Advertisement

Visualizing the Dynamics of Inactive X Chromosomes in Living Cells Using Antibody-Based Fluorescent Probes

  • Yuko Sato
  • Timothy J. Stasevich
  • Hiroshi Kimura
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1861)

Abstract

The inactive X chromosome (Xi) harbors characteristic epigenetic features, including the enrichment of histone H3 lysine 27 trimethylation (H3K27me3) and H4 lysine 20 monomethylation (H4K20me1) as well as a lack of histone acetylation. Recently, these modifications have been visualized not only in fixed specimen, but also in living cells via probes derived from modification-specific antibodies. The probes include fluorescently labeled antigen binding fragments (Fabs), which can be loaded into cells, as well as genetically encoded single-chain variable fragments tagged with the green fluorescent protein. We refer to the latter as modification specific intracellular antibodies, or “mintbodies” for short. By using Fabs or mintbodies to target Xi-specific modifications, the dynamics of Xi in living cells can be visualized.

Key words

Histone modifications Live-cell imaging Mintbody Monoclonal antibody 

Notes

Acknowledgment

This work was supported by JSPS KAKENHI Grants JP15K07157 (to Y.S.) and JP25116005, JP26291071, and JP17H01417 (to H.K.).

References

  1. 1.
    Ng K, Daigle N, Bancaud A, Ohhata T, Humphreys P, Walker R, Ellenberg J, Wutz A (2011) A system for imaging the regulatory noncoding Xist RNA in living mouse embryonic stem cells. Mol Biol Cell 22:2634–2645CrossRefPubMedCentralGoogle Scholar
  2. 2.
    Perche PY, Vourc'h C, Konecny L, Souchier C, Robert-Nicoud M, Dimitrov S, Khochbin S (2000) Higher concentrations of histone macroH2A in the Barr body are correlated with higher nucleosome density. Curr Biol 10:1531–1534CrossRefPubMedCentralGoogle Scholar
  3. 3.
    Soma A, Sato K, Nakanishi T (2013) Visualization of inactive X chromosome in preimplantation embryos utilizing MacroH2A-EGFP transgenic mouse. Genesis 51:259–267CrossRefPubMedCentralGoogle Scholar
  4. 4.
    Kimura H, Hayashi-Takanaka Y, Stasevich TJ, Sato Y (2015) Visualizing posttranslational and epigenetic modifications of endogenous proteins in vivo. Histochem Cell Biol 144:101–109CrossRefPubMedCentralGoogle Scholar
  5. 5.
    Hayashi-Takanaka Y, Yamagata K, Wakayama T, Stasevich TJ, Kainuma T, Tsurimoto T, Tachibana M, Shinkai Y, Kurumizaka H, Nozaki N, Kimura H (2011) Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling. Nucleic Acids Res 39:6475–6488CrossRefPubMedCentralGoogle Scholar
  6. 6.
    Sato Y, Mukai M, Ueda J, Muraki M, Stasevich TJ, Horikoshi N, Kujirai T, Kita H, Kimura T, Hira S, Okada Y, Hayashi-Takanaka Y, Obuse C, Kurumizaka H, Kawahara A, Yamagata K, Nozaki N, Kimura H (2013) Genetically encoded system to track histone modification in vivo. Sci Rep 3:2436CrossRefPubMedCentralGoogle Scholar
  7. 7.
    Sato Y, Kujirai T, Arai R, Asakawa H, Ohtsuki C, Horikoshi N, Yamagata K, Ueda J, Nagase T, Haraguchi T, Hiraoka Y, Kimura A, Kurumizaka H, Kimura H (2016) A genetically encoded probe for live-cell imaging of H4K20 Monomethylation. J Mol Biol 428:3885–3902CrossRefPubMedCentralGoogle Scholar
  8. 8.
    Kimura H, Yamagata K (2014) Visualization of epigenetic modifications in Preimplantation embryos. Nuclear Reprogramming 348:229–236Google Scholar
  9. 9.
    Zhou H, Fisher RJ, Papas TS (1994) Optimization of primer sequences for mouse scFv repertoire display library construction. Nucleic Acids Res 22:888–889CrossRefPubMedCentralGoogle Scholar
  10. 10.
    Hayashi-Takanaka Y, Stasevich TJ, Kurumizaka H, Nozaki N, Kimura H (2014) Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging. PLoS One 9:e106271CrossRefPubMedCentralGoogle Scholar
  11. 11.
    Kuniyoshi Y, Maehara K, Iwasaki T, Hayashi M, Semba Y, Fujita M, Sato Y, Kimura H, Harada A, Ohkawa Y (2016) Identification of immunoglobulin gene sequences from a small read number of mRNA-Seq using Hybridomas. PLoS One 11:e0165473CrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Yuko Sato
    • 1
  • Timothy J. Stasevich
    • 2
    • 3
  • Hiroshi Kimura
    • 1
    • 2
  1. 1.Cell Biology Center, Institute of Innovative ResearchTokyo Institute of TechnologyYokohamaJapan
  2. 2.World Research Hub Initiative, Institute of Innovative ResearchTokyo Institute of TechnologyYokohamaJapan
  3. 3.Department of Biochemistry and Molecular BiologyColorado State UniversityFort CollinsUSA

Personalised recommendations