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Understanding paternal genome demethylation through live-cell imaging and siRNA

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Abstract

Identification of a DNA demethylase responsible for zygotic paternal DNA demethylation has been one of the most challenging goals in the field of epigenetics. Several candidate molecules have been proposed, but their involvement in the demethylation remains controversial, partly due to the difficulty of preparing a sufficient quantity of materials for biochemical analysis. In this review, we utilize a recently developed method for live-cell imaging of mouse zygotes combined with RNA interference (RNAi) to search for factors that affect zygotic paternal DNA demethylation. The combined use of various fluorescent probes and RNAi is a useful approach for the study of not only DNA demethylation but also the spatiotemporal dynamics of histone depositions in zygotes, although it is not appropriate for large-scale screening or knockdown of genes that are abundantly expressed before fertilization. This new technique enables us to understand the epigenetic hierarchy during cellular response and differentiation in preimplantation embryos.

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References

  1. Okada Y, Yamagata K, Hong K, Wakayama T, Zhang Y (2010) A role for the elongator complex in zygotic paternal genome demethylation. Nature 463(7280):554–558

    Article  PubMed  CAS  Google Scholar 

  2. Mayer W, Niveleau A, Walter J, Fundele R, Haaf T (2000) Demethylation of the zygotic paternal genome. Nature 403(6769):501–502

    Article  PubMed  CAS  Google Scholar 

  3. Santos F, Hendrich B, Reik W, Dean W (2002) Dynamic reprogramming of DNA methylation in the early mouse embryo. Dev Biol 241(1):172–182

    Article  PubMed  CAS  Google Scholar 

  4. Gehring M, Reik W, Henikoff S (2009) DNA demethylation by DNA repair. Trends Genet 25(2):82–90

    Article  PubMed  CAS  Google Scholar 

  5. Ooi SK, Bestor TH (2008) The colorful history of active DNA demethylation. Cell 133(7):1145–1148

    Article  PubMed  CAS  Google Scholar 

  6. Bhutani N, Brady JJ, Damian M, Sacco A, Corbel SY, Blau HM (2010) Reprogramming towards pluripotency requires AID-dependent DNA demethylation. Nature 463(7284):1042–1047

    Article  PubMed  CAS  Google Scholar 

  7. Popp C, Dean W, Feng S, Cokus SJ, Andrews S, Pellegrini M, Jacobsen SE, Reik W (2010) Genome-wide erasure of DNA methylation in mouse primordial germ cells is affected by AID deficiency. Nature 463(7284):1101–1105

    Article  PubMed  CAS  Google Scholar 

  8. Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brudno Y, Agarwal S, Iyer LM, Liu DR, Aravind L, Rao A (2009) Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324(5929):930–935

    Article  PubMed  CAS  Google Scholar 

  9. Ito S, D’Alessio AC, Taranova OV, Hong K, Sowers LC, Zhang Y (2010) Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature 466(7310):1129–1133

    Google Scholar 

  10. Shimomura O, Johnson FH, Saiga Y (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 59:223–239

    Article  PubMed  CAS  Google Scholar 

  11. Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ (1992) Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111(2):229–233

    Article  PubMed  CAS  Google Scholar 

  12. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263(5148):802–805

    Article  PubMed  CAS  Google Scholar 

  13. Amsterdam A, Lin S, Hopkins N (1995) The Aequorea victoria green fluorescent protein can be used as a reporter in live zebrafish embryos. Dev Biol 171(1):123–129

    Article  PubMed  CAS  Google Scholar 

  14. Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y (1997) ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett 407(3):313–319

    Article  PubMed  CAS  Google Scholar 

  15. Ikawa M, Kominami K, Yoshimura Y, Tanaka K, Nishimune Y, Okabe M (1995) A rapid and non-invasive selection of transgenic embryos before implantation using green fluorescent protein (GFP). FEBS Lett 375(1–2):125–128

    Article  PubMed  CAS  Google Scholar 

  16. Zernicka-Goetz M, Pines J, McLean Hunter S, Dixon JP, Siemering KR, Haseloff J, Evans MJ (1997) Following cell fate in the living mouse embryo. Development 124(6):1133–1137

    PubMed  CAS  Google Scholar 

  17. Brunet S, Polanski Z, Verlhac MH, Kubiak JZ, Maro B (1998) Bipolar meiotic spindle formation without chromatin. Curr Biol 8(22):1231–1234

    Article  PubMed  CAS  Google Scholar 

  18. Hadjantonakis AK, Gertsenstein M, Ikawa M, Okabe M, Nagy A (1998) Non-invasive sexing of preimplantation stage mammalian embryos. Nat Genet 19(3):220–222

    Article  PubMed  CAS  Google Scholar 

  19. Dard N, Louvet S, Santa-Maria A, Aghion J, Martin M, Mangeat P, Maro B (2001) In vivo functional analysis of ezrin during mouse blastocyst formation. Dev Biol 233(1):161–173

    Article  PubMed  CAS  Google Scholar 

  20. Plusa B, Hadjantonakis AK, Gray D, Piotrowska-Nitsche K, Jedrusik A, Papaioannou VE, Glover DM, Zernicka-Goetz M (2005) The first cleavage of the mouse zygote predicts the blastocyst axis. Nature 434(7031):391–395

    Article  PubMed  CAS  Google Scholar 

  21. Kurotaki Y, Hatta K, Nakao K, Nabeshima Y, Fujimori T (2007) Blastocyst axis is specified independently of early cell lineage but aligns with the ZP shape. Science 316(5825):719–723

    Article  PubMed  CAS  Google Scholar 

  22. Yamagata K, Yamazaki T, Miki H, Ogonuki N, Inoue K, Ogura A, Baba T (2007) Centromeric DNA hypomethylation as an epigenetic signature discriminates between germ and somatic cell lineages. Dev Biol 312(1):419–426

    Article  PubMed  CAS  Google Scholar 

  23. Cavaleri FM, Balbach ST, Gentile L, Jauch A, Bohm-Steuer B, Han YM, Scholer HR, Boiani M (2008) Subsets of cloned mouse embryos and their non-random relationship to development and nuclear reprogramming. Mech Dev 125(1–2):153–166

    Article  PubMed  CAS  Google Scholar 

  24. Yamazaki T, Yamagata K, Baba T (2007) Time-lapse and retrospective analysis of DNA methylation in mouse preimplantation embryos by live cell imaging. Dev Biol 304(1):409–419

    Article  PubMed  CAS  Google Scholar 

  25. Nakao M, Matsui S, Yamamoto S, Okumura K, Shirakawa M, Fujita N (2001) Regulation of transcription and chromatin by methyl-CpG binding protein MBD1. Brain Dev 23(1):S174–S176

    Article  PubMed  Google Scholar 

  26. Hendrich B, Bird A (1998) Identification and characterization of a family of mammalian methyl-CpG binding proteins. Mol Cell Biol 18(11):6538–6547

    PubMed  CAS  Google Scholar 

  27. Jorgensen HF, Ben-Porath I, Bird AP (2004) Mbd1 is recruited to both methylated and nonmethylated CpGs via distinct DNA binding domains. Mol Cell Biol 24(8):3387–3395

    Article  PubMed  CAS  Google Scholar 

  28. Klose RJ, Bird AP (2006) Genomic DNA methylation: the mark and its mediators. Trends Biochem Sci 31(2):89–97

    Article  PubMed  CAS  Google Scholar 

  29. Sasai N, Defossez PA (2009) Many paths to one goal? The proteins that recognize methylated DNA in eukaryotes. Int J Dev Biol 53(2–3):323–334

    Article  PubMed  CAS  Google Scholar 

  30. Fujita N, Takebayashi S, Okumura K, Kudo S, Chiba T, Saya H, Nakao M (1999) Methylation-mediated transcriptional silencing in euchromatin by methyl-CpG binding protein MBD1 isoforms. Mol Cell Biol 19(9):6415–6426

    PubMed  CAS  Google Scholar 

  31. Ohki I, Shimotake N, Fujita N, Nakao M, Shirakawa M (1999) Solution structure of the methyl-CpG-binding domain of the methylation-dependent transcriptional repressor MBD1. EMBO J 18(23):6653–6661

    Article  PubMed  CAS  Google Scholar 

  32. Ohki I, Shimotake N, Fujita N, Jee J, Ikegami T, Nakao M, Shirakawa M (2001) Solution structure of the methyl-CpG binding domain of human MBD1 in complex with methylated DNA. Cell 105(4):487–497

    Article  PubMed  CAS  Google Scholar 

  33. Sarraf SA, Stancheva I (2004) Methyl-CpG binding protein MBD1 couples histone H3 methylation at lysine 9 by SETDB1 to DNA replication and chromatin assembly. Mol Cell 15(4):595–605

    Article  PubMed  CAS  Google Scholar 

  34. Tsumura A, Hayakawa T, Kumaki Y, Takebayashi S, Sakaue M, Matsuoka C, Shimotohno K, Ishikawa F, Li E, Ueda HR, Nakayama J, Okano M (2006) Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Genes Cells 11(7):805–814

    Article  PubMed  CAS  Google Scholar 

  35. Devgan V, Rao MR, Seshagiri PB (2004) Impact of embryonic expression of enhanced green fluorescent protein on early mouse development. Biochem Biophys Res Commun 313(4):1030–1036

    Article  PubMed  CAS  Google Scholar 

  36. Richter JD (1999) Cytoplasmic polyadenylation in development and beyond. Microbiol Mol Biol Rev 63(2):446–456

    PubMed  CAS  Google Scholar 

  37. Yamagata K, Yamazaki T, Yamashita M, Hara Y, Ogonuki N, Ogura A (2005) Noninvasive visualization of molecular events in the mammalian zygote. Genesis 43(2):71–79

    Article  PubMed  CAS  Google Scholar 

  38. Kobayakawa S, Miike K, Nakao M, Abe K (2007) Dynamic changes in the epigenomic state and nuclear organization of differentiating mouse embryonic stem cells. Genes Cells 12(4):447–460

    Article  PubMed  CAS  Google Scholar 

  39. Yamazaki T, Kobayakawa S, Yamagata K, Abe K, Baba T (2007) Molecular dynamics of heterochromatin protein 1beta, HP1beta, during mouse preimplantation development. J Reprod Dev 53(5):1035–1041

    Article  PubMed  CAS  Google Scholar 

  40. Yamagata K, Suetsugu R, Wakayama T (2009) Long-term, six-dimensional live-cell imaging for the mouse preimplantation embryo that does not affect full-term development. J Reprod Dev 55(3):343–350

    Article  PubMed  Google Scholar 

  41. Yamagata K, Suetsugu R, Wakayama T (2009) Assessment of chromosomal integrity using a novel live-cell imaging technique in mouse embryos produced by intracytoplasmic sperm injection. Hum Reprod 24(10):2490–2499

    Article  PubMed  CAS  Google Scholar 

  42. Fujita N, Watanabe S, Ichimura T, Tsuruzoe S, Shinkai Y, Tachibana M, Chiba T, Nakao M (2003) Methyl-CpG binding domain 1 (MBD1) interacts with the Suv39h1-HP1 heterochromatic complex for DNA methylation-based transcriptional repression. J Biol Chem 278(26):24132–24138

    Article  PubMed  CAS  Google Scholar 

  43. Wianny F, Zernicka-Goetz M (2000) Specific interference with gene function by double-stranded RNA in early mouse development. Nat Cell Biol 2(2):70–75

    Article  PubMed  CAS  Google Scholar 

  44. Sonn S, Oh GT, Rhee K (2010) Nek2 and its substrate, centrobin/Nip2, are required for proper meiotic spindle formation of the mouse oocytes. Zygote (in press)

  45. Amanai M, Shoji S, Yoshida N, Brahmajosyula M, Perry AC (2006) Injection of mammalian metaphase II oocytes with short interfering RNAs to dissect meiotic and early mitotic events. Biol Reprod 75(6):891–898

    Article  PubMed  CAS  Google Scholar 

  46. Wong LH, Ren H, Williams E, McGhie J, Ahn S, Sim M, Tam A, Earle E, Anderson MA, Mann J, Choo KH (2009) Histone H3.3 incorporation provides a unique and functionally essential telomeric chromatin in embryonic stem cells. Genome Res 19(3):404–414

    Article  PubMed  CAS  Google Scholar 

  47. Matsuoka T, Sato M, Tokoro M, Shin SW, Uenoyama A, Ito K, Hitomi S, Amano T, Anzai M, Kato H, Mitani T, Saeki K, Hosoi Y, Iritani A, Matsumoto K (2008) Identification of ZAG1, a novel protein expressed in mouse preimplantation, and its putative roles in zygotic genome activation. J Reprod Dev 54(3):192–197

    Article  PubMed  CAS  Google Scholar 

  48. Talbert PB, Henikoff S (2010) Histone variants–ancient wrap artists of the epigenome. Nat Rev Mol Cell Biol 11(4):264–275

    Article  PubMed  CAS  Google Scholar 

  49. Trostle-Weige PK, Meistrich ML, Brock WA, Nishioka K (1984) Isolation and characterization of TH3, a germ cell-specific variant of histone 3 in rat testis. J Biol Chem 259(14):8769–8776

    PubMed  CAS  Google Scholar 

  50. Loppin B, Bonnefoy E, Anselme C, Laurencon A, Karr TL, Couble P (2005) The histone H3.3 chaperone HIRA is essential for chromatin assembly in the male pronucleus. Nature 437(7063):1386–1390

    Article  PubMed  CAS  Google Scholar 

  51. Torres-Padilla ME, Bannister AJ, Hurd PJ, Kouzarides T, Zernicka-Goetz M (2006) Dynamic distribution of the replacement histone variant H3.3 in the mouse oocyte and preimplantation embryos. Int J Dev Biol 50(5):455–461

    PubMed  CAS  Google Scholar 

  52. Santenard A, Ziegler-Birling C, Koch M, Tora L, Bannister AJ, Torres-Padilla ME (2010) Heterochromatin formation in the mouse embryo requires critical residues of the histone variant H3.3. Nat Cell Biol 12(9):853–862

    Article  PubMed  CAS  Google Scholar 

  53. Nakano A (2002) Spinning-disk confocal microscopy—a cutting-edge tool for imaging of membrane traffic. Cell Struct Funct 27(5):349–355

    Article  PubMed  Google Scholar 

  54. Toomre D, Pawly JB (2006) Disk-scanning confocal microscope. Springer, New York

    Google Scholar 

  55. Walter T, Shattuck DW, Baldock R, Bastin ME, Carpenter AE, Duce S, Ellenberg J, Fraser A, Hamilton N, Pieper S, Ragan MA, Schneider JE, Tomancak P, Heriche JK (2010) Visualization of image data from cells to organisms. Nat Methods 7(3 Suppl):S26–S41

    Article  PubMed  CAS  Google Scholar 

  56. Held M, Schmitz MH, Fischer B, Walter T, Neumann B, Olma MH, Peter M, Ellenberg J, Gerlich DW (2010) CellCognition: time-resolved phenotype annotation in high-throughput live cell imaging. Nat Methods 7(9):747–754

    Article  PubMed  CAS  Google Scholar 

  57. Barreto G, Schafer A, Marhold J, Stach D, Swaminathan SK, Handa V, Doderlein G, Maltry N, Wu W, Lyko F, Niehrs C (2007) Gadd45a promotes epigenetic gene activation by repair-mediated DNA demethylation. Nature 445(7128):671–675

    Article  PubMed  CAS  Google Scholar 

  58. Hayashi-Takanaka Y, Yamagata K, Nozaki N, Kimura H (2009) Visualizing histone modifications in living cells: spatiotemporal dynamics of H3 phosphorylation during interphase. J Cell Biol 187(6):781–790

    Article  PubMed  CAS  Google Scholar 

  59. Kimura H, Hayashi-Takanaka Y, Goto Y, Takizawa N, Nozaki N (2008) The organization of histone H3 modifications as revealed by a panel of specific monoclonal antibodies. Cell Struct Funct 33(1):61–73

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We are grateful to Drs. Yi Zhang and Teruhiko Wakayama for their help in realizing this work. This review was supported by a Special Coordination Fund for Promoting Science and Technology and JST PRESTO program (to Y.O.), and in part by a Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to K.Y.).

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Authors and Affiliations

  1. Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan

    Kazuo Yamagata

  2. Career-Path Promotion Unit for Young Life Scientists, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan

    Yuki Okada

  3. PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho Kawaguchi, Saitama, 332-0012, Japan

    Yuki Okada

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  1. Kazuo Yamagata
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  2. Yuki Okada
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Correspondence to Yuki Okada.

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Yamagata, K., Okada, Y. Understanding paternal genome demethylation through live-cell imaging and siRNA. Cell. Mol. Life Sci. 68, 1669–1679 (2011). https://doi.org/10.1007/s00018-010-0623-0

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