Advertisement

Manipulation of Promoter-Associated Noncoding RNAs in Mouse Early Embryos for Controlling Sequence-Specific Epigenetic Status

  • Nobuhiko Hamazaki
  • Kinichi Nakashima
  • Takuya ImamuraEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1543)

Abstract

In mammals, transcription in the zygote begins after fertilization. This transcriptional wave is called zygotic gene activation (ZGA). During ZGA, epigenetic modifications, such as DNA methylation and histone modifications, are dynamically and drastically reconstructed in a sequence-specific manner. However, how such orchestrated gene upregulation is regulated remains unknown. Recently, using microinjection techniques, we have revealed that a class of long noncoding RNAs, named promoter-associated noncoding RNAs (pancRNAs), mediates specific gene upregulation through promoter DNA demethylation during ZGA. Here, we describe the experimental methods available to control the expression levels of pancRNAs and to evaluate epigenetic status after pancRNA manipulation.

Key words

Microinjection Long noncoding RNA Mouse DNA methylation 

Notes

Acknowledgments

We thank Dr. Elizabeth Nakajima for proofreading the manuscript. We thank Katsuhiko Hayashi for discussion. This work was supported by Grants-in-Aid [No. 15H04603] to T.I. from Japan Society for the Promotion of Science (JSPS). N.H. is supported by a JSPS Research Fellowship.

References

  1. 1.
    Aoki F, Worrad DM, Schultz RM (1997) Regulation of transcriptional activity during the first and second cell cycles in the preimplantation mouse embryo. Dev Biol 181:296–307CrossRefPubMedGoogle Scholar
  2. 2.
    Latham KE, Garrels JI, Chang C et al (1991) Quantitative analysis of protein synthesis in mouse embryos. I. Extensive reprogramming at the one-and two-cell stages. Development 112:921–932PubMedGoogle Scholar
  3. 3.
    Li E (2002) Chromatin modification and epigenetic reprogramming in mammalian development. Nat Rev Genet 3:662–673CrossRefPubMedGoogle Scholar
  4. 4.
    Farthing CR, Ficz G, Ng RK et al (2008) Global mapping of DNA methylation in mouse promoters reveals epigenetic reprogramming of pluripotency genes. PLoS Genet 4:e1000116CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Mayer W, Niveleau A, Walter J et al (2000) Demethylation of the zygotic paternal genome. Nature 403:501–502CrossRefPubMedGoogle Scholar
  6. 6.
    Oswald J, Engemann S, Lane N et al (2000) Active demethylation of the paternal genome in the mouse zygote. Curr Biol 10:475–478CrossRefPubMedGoogle Scholar
  7. 7.
    Borgel J, Guibert S, Li Y et al (2010) Targets and dynamics of promoter DNA methylation during early mouse development. Nat Genet 42:1093–1100CrossRefPubMedGoogle Scholar
  8. 8.
    Smallwood SA, Tomizawa S-I, Krueger F et al (2011) Dynamic CpG island methylation landscape in oocytes and preimplantation embryos. Nat Genet 43:811–814CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Smith ZD, Chan MM, Mikkelsen TS et al (2012) A unique regulatory phase of DNA methylation in the early mammalian embryo. Nature 484:339–344CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Imamura T, Yamamoto S, Ohgane J et al (2004) Non-coding RNA directed DNA demethylation of Sphk1 CpG island. Biochem Biophys Res Commun 322:593–600CrossRefPubMedGoogle Scholar
  11. 11.
    Tomikawa J, Shimokawa H, Uesaka M et al (2011) Single-stranded noncoding RNAs mediate local epigenetic alterations at gene promoters in rat cell lines. J Biol Chem 286:34788–34799CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Hamazaki N, Uesaka M, Nakashima K et al (2015) Gene activation-associated long noncoding RNAs function in mouse preimplantation development. Development 142:910–920CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Zhang Y, Rohde C, Tierling S et al (2009) DNA methylation analysis by bisulfite conversion, cloning, and sequencing of individual clones. Methods Mol Biol 507:177–187Google Scholar
  14. 14.
    Li L-C, Dahiya R (2002) MethPrimer: Designing primers for methylation PCRs. Bioinformatics (Oxford, England) 18:1427–1431CrossRefGoogle Scholar
  15. 15.
    Kline D (2009) Quantitative microinjection of mouse oocytes and eggs. Methods Mol Biol 518:135–156CrossRefPubMedGoogle Scholar
  16. 16.
    Kumaki Y, Oda M, Okano M (2008) QUMA: quantification tool for methylation analysis. Nucl Acids Res 36:W170–5Google Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Nobuhiko Hamazaki
    • 1
  • Kinichi Nakashima
    • 1
  • Takuya Imamura
    • 1
    Email author
  1. 1.Department of Stem Cell Biology and MedicineGraduate School of Medical Sciences, Kyushu UniversityFukuokaJapan

Personalised recommendations