Skip to main content
SpringerLink
Log in

Targeted DNA Methylation in Mouse Early Embryos

  • Protocol
  • First Online:
Epigenomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2577))

  • 1814 Accesses

Abstract

In germ cell lines, including early preimplantation embryos, centromeres and pericentromeres are known to show a marked hypomethylation pattern compared to somatic cells. Elucidation of the biological function of this region-specific DNA hypomethylation state, region-specific epigenomic manipulation is essential as an analytical method. We have applied genome editing to show that region-specific DNA methylation can be effectively introduced by a fusion protein, TALE, which recognizes pericentromeres, and SssI, a bacterial CpG methyltransferase. This makes it possible to increase the DNA methylation state of the pericentromeres, which is normally about 20%, to about 60–75%, enabling comparative analysis of the developmental processes of normal embryos with hypomethylated pericentromeres and embryos that have been epigenetically edited to be hypermethylated. In this chapter, we describe a method for introducing DNA methylation into pericentromeres of early mouse embryos by expressing TALE-SssI fusion protein and a method for detecting DNA methylation.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Smith ZD, Meissner A (2013) DNA methylation: roles in mammalian development. Nat Rev Genet 14(3):204–220. https://doi.org/10.1038/nrg3354

    Article  CAS  PubMed  Google Scholar 

  2. Bergman Y, Cedar H (2013) DNA methylation dynamics in health and disease. Nat Struct Mol Biol 20(3):274–281. https://doi.org/10.1038/nsmb.2518

    Article  CAS  PubMed  Google Scholar 

  3. Lister R, Pelizzola M, Dowen RH, Hawkins RD, Hon G, Tonti-Filippini J, Nery JR, Lee L, Ye Z, Ngo QM, Edsall L, Antosiewicz-Bourget J, Stewart R, Ruotti V, Millar AH, Thomson JA, Ren B, Ecker JR (2009) Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462(7271):315–322. https://doi.org/10.1038/nature08514

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Wilson AS, Power BE, Molloy PL (2007) DNA hypomethylation and human diseases. Biochim Biophys Acta 1775(1):138–162. https://doi.org/10.1016/j.bbcan.2006.08.007

    Article  CAS  PubMed  Google Scholar 

  5. Ehrlich M (2009) DNA hypomethylation in cancer cells. Epigenomics 1(2):239–259. https://doi.org/10.2217/epi.09.33

    Article  CAS  PubMed  Google Scholar 

  6. 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. https://doi.org/10.1016/j.ydbio.2007.09.041

    Article  CAS  PubMed  Google Scholar 

  7. Ueda J, Maehara K, Mashiko D, Ichinose T, Yao T, Hori M, Sato Y, Kimura H, Ohkawa Y, Yamagata K (2014) Heterochromatin dynamics during the differentiation process revealed by the DNA methylation reporter mouse, MethylRO. Stem Cell Reports 2(6):910–924. https://doi.org/10.1016/j.stemcr.2014.05.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Yamazaki T, Hatano Y, Taniguchi R, Kobayashi N, Yamagata K (2020) Editing DNA methylation in mammalian embryos. Int J Mol Sci 21(2):doi:10.3390/ijms21020637

    Article  Google Scholar 

  9. Nakamura M, Gao Y, Dominguez AA, Qi LS (2021) CRISPR technologies for precise epigenome editing. Nat Cell Biol 23(1):11–22. https://doi.org/10.1038/s41556-020-00620-7

    Article  CAS  PubMed  Google Scholar 

  10. Yamazaki T, Hatano Y, Handa T, Kato S, Hoida K, Yamamura R, Fukuyama T, Uematsu T, Kobayashi N, Kimura H, Yamagata K (2017) Targeted DNA methylation in pericentromeres with genome editing-based artificial DNA methyltransferase. PLoS One 12(5):e0177764. https://doi.org/10.1371/journal.pone.0177764

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Wei Y, Lang J, Zhang Q, Yang CR, Zhao ZA, Zhang Y, Du Y, Sun Y (2019) DNA methylation analysis and editing in single mammalian oocytes. Proc Natl Acad Sci U S A 116(20):9883–9892. https://doi.org/10.1073/pnas.1817703116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Horii T, Morita S, Hino S, Kimura M, Hino Y, Kogo H, Nakao M, Hatada I (2020) Successful generation of epigenetic disease model mice by targeted demethylation of the epigenome. Genome Biol 21(1):77. https://doi.org/10.1186/s13059-020-01991-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Martens JH, O’Sullivan RJ, Braunschweig U, Opravil S, Radolf M, Steinlein P, Jenuwein T (2005) The profile of repeat-associated histone lysine methylation states in the mouse epigenome. EMBO J 24(4):800–812. https://doi.org/10.1038/sj.emboj.7600545

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Miyanari Y, Ziegler-Birling C, Torres-Padilla ME (2013) Live visualization of chromatin dynamics with fluorescent TALEs. Nat Struct Mol Biol 20(11):1321–1324. https://doi.org/10.1038/nsmb.2680

    Article  CAS  PubMed  Google Scholar 

  15. Kanda T, Sullivan KF, Wahl GM (1998) Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells. Curr Biol 8(7):377–385. https://doi.org/10.1016/s0960-9822(98)70156-3

    Article  CAS  PubMed  Google Scholar 

  16. Toyoda Y, Yokoyama M, Hosi T (1971) Studies on the fertilization of mouse eggs in vitro I. in vitro fertilization of eggs by fresh epididymal sperm. Jpn J Anim Reprod 16(4):147–151. https://doi.org/10.1262/jrd1955.16.147

    Article  Google Scholar 

  17. Biggers JD (2002) Thoughts on embryo culture conditions. Reprod Biomed Online 4:30–38. https://doi.org/10.1016/s1472-6483(12)60009-1

    Article  CAS  PubMed  Google Scholar 

  18. 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. https://doi.org/10.1002/gene.20158

    Article  CAS  PubMed  Google Scholar 

  19. Kumaki Y, Oda M, Okano M (2008) QUMA: quantification tool for methylation analysis. Nucleic Acids Res 36(Web Server issue):W170–W175. https://doi.org/10.1093/nar/gkn294

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was supported by JSPS KAKENHI Grant Numbers JP19K05956, JP22K05562 (TY) and JP18H02357, JP18H05528 (KY). This work was also supported by a Grant-in-Aid for Research from the Kitasato University Medical Center and Kawano Masanori Memorial Public Interest Incorporated Foundation for Promotion of Pediatrics (TY).

Author information

Authors and Affiliations

  1. Division of Biomedical Research, Kitasato University Medical Center, Kitasato University, Kitamoto, Saitama, Japan

    Taiga Yamazaki & Noritada Kobayashi

  2. Faculty of Biology-Oriented Science and Technology, Kindai University, Kinokawa, Wakayama, Japan

    Yu Hatano & Kazuo Yamagata

Authors
  1. Taiga Yamazaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yu Hatano
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Noritada Kobayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kazuo Yamagata
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Taiga Yamazaki or Kazuo Yamagata .

Editor information

Editors and Affiliations

  1. Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, Japan

    Izuho Hatada

  2. Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, Japan

    Takuro Horii

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Yamazaki, T., Hatano, Y., Kobayashi, N., Yamagata, K. (2023). Targeted DNA Methylation in Mouse Early Embryos. In: Hatada, I., Horii, T. (eds) Epigenomics. Methods in Molecular Biology, vol 2577. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2724-2_17

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2724-2_17

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2723-5

  • Online ISBN: 978-1-0716-2724-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation