Skip to main content
NCBI home page
EMBO Reports logoLink to EMBO Reports
. 2015 Feb 18;16(5):582–589. doi: 10.15252/embr.201439427

Stella preserves maternal chromosome integrity by inhibiting 5hmC-induced γH2AX accumulation

Tsunetoshi Nakatani 1, Kazuo Yamagata 2, Tohru Kimura 1,3,4, Masaaki Oda 1,3, Hiroyuki Nakashima 3, Mayuko Hori 2, Yoichi Sekita 1,4, Tatsuhiko Arakawa 3, Toshinobu Nakamura 5,6, Toru Nakano 1,3,6,*
PMCID: PMC4428038  PMID: 25694116

Abstract

In the mouse zygote, Stella/PGC7 protects 5-methylcytosine (5mC) of the maternal genome from Tet3-mediated oxidation to 5-hydroxymethylcytosine (5hmC). Although ablation of Stella causes early embryonic lethality, the underlying molecular mechanisms remain unknown. In this study, we report impaired DNA replication and abnormal chromosome segregation (ACS) of maternal chromosomes in Stella-null embryos. In addition, phosphorylation of H2AX (γH2AX), which has been reported to inhibit DNA replication, accumulates in the maternal chromatin of Stella-null zygotes in a Tet3-dependent manner. Cell culture assays verified that ectopic appearance of 5hmC induces abnormal accumulation of γH2AX and subsequent growth retardation. Thus, Stella protects maternal chromosomes from aberrant epigenetic modifications to ensure early embryogenesis.

Keywords: 5-hydroxymethylcytosine, gammaH2AX, Stella, Tet3, zygote

Introduction

Global DNA demethylation takes place soon after fertilization and is essential for reprogramming 1. Although both paternal and maternal genomes are demethylated, the molecular mechanisms underlying these processes are different 2. In the paternal genome, 5-methylcytosine (5mC) rapidly disappears before DNA replication by active DNA demethylation following the conversion to 5-hydroxymethylcytosine (5hmC) by the dioxygenase Tet3 3, 4, 5. In contrast, 5mC of the maternal genome is protected from this process 6, and 5mC and 5mC derivatives, such as 5hmC, become diluted in a DNA replication-dependent manner 7, 8.

Stella, also known as PGC7 and Dppa3, is a maternal factor essential for early development. Stella-deficient females were infertile due to developmental arrest before implantation 6, 9. We showed previously that Stella was essential for protecting the maternal genome from oxidative conversion in zygotes. Stella binds to chromatin containing dimethylated histone 3 lysine 9 (H3K9me2) and inhibits the function of Tet3 by altering localization of the enzyme 10. Thus, Stella-dependent maintenance of epigenetic asymmetry is required for early embryogenesis.

Phosphorylation of the histone variant H2AX (γH2AX) localizes to DNA double-strand breaks (DSBs) to recruit DSB repair factors 11. Recently, it has been demonstrated that γH2AX accumulates without DSBs as a regulator of cell cycle progression by inhibiting DNA replication 12, 13, 14. γH2AX is predominantly localized in the paternal pronucleus of zygotes, but the mechanisms and functions of γH2AX accumulation are unknown.

How the protection of DNA methylation or the inhibition of 5hmC production relates to early development remains unclear. To focus on this issue, we analyzed the abnormal phenotype of Stella-null early embryos using in vivo imaging. Based on detailed observations, we acquired important clues suggesting that delayed DNA replication and abnormal chromosome segregation (ACS) derived from maternal chromatin would be crucial for the developmental arrest. In addition, we revealed that Tet3-dependent aberrant γH2AX formation could be the underlying molecular mechanism.

Results and Discussion

In vivo imaging analysis of Stella-null embryos

To examine the consequences of the Stella-null mutation in detail, we performed live cell imaging of chromosomal dynamics using the monomeric red fluorescent protein 1 coupled to histone H2B (H2B-mRFP1) as a marker. After 4 days in culture, 80% of control and < 10% of Stella-null embryos developed into blastocysts (Fig1A and B and Supplementary Movie S1). Time-lapse imaging demonstrated the delay in cell division of Stella-null embryos; this was verified by calculating the numbers of nuclei during early embryogenesis (Fig1C). Timing of the first cell division was similar in control and Stella-null embryos, but developmental retardation in Stella-null embryos began at the 2- to 4-cell transition (Fig1C).

Figure 1. Abnormal cell division, chromosome segregation, and chromosome integrity of Stella-null embryos.

Figure 1

Open in a new tab
  • A, B Abnormal cell division of Stella-null embryos. Representative live cell imaging photographs from fertilization to 91 h (A). Developmental stages of the embryos at 91 h after in vitro fertilization were categorized into four groups based on the morphology (B). Fifteen control and 22 Stella-null zygotes were obtained from Stella+/− and Stella−/− female mice from a single experiment for time-lapse imaging, respectively.
  • C–E Retarded cell division and abnormal chromosome segregation of Stella-null embryos. The numbers of nuclei visualized by exogenous H2B-mRFP in control (blue) and Stella-null (red) developing embryos were analyzed from 6–66 h after in vitro fertilization (C). Representative images of normal chromosome segregation (NCS) and abnormal chromosome segregation (ASC) in early embryos are shown (D). ACS is represented by ectopic micronuclei, indicated by yellow arrowheads. Embryos cultured until 91 h after in vitro fertilization were categorized to NCS or four ACS groups based on the timing of micronuclei formation (E). Fifteen control and 22 Stella-null zygotes were obtained from Stella+/− and Stella−/− female mice from a single experiment for time-lapse imaging, respectively.
  • F, G Abnormal chromosome integrity of intracytoplasmic sperm injection (ICSI)-derived and Stella-null 2-cell embryos. Representative images of ICSI-derived and Stella-null 2-cell embryos (F). The percentages of H3K9me2-positive micronuclei are shown (G). Total of 31 ICSI embryos from a single experiment and 17 Stella-null embryos from two independent experiments were analyzed.

Data information: Scale bars: 20 μm.

During the course of these experiments, we observed that the early mitotic Stella-null blastomere frequently exhibited abnormal chromosome segregation (ACS) resembling ectopic micronuclei formation (Fig1D and Supplementary Movie S2). The frequencies of ACS in Stella-null embryos from the 1- to 8-cell stages were significantly higher than those of control embryos (Fig1E). Ectopic micronuclei, which are produced in response to the damaged genome, are frequently present in intracytoplasmic sperm injection (ICSI)-derived embryos 15. Micronuclei in ICSI-derived embryos should be derived from paternal chromosomes as they are thought to be formed from the artificially introduced sperm. As expected, the vast majority of micronuclei in the ICSI-derived embryos were H3K9me2 negative, confirming that they were derived from paternal chromosomes (Fig1F and G). Only the maternal pronuclei were stained with the anti-H3K9me2 antibody not only in the control but also in the Stella-null zygotes 6. In contrast, > 80% of the ectopic micronuclei in Stella-null embryos were H3K9me2 positive (Fig1F and G and Supplementary Fig S1), which demonstrated that these micronuclei were derived from impaired maternal chromatin. Thus, chromosomal aberration and subsequent retarded cell division take place in the Stella-null early embryos, presumably due to abnormal integrity of maternal chromatin, as previously reported 16.

Impairment of DNA replication and aberrant accumulation of γH2AX

Mitotic catastrophe occurs following premature entry of cells into mitosis prior to completion of DNA synthesis, and chromosome missegregations could largely be caused by replication errors 17, 18, 19. Therefore, it is conceivable that the chromosomal abnormality of Stella-null early embryos would be caused by incoordination between the timing of cell division and DNA synthesis. We performed a detailed time-course analysis of DNA synthesis in embryos cultured in the presence of 5-bromo-2′-deoxyuridine (BrdU) for every 2-h interval. As shown in Fig2A, the timing of Stella-null zygotes entering into S-phase was similar to control embryos and the majority of control embryos completed DNA replication 12 h after in vitro fertilization (IVF). To the contrary, in the Stella-null embryos, the percentage of the cells with the strong BrdU incorporation was reduced between 8 and 10 h after IVF which suggests that DNA replication had ceased at that time point. In addition, DNA replication was delayed in Stella-null embryos at 2-cell stage (Supplementary Fig S2A and B). These data showed that DNA replication was impaired in the Stella-null embryos. We hypothesized that accumulation of γH2AX, the phosphorylated form of H2AX, which was asymmetrically enriched in paternal chromatin would be an essentially important factor 20, 21, since γH2AX had been reported to inhibit DNA replication 12, 14.

Figure 2. Delayed DNA replication and aberrant γH2AX accumulation in Stella-null embryos.

Figure 2

Open in a new tab
  • A Delayed DNA replication timing in Stella-null embryos. The control and Stella-null zygotes produced by IVF were incubated in KSOM containing BrdU at the indicated intervals. BrdU signals were classified as shown in the Supplementary Materials and Methods. The experiments were repeated at least twice for each interval. Total numbers of samples in each period (4–6, 6–8, 8–10, 10–12, 12–14, 14–16, 16–18, 18–20 hpf) were 7, 13, 13, 10, 9, 4, 8, and 2 in the control and 9, 11, 10, 9, 11, 13, 5, and 2 in Stella-null embryos, respectively.
  • B, C γH2AX accumulation in control and Stella-null embryos during zygotic development. Pronuclei were stained with an anti-Ser139-phospho-H2AX antibody and counterstained with DAPI (B). The numbers of γH2AX foci in maternal and paternal pronuclei are shown (C). The experiments were repeated twice for PN1–2 and PN5–syngamy, and three times for PN3–4. Total numbers of samples in each stage (PN1–2, PN3–4, PN5–syngamy) were 12, 32, and 9 in the control and 8, 24, and 7 in Stella-null embryos, respectively. The median was indicated with a vertical line in the interior of the box, and the maximum and minimum are at the ends of the whiskers. *P < 0.001, **P < 0.0001, n.s., not significant by t-test.
  • D Distribution of H3K9me2 and γH2AX in 2-cell embryos. Total of 12 control and 14 Stella-null embryos from two independent experiments were analyzed.
  • E Distribution of H3K9me2 and 5hmC in 2-cell embryos. Total of 12 control and 11 Stella-null embryos from two independent experiments were analyzed.

Data information: m, maternal pronuleus; p, paternal pronucleus; pb, polar body; Scale bars: 20 μm.

γH2AX foci were detected at PN3–4 stages, predominantly in paternal pronuclei (Fig2B and C and Supplementary Table S1), as reported previously 20, 21. In contrast, γH2AX foci were increased in maternal pronuclei of Stella-null zygotes at this stage (Fig2B and C and Supplementary Table S1). To examine the effect of γH2AX in the development of Stella-null embryos, we inhibited the γH2AX accumulation using various kinase inhibitors 22, 23. Only ataxia telangiectasia and Rad3-related (ATR) inhibitor suppressed the accumulation of γH2AX (Supplementary Fig S3A and B). However, developmental defect of Stella-null embryos was not restored, because the function of ATR other than the γH2AX accumulation was essential for early development (Supplementary Fig S3C) 24.

The timing of γH2AX accumulation was identical to that of 5hmC, which was produced by oxidation from 5mC by Tet3 3, 4, 5 (Fig2B). This concomitant appearance and the data from Stella-null embryos strongly suggested a functional relationship between 5hmC and γH2AX. Next, we examined the pattern of γH2AX at the 2-cell stage, because the delay of cell division and DNA replication of the Stella-null embryos were observed at 2-cell stage (Fig1C and Supplementary Fig S2B). At 2-cell stage, when Tet3 protein was no longer expressed, both γH2AX foci and 5hmC were detectable only in the H3K9me2-negative paternal chromatin (Fig2D and E and Supplementary Fig S4). In contrast, γH2AX foci and 5hmC were distributed evenly in the H3K9me2-positive chromatin of Stella-null 2-cell embryos (Fig2D and E). These data support the notion that the accumulation of γH2AX foci well correlates with the induction of 5hmC.

To exclude the possibility that the other 5mC oxidative derivatives, 5fC and 5caC, were involved in the induction of γH2AX accumulation (Fig3A), we analyzed 5fC and 5caC in zygotes after expression of thymine DNA glycosylase (TDG). Microinjection of Tdg mRNA into zygotes decreased 5fC and 5caC, but did not change the amount of 5hmC 25 (Fig3B and C and Supplementary Fig S5). In addition, although the number of γH2AX foci was not affected by the expression of TDG, it was reduced by the knockdown of Tet3 (Fig3D–F and Supplementary Table S2). These results strongly suggest a high correlation between the appearance of 5hmC and the accumulation of γH2AX.

Figure 3. Accumulation of 5hmC and γH2AX in zygotes.

Figure 3

Open in a new tab
  • A Distribution of 5hmC, 5fC, and 5caC in early embryos. Control and Stella-null embryos at the PN3–4 stage were stained with anti-5hmC, anti-5fC, and anti-5caC antibodies. Total numbers of samples in each staining (5hmC, 5fC, and 5caC) from two independent experiments were 5, 7, and 5 in the control and 7, 7, and 7 in Stella-null embryos, respectively.
  • B, C Distribution of 5hmC, 5fC, and 5caC in TDG-expressing zygotes. Zygotes were injected with Myc-tagged Tdg mRNA and cultured for 5 h in KSOM. Representative images of 5mC derivatives (B) and the fluorescence intensities (C) are shown. Total numbers of samples in each staining (5hmC, 5fC, and 5caC) from three independent experiments were 17, 14, and 7 in the control and 13, 15, and 8 in TDG-expressing zygotes, respectively. Bar shows the mean ± SEM. *P < 0.01, **P < 0.001, ***P < 0.0001, n.s., not significant by t-test.
  • D, E γH2AX localization in the TDG-expressing zygotes. Representative images of γH2AX (D) and the numbers of γH2AX foci are shown (E). Total of 13 and 15 TDG-expressing zygotes from three independent experiments were analyzed. The median was indicated with a vertical line in the interior of the box, and the maximum and minimum are at the ends of the whiskers. *P < 0.005, n.s., not significant by t-test.
  • F γH2AX localization in the Tet3 knockdown zygotes. Representative images of γH2AX and Tet3 are shown. The numbers of samples from a single experiment for each staining (γH2AX and Tet3) were 6 and 10 in the control and 11 and 4 in TDG-expressing zygotes, respectively.

Data information: m, maternal pronuleus; p, paternal pronucleus; pb, polar body; Scale bars: 20 μm.

Accumulation of γH2AX and abnormal DNA replication by Tet-dependent production of 5hmC in culture

To understand the molecular mechanism underlying the effect of 5hmC on γH2AX and DNA replication in more detail, we performed in vitro experiments using NIH-3T3 cells. Enforced expression of Tet3 or Tet2 induced only 5hmC among the three oxidative derivatives (Fig4A). Meanwhile, expression of the Tet3 mutant (H950Y, D952A), in which enzymatic activity was abolished, induced none of the oxidative derivatives 26 (Fig4A). γH2AX was induced in the Tet3- and Tet2-expressing cells but not in the Tet3 mutant-expressing cells, indicating that γH2AX formation was induced by 5hmC (Fig4B). Overexpression of Tet markedly decreased BrdU incorporation, S and G2/M population, and growth rate, showing that Tet negatively affected the DNA replication, cell cycle, and cell proliferation (Fig4C–F, and Supplementary Table S3).

Figure 4. γH2AX accumulation and delayed DNA replication by Tet3-induced 5hmC.

Figure 4

Open in a new tab
  • A 5hmC induction by Tet3 in NIH-3T3 cells. Immunostaining analysis was performed at 48 h after transfection with FLAG-Tet2, FLAG-Tet3, and a catalytic domain-defective FLAG-Tet3 mutant-expressing vector. In vitro experiments were repeated at least three times, and similar results were obtained.
  • B γH2AX induction by Tet3-induced 5hmC in NIH-3T3 cells. Immunostaining was performed at 48 h after transfection with Tet-expressing vectors. Treatment with 500 μM H2O2 for 3 h was used as a positive control.
  • C, D Inhibition of DNA replication by Tet3-induced 5hmC in NIH-3T3 cells. Representative immunostaining images (C) and percentages of BrdU-positive cells (D) are shown. Two hundred and fourteen control, 29 FLAG-Tet2-, 37 FLAG-Tet3-, and 43 FLAG-Tet3 mutant-expressing cells were analyzed. *P < 0.001 by chi-square test.
  • E Delayed cell cycle progression in the FLAG-Tet3-expressing NIH-3T3 cells. Expressions of the EGFP, FLAG-Tet3, and FLAG-Tet3 mutant were introduced by lentiviral vectors.
  • F Growth retardation in the FLAG-Tet3-overexpressing NIH-3T3 cells. The number of cells in control (blue), FALG-Tet3-expressing (red), and FLAG-Tet3 mutant-expressing (green) were counted every 3 days in culture. Cell proliferation data were determined by the average of four independent experiments.

Data information: Scale bars: 10 μm.

Conclusions

In this study, a novel molecular link between two epigenetic modifications, 5hmC and γH2AX, was analyzed. Expression analysis using Tet2, Tet3, and Tet3 catalytic mutants showed that the enzymatic activity of Tet, primarily characterized by the oxidation of 5mC to 5hmC, gave rise to the accumulation of γH2AX (Fig4). The data obtained in vitro were consistent with those observed in the maternal pronuclei of Stella-null zygotes (Fig2). These processes in both cultured cells and zygotes were induced by 5hmC, but not by 5fC or 5caC, since the enzymatic activity of Tet3 and Tet2 did not increase 5fC and 5caC (Fig4). In addition, the accumulation of γH2AX was suppressed by the knockdown of Tet3 in vivo (Fig3F). Although epigenetic asymmetry, defined as the difference in epigenetic modifications between parental genomes, is widely observed in mammals and believed to be essential for early embryonic development, the underlying molecular mechanisms and their roles remain elusive. Maternal chromatin and paternal chromatin in zygotes differ in several respects, such as histone modification 27 and susceptibility to Tet3 10, which are dependent on the function of Stella. We reported here that Stella-null embryos, which acquired aberrant 5hmC and γH2AX in maternal chromatin, showed biological phenomena such as ACS and delay of DNA replication. It has been demonstrated that accumulation of γH2AX induces the inhibition of DNA replication and the induction of mitotic catastrophe by replication stress 12, 19, 28. Taken together, our results suggest that the lack of epigenetic asymmetry by Stella deletion would be the cause of deleterious effects on early development by the ACS and delay of DNA replication.

A global alteration in DNA methylation soon after fertilization plays a pivotal role in reprogramming the epigenetic status of chromatin. Although 5hmC has emerged as an intermediate for active DNA demethylation 29, it exists stably from zygotes to 2-cell stage embryos and, therefore, has been postulated to play a functional role in chromatin 3. Based on our NIH-3T3 data, we suggest that 5hmC would perturb the integrity of maternal chromosomes and that the protection of the maternal chromatin integration by Stella is critical for normal development. Our data would provide novel insight into the function of Stella in DNA replication and chromosome integrity in early embryogenesis.

Materials and Methods

Mouse zygotes were obtained from in vitro fertilization, ICSI or natural matings between superovulated B6D2F1, Stella+/−, or Stella−/− female mice and B6D2F1 male mice. Zygotes and in vitro cultured embryos were used for time-lapse imaging, BrdU labeling, and immunohistochemistry. NIH-3T3 cells were grown under standard conditions. Full Materials and Methods are available in the Supplementary Information.

Acknowledgments

We thank Guo-Liang Xu (Institute of Biochemistry And Cell Biology, Shanghai, China) for providing the rabbit anti-Tet3 antibody and Hiroyuki Miyoshi (RIKEN BRC, Tsukuba, Japan) for providing the pCAG-HIVgp, pCMV-VSV-G-RSV-Rev, and CSII-EF-MCS-IRES2-Venus plasmid. The work was funded by JST CREST, Ministry of Education, Science, Sports, Culture, and Technology of Japan.

Author contributions

TsuN, MO, and TorN conceived the project and wrote the manuscript. TsuN designed and performed the experiments and evaluated the results. KY, MH, TK, HN, YS, TA, and TosN performed some experiments.

Conflict of interest

The authors declare that they have no conflict of interest.

Supporting Information

Supplementary Figure S1

embr0016-0582-sd1.pdf (13.2MB, pdf)

Supplementary Figure S2

embr0016-0582-sd2.pdf (1.6MB, pdf)

Supplementary Figure S3

embr0016-0582-sd3.pdf (13.6MB, pdf)

Supplementary Figure S4

embr0016-0582-sd4.pdf (8.1MB, pdf)

Supplementary Figure S5

embr0016-0582-sd5.pdf (52.5MB, pdf)

Supplementary Tables

embr0016-0582-sd6.docx (24.5KB, docx)

Supplementary Movie S1

Download video file (5.9MB, avi)

Supplementary Movie S2

Download video file (3.9MB, avi)

Supplementary Information

embr0016-0582-sd9.docx (37.9KB, docx)

Review Process File

embr0016-0582-sd10.pdf (290.4KB, pdf)

References

  1. Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development. Science. 2001;293:1089–1093. doi: 10.1126/science.1063443. [DOI] [PubMed] [Google Scholar]
  2. Oswald J, Engemann S, Lane N, Mayer W, Olek A, Fundele R, Dean W, Reik W, Walter J. Active demethylation of the paternal genome in the mouse zygote. Curr Biol. 2000;10:475–478. doi: 10.1016/s0960-9822(00)00448-6. [DOI] [PubMed] [Google Scholar]
  3. Iqbal K, Jin SG, Pfeifer GP, Szabo PE. Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine. Proc Natl Acad Sci U S A. 2011;108:3642–3647. doi: 10.1073/pnas.1014033108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Wossidlo M, Nakamura T, Lepikhov K, Marques CJ, Zakhartchenko V, Boiani M, Arand J, Nakano T, Reik W, Walter J. 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming. Nat Commun. 2011;2:241. doi: 10.1038/ncomms1240. [DOI] [PubMed] [Google Scholar]
  5. Gu TP, Guo F, Yang H, Wu HP, Xu GF, Liu W, Xie ZG, Shi L, He X, Jin SG, et al. The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature. 2012;477:606–610. doi: 10.1038/nature10443. [DOI] [PubMed] [Google Scholar]
  6. Nakamura T, Arai Y, Umehara H, Masuhara M, Kimura T, Taniguchi H, Sekimoto T, Ikawa M, Yoneda Y, Okabe M, et al. PGC7/Stella protects against DNA demethylation in early embryogenesis. Nat Cell Biol. 2007;9:64–71. doi: 10.1038/ncb1519. [DOI] [PubMed] [Google Scholar]
  7. Inoue A, Shen L, Dai Q, He C, Zhang Y. Generation and replication-dependent dilution of 5fC and 5caC during mouse preimplantation development. Cell Res. 2011;21:1670–1676. doi: 10.1038/cr.2011.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Inoue A, Zhang Y. Replication-dependent loss of 5-hydroxymethylcytosine in mouse preimplantation embryos. Science. 2011;334:194. doi: 10.1126/science.1212483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Payer B, Saitou M, Barton SC, Thresher R, Dixon JP, Zahn D, Colledge WH, Carlton MB, Nakano T, Surani MA. Stella is a maternal effect gene required for normal early development in mice. Curr Biol. 2003;13:2110–2117. doi: 10.1016/j.cub.2003.11.026. [DOI] [PubMed] [Google Scholar]
  10. Nakamura T, Liu YJ, Nakashima H, Umehara H, Inoue K, Matoba S, Tachibana M, Ogura A, Shinkai Y, Nakano T. PGC7 binds histone H3K9me2 to protect against conversion of 5mC to 5hmC in early embryos. Nature. 2012;486:415–419. doi: 10.1038/nature11093. [DOI] [PubMed] [Google Scholar]
  11. Fernandez-Capetillo O, Lee A, Nussenzweig M, Nussenzweig A. H2AX: the histone guardian of the genome. DNA Repair (Amst) 2004;3:959–967. doi: 10.1016/j.dnarep.2004.03.024. [DOI] [PubMed] [Google Scholar]
  12. Andang M, Hjerling-Leffler J, Moliner A, Lundgren TK, Castelo-Branco G, Nanou E, Pozas E, Bryja V, Halliez S, Nishimaru H, et al. Histone H2AX-dependent GABA(A) receptor regulation of stem cell proliferation. Nature. 2008;451:460–464. doi: 10.1038/nature06488. [DOI] [PubMed] [Google Scholar]
  13. Turinetto V, Orlando L, Sanchez-Ripoll Y, Kumpfmueller B, Storm MP, Porcedda P, Minieri V, Saviozzi S, Accomasso L, Cibrario Rocchietti E, et al. High basal gammaH2AX levels sustain self-renewal of mouse embryonic and induced pluripotent stem cells. Stem Cells. 2012;30:1414–1423. doi: 10.1002/stem.1133. [DOI] [PubMed] [Google Scholar]
  14. Fernando RN, Eleuteri B, Abdelhady S, Nussenzweig A, Andang M, Ernfors P. Cell cycle restriction by histone H2AX limits proliferation of adult neural stem cells. Proc Natl Acad Sci U S A. 2011;108:5837–5842. doi: 10.1073/pnas.1014993108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Yamagata K, Suetsugu R, Wakayama T. Assessment of chromosomal integrity using a novel live-cell imaging technique in mouse embryos produced by intracytoplasmic sperm injection. Hum Reprod. 2009;24:2490–2499. doi: 10.1093/humrep/dep236. [DOI] [PubMed] [Google Scholar]
  16. Mizutani E, Yamagata K, Ono T, Akagi S, Geshi M, Wakayama T. Abnormal chromosome segregation at early cleavage is a major cause of the full-term developmental failure of mouse clones. Dev Biol. 2012;364:56–65. doi: 10.1016/j.ydbio.2012.01.001. [DOI] [PubMed] [Google Scholar]
  17. Heald R, McLoughlin M, McKeon F. Human wee1 maintains mitotic timing by protecting the nucleus from cytoplasmically activated Cdc2 kinase. Cell. 1993;74:463–474. doi: 10.1016/0092-8674(93)80048-j. [DOI] [PubMed] [Google Scholar]
  18. Schlegel R, Pardee AB. Caffeine-induced uncoupling of mitosis from the completion of DNA replication in mammalian cells. Science. 1986;232:1264–1266. doi: 10.1126/science.2422760. [DOI] [PubMed] [Google Scholar]
  19. Burrell RA, McClelland SE, Endesfelder D, Groth P, Weller MC, Shaikh N, Domingo E, Kanu N, Dewhurst SM, Gronroos E, et al. Replication stress links structural and numerical cancer chromosomal instability. Nature. 2013;494:492–496. doi: 10.1038/nature11935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wossidlo M, Arand J, Sebastiano V, Lepikhov K, Boiani M, Reinhardt R, Scholer H, Walter J. Dynamic link of DNA demethylation, DNA strand breaks and repair in mouse zygotes. EMBO J. 2010;29:1877–1888. doi: 10.1038/emboj.2010.80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ziegler-Birling C, Helmrich A, Tora L, Torres-Padilla ME. Distribution of p53 binding protein 1 (53BP1) and phosphorylated H2A.X during mouse preimplantation development in the absence of DNA damage. Int J Dev Biol. 2009;53:1003–1011. doi: 10.1387/ijdb.082707cz. [DOI] [PubMed] [Google Scholar]
  22. An J, Huang YC, Xu QZ, Zhou LJ, Shang ZF, Huang B, Wang Y, Liu XD, Wu DC, Zhou PK. DNA-PKcs plays a dominant role in the regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression. BMC Mol Biol. 2010;11:18. doi: 10.1186/1471-2199-11-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Reaper PM, Griffiths MR, Long JM, Charrier JD, Maccormick S, Charlton PA, Golec JM, Pollard JR. Selective killing of ATM- or p53-deficient cancer cells through inhibition of ATR. Nat Chem Biol. 2011;7:428–430. doi: 10.1038/nchembio.573. [DOI] [PubMed] [Google Scholar]
  24. Brown EJ, Baltimore D. ATR disruption leads to chromosomal fragmentation and early embryonic lethality. Genes Dev. 2000;14:397–402. [PMC free article] [PubMed] [Google Scholar]
  25. Nabel CS, Jia H, Ye Y, Shen L, Goldschmidt HL, Stivers JT, Zhang Y, Kohli RM. AID/APOBEC deaminases disfavor modified cytosines implicated in DNA demethylation. Nat Chem Biol. 2012;8:751–758. doi: 10.1038/nchembio.1042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ito S, D'Alessio AC, Taranova OV, Hong K, Sowers LC, Zhang Y. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature. 2010;466:1129–1133. doi: 10.1038/nature09303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Arney KL, Bao S, Bannister AJ, Kouzarides T, Surani MA. Histone methylation defines epigenetic asymmetry in the mouse zygote. Int J Dev Biol. 2002;46:317–320. [PubMed] [Google Scholar]
  28. Fernandez-Capetillo O, Mahadevaiah SK, Celeste A, Romanienko PJ, Camerini-Otero RD, Bonner WM, Manova K, Burgoyne P, Nussenzweig A. H2AX is required for chromatin remodeling and inactivation of sex chromosomes in male mouse meiosis. Dev Cell. 2003;4:497–508. doi: 10.1016/s1534-5807(03)00093-5. [DOI] [PubMed] [Google Scholar]
  29. Hajkova P, Jeffries SJ, Lee C, Miller N, Jackson SP, Surani MA. Genome-wide reprogramming in the mouse germ line entails the base excision repair pathway. Science. 2010;329:78–82. doi: 10.1126/science.1187945. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figure S1

embr0016-0582-sd1.pdf (13.2MB, pdf)

Supplementary Figure S2

embr0016-0582-sd2.pdf (1.6MB, pdf)

Supplementary Figure S3

embr0016-0582-sd3.pdf (13.6MB, pdf)

Supplementary Figure S4

embr0016-0582-sd4.pdf (8.1MB, pdf)

Supplementary Figure S5

embr0016-0582-sd5.pdf (52.5MB, pdf)

Supplementary Tables

embr0016-0582-sd6.docx (24.5KB, docx)

Supplementary Movie S1

Download video file (5.9MB, avi)

Supplementary Movie S2

Download video file (3.9MB, avi)

Supplementary Information

embr0016-0582-sd9.docx (37.9KB, docx)

Review Process File

embr0016-0582-sd10.pdf (290.4KB, pdf)

Articles from EMBO Reports are provided here courtesy of Nature Publishing Group

RESOURCES