Advertisement

Single Cell Restriction Enzyme-Based Analysis of Methylation at Genomic Imprinted Regions in Preimplantation Mouse Embryos

  • Ka Yi Ling
  • Lih Feng Cheow
  • Stephen R. Quake
  • William F. Burkholder
  • Daniel M. Messerschmidt
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1605)

Abstract

The methylation of cytosines in DNA is a fundamental epigenetic regulatory mechanism. During preimplantation development, mammalian embryos undergo extensive epigenetic reprogramming, including the global erasure of germ cell-specific DNA methylation marks, to allow for the establishment of the pluripotent state of the epiblast. However, DNA methylation marks at specific regions, such as imprinted gene regions, escape this reprogramming process, as their inheritance from germline to soma is paramount for proper development. To study the dynamics of DNA methylation marks in single blastomeres of mouse preimplantation embryos, we devised a new approach—single cell restriction enzyme analysis of methylation (SCRAM). SCRAM allows for reliable, fast, and high-throughput analysis of DNA methylation states of multiple regions of interest from single cells. In the method described below, SCRAM is specifically used to address loss of DNA methylation at genomic imprints or other highly methylated regions of interest.

Key words

Epigenetics DNA methylation Single cell Oocyte Blastomere SCRAM Imprinted genes MSRE 

References

  1. 1.
    Messerschmidt DM, Knowles BB, Solter D (2014) DNA methylation dynamics during epigenetic reprogramming in the germline and preimplantation embryos. Genes Dev 28(8):812–828CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Wu TP et al (2016) DNA methylation on N6-adenine in mammalian embryonic stem cells. Nature 532(7599):329–333CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Li E, Bestor TH, Jaenisch R (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69(6):915–926CrossRefPubMedGoogle Scholar
  4. 4.
    Okano M, Xie S, Li E (1998) Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nat Genet 19(3):219–220CrossRefPubMedGoogle Scholar
  5. 5.
    Okano M et al (1999) DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99(3):247–257CrossRefPubMedGoogle Scholar
  6. 6.
    Bourc'his D, Bestor TH (2004) Meiotic catastrophe and retrotransposon reactivation in male germ cells lacking Dnmt3L. Nature 431(7004):96–99CrossRefPubMedGoogle Scholar
  7. 7.
    Bourc'his D et al (2001) Dnmt3L and the establishment of maternal genomic imprints. Science 294(5551):2536–2539CrossRefPubMedGoogle Scholar
  8. 8.
    Hirasawa R et al (2008) Maternal and zygotic Dnmt1 are necessary and sufficient for the maintenance of DNA methylation imprints during preimplantation development. Genes Dev 22(12):1607–1616CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Kaneda M et al (2004) Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting. Nature 429(6994):900–903CrossRefPubMedGoogle Scholar
  10. 10.
    Gjerset RA, Martin D (1982) Presence of a DNA demethylating activity in the nucleus of murine erythroleukemic cells. J Biol Chem 257(15):8581–8583PubMedGoogle Scholar
  11. 11.
    Kurimoto K et al (2008) Complex genome-wide transcription dynamics orchestrated by Blimp1 for the specification of the germ cell lineage in mice. Genes Dev 22(12):1617–1635CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Kagiwada S et al (2013) Replication-coupled passive DNA demethylation for the erasure of genome imprints in mice. EMBO J 32(3):340–353CrossRefPubMedGoogle Scholar
  13. 13.
    He Y-F et al (2011) Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333(6047):1303–1307CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Inoue A et al (2011) Generation and replication-dependent dilution of 5fC and 5caC during mouse preimplantation development. Cell Res 21(12):1670–1676CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Ito S et al (2011) Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333(6047):1300–1303CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Okamoto Y et al (2016) DNA methylation dynamics in mouse preimplantation embryos revealed by mass spectrometry. Sci Rep 6:19134CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Popp C et al (2010) Genome-wide erasure of DNA methylation in mouse primordial germ cells is affected by AID deficiency. Nature 463(7284):1101–1105CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Santos F et al (2002) Dynamic reprogramming of DNA methylation in the early mouse embryo. Dev Biol 241(1):172–182CrossRefPubMedGoogle Scholar
  19. 19.
    Rougier N et al (1998) Chromosome methylation patterns during mammalian preimplantation development. Genes Dev 12(14):2108–2113CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Mayer W et al (2000) Embryogenesis: demethylation of the zygotic paternal genome. Nature 403(6769):501–502CrossRefPubMedGoogle Scholar
  21. 21.
    Oswald J et al (2000) Active demethylation of the paternal genome in the mouse zygote. Curr Biol 10(8):475–478CrossRefPubMedGoogle Scholar
  22. 22.
    Howell CY et al (2001) Genomic imprinting disrupted by a maternal effect mutation in the Dnmt1 gene. Cell 104(6):829–838CrossRefPubMedGoogle Scholar
  23. 23.
    Ratnam S et al (2002) Dynamics of Dnmt1 methyltransferase expression and intracellular localization during oogenesis and preimplantation development. Dev Biol 245(2):304–314CrossRefPubMedGoogle Scholar
  24. 24.
    Peaston AE et al (2004) Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos. Dev Cell 7(4):597–606CrossRefPubMedGoogle Scholar
  25. 25.
    Macfarlan TS et al (2012) Embryonic stem cell potency fluctuates with endogenous retrovirus activity. Nature 487(7405):57–63PubMedPubMedCentralGoogle Scholar
  26. 26.
    Messerschmidt DM et al (2012) Trim28 is required for epigenetic stability during mouse oocyte to embryo transition. Science 335(6075):1499–1502CrossRefPubMedGoogle Scholar
  27. 27.
    Quenneville S et al (2012) The KRAB-ZFP/KAP1 system contributes to the early embryonic establishment of site-specific DNA methylation patterns maintained during development. Cell Rep 2(4):766–773CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Ferguson-Smith AC (2011) Genomic imprinting: the emergence of an epigenetic paradigm. Nat Rev Genet 12(8):565–575CrossRefPubMedGoogle Scholar
  29. 29.
    Tomizawa S-i, Sasaki H (2012) Genomic imprinting and its relevance to congenital disease, infertility, molar pregnancy and induced pluripotent stem cell. J Hum Genet 57(2):84–91CrossRefPubMedGoogle Scholar
  30. 30.
    Lee JT, Bartolomei MS (2013) X-inactivation, imprinting, and long noncoding RNAs in health and disease. Cell 152(6):1308–1323CrossRefPubMedGoogle Scholar
  31. 31.
    Bird A (2002) DNA methylation patterns and epigenetic memory. Genes Dev 16(1):6–21CrossRefPubMedGoogle Scholar
  32. 32.
    Colella S et al (2003) Sensitive and quantitative universal pyrosequencing™ methylation analysis of CpG sites. BioTechniques 35(1):146–151PubMedGoogle Scholar
  33. 33.
    Guo H et al (2013) Single-cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing. Genome Res 23(12):2126–2135CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Bibikova M et al (2006) Human embryonic stem cells have a unique epigenetic signature. Genome Res 16(9):1075–1083CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    El Hajj N et al (2011) Limiting dilution bisulfite (pyro) sequencing reveals parent-specific methylation patterns in single early mouse embryos and bovine oocytes. Epigenetics 6(10):1176–1188CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Smallwood SA et al (2014) Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nat Methods 11(8):817–820CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Farlik M et al (2015) Single-cell DNA methylome sequencing and bioinformatic inference of epigenomic cell-state dynamics. Cell Rep 10(8):1386–1397CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Eads CA et al (2000) MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res 28(8):E32CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Ehrich M et al (2005) Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry. Proc Natl Acad Sci U S A 102(44):15785–15790CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Down TA et al (2008) A Bayesian deconvolution strategy for immunoprecipitation-based DNA methylome analysis. Nat Biotechnol 26(7):779–785CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Weber M et al (2005) Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet 37(8):853–862CrossRefPubMedGoogle Scholar
  42. 42.
    Melnikov AA et al (2005) MSRE-PCR for analysis of gene-specific DNA methylation. Nucleic Acids Res 33(10):e93CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Kantlehner M et al (2011) A high-throughput DNA methylation analysis of a single cell. Nucleic Acids Res 39(7):e44CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Lorthongpanich C et al (2013) Single-cell DNA-methylation analysis reveals epigenetic chimerism in preimplantation embryos. Science 341(6150):1110–1112CrossRefPubMedGoogle Scholar
  45. 45.
    Cheow LF et al (2015) Multiplexed locus-specific analysis of DNA methylation in single cells. Nat Protoc 10(4):619–631CrossRefPubMedGoogle Scholar
  46. 46.
    Smith ZD et al (2012) A unique regulatory phase of DNA methylation in the early mammalian embryo. Nature 484(7394):339–344CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Smallwood SA et al (2011) Dynamic CpG island methylation landscape in oocytes and preimplantation embryos. Nat Genet 43(8):811–814CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Olek A, Oswald J, Walter J (1996) A modified and improved method for bisulphite based cytosine methylation analysis. Nucleic Acids Res 24(24):5064–5066CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Barrera V, Peinado MA (2012) Evaluation of single CpG sites as proxies of CpG island methylation states at the genome scale. Nucleic Acids Res 40(22):11490–11498CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Cheow LF et al (2016) Single-cell multimodal profiling reveals cellular epigenetic heterogeneity. Nat Methods 13(10):833–836Google Scholar
  51. 51.
    Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292(5819):154–156Google Scholar
  52. 52.
    Handyside A, Barton S (1977) Evaluation of the technique of immunosurgery for the isolation of inner cell masses from mouse blastocysts. J Embryol Exp Morphol 37(1):217–226Google Scholar
  53. 53.
    Shapiro HM (2005) Practical flow cytometry. John Wiley & Sons, Hoboken, NJGoogle Scholar
  54. 54.
    Suarez-Quian C et al (1999) Laser capture microdissection of single cells from complex tissues. BioTechniques 26(2):328–335PubMedGoogle Scholar
  55. 55.
    Suarez-Quian C et al (1999) Laser capture microdissection of single cells from complex tissues. BioTechniques 26(2):328–335Google Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Ka Yi Ling
    • 1
  • Lih Feng Cheow
    • 2
  • Stephen R. Quake
    • 3
    • 4
  • William F. Burkholder
    • 2
  • Daniel M. Messerschmidt
    • 1
  1. 1.Developmental Epigenetics and Disease Laboratory, Institute of Molecular and Cell Biology, Agency for ScienceTechnology and Research (A*STAR)SingaporeSingapore
  2. 2.Microfluidics Systems Biology Laboratory, Institute of Molecular and Cell Biology, Agency for ScienceTechnology and Research (A*STAR)SingaporeSingapore
  3. 3.Department of Bioengineering and Applied PhysicsStanford UniversityStanfordUSA
  4. 4.Howard Hughes Medical InstituteStanfordUSA

Personalised recommendations