3003–3011.
38. Shirahige, K., Y. Hori, K. Shiraishi, M. Yamashita, K. Takahashi, C. Obuse,
T. Tsurimoto, and H. Yoshikawa. 1998. Regulation of DNA-replication
origins during cell-cycle progression. Nature 395:618–621.
39. Smith, D. B., and K. S. Johnson. 1988. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase.
Gene 67:31–40.
40. Taga, M., K. Shiraishi, T. Shimura, N. Uematsu, T. Kato, Y. Nishimune, S.
Aizawa, M. Oshimura, and O. Niwa. 2000. The effect of caffeine on p53dependent radioresponses in undifferentiated mouse embryonal carcinoma
cells after X-ray and UV-irradiations. J. Radiat. Res. 41:227–241.
41. Tchang, F., and M. Mechali. 1999. Nuclear import of p53 during Xenopus
laevis early development in relation to DNA replication and DNA repair.
Exp. Cell Res. 251:46–56.
42. Tsukada, T., Y. Tomooka, S. Takai, Y. Ueda, S. I. Nishikawa, T. Yagi, T.
Tokunaga, N. Takeda, Y. Suda, S. Abe, I. Matsuo, Y. Ikawa, and S. Aizawa.
1993. Enhanced proliferative potential in culture of cells from p53-deficient
mice. Oncogene 8:3313–3322.
43. Xie, G., R. C. Habbersett, Y. Jia, S. R. Peterson, B. E. Lehnert, E. M. Bradbury,
and J. A. D’Anna. 1998. Requirements for p53 and the ATM gene product in the
regulation of G1/S and S phase checkpoints. Oncogene 16:721–736.
44. Yamada, T., O. Yukawa, K. Asami, and T. Nakazawa. 1982. Effect of chronic
HTO ␤ or 60CO ␥ radiation on preimplantation mouse development in vitro.
Radiat. Res. 92:359–369.
Downloaded from http://mcb.asm.org/ on April 27, 2015 by guest
ACKNOWLEDGMENTS
We thank C. Streffer, Y. Terada, T. Matsumoto, and S. Takeda for
critical reading of the manuscript. Microinjection of the reporter plasmid was done by K. Yokoyama.
This work was supported by a grant-in-aid from the Ministry of
Education, Culture, Sports, Science, and Technology of Japan. The
work was also supported by the Nuclear Safety Research Association.
MOL. CELL. BIOL.