Skip to main content
SpringerLink
Log in

Methods for Preparing Nucleosomes Containing Histone Variants

  • Protocol
  • First Online:
Histone Variants

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1832))

Abstract

Histone variants are key epigenetic players that regulate transcription, repair, replication, and recombination of genomic DNA. Histone variant incorporation into nucleosomes induces structural diversity of nucleosomes, consequently leading to the structural versatility of chromatin. Such chromatin diversity created by histone variants may play a central role in the epigenetic regulation of genes. Each histone variant possesses specific biochemical and physical characteristics, and thus the preparation methods are complicated. Here, we introduce the methods for the purification of human histone variants as recombinant proteins, and describe the preparation methods for histone complexes and nucleosomes containing various histone variants. We also describe the detailed method for the preparation of heterotypic nucleosomes, which may function in certain biological phenomena. These methods are useful for biochemical, structural, and biophysical studies.

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 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
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. Wolffe A (1998) Chromatin: structure and function, 3rd edn. Academic Press, San Diego

    Google Scholar 

  2. Luger K, Dechassa ML, Tremethick DJ et al (2012) New insights into nucleosome and chromatin structure: an ordered state or a disordered affair? Nat Rev Mol Cell Biol 13:436–447

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Venkatesh S, Workman JL (2015) Histone exchange, chromatin structure and the regulation of transcription. Nat Rev Mol Cell Biol 16:178–189

    Article  CAS  PubMed  Google Scholar 

  4. Campos EI, Stafford JM, Reinberg D (2014) Epigenetic inheritance: histone bookmarks across generations. Trends Cell Biol 24:664–674

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Luger K, Mäder AW, Richmond RK et al (1997) Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature 389:251–260

    Article  CAS  PubMed  Google Scholar 

  6. Strahl BD, Allis CD (2000) The language of covalent histone modifications. Nature 403:41–45

    Article  CAS  PubMed  Google Scholar 

  7. Kouzarides T (2007) Chromatin modifications and their function. Cell 128:693–705

    Article  CAS  PubMed  Google Scholar 

  8. Bhaumik SR, Smith E, Shilatifard A (2007) Covalent modifications of histones during development and disease pathogenesis. Nat Struct Mol Biol 14:1008–1016

    Article  CAS  PubMed  Google Scholar 

  9. Bannister AJ, Kouzarides T (2011) Regulation of chromatin by histone modifications. Cell Res 21:381–395

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Rousseaux S, Khochbin S (2015) Histone acylation beyond acetylation: terra incognita in chromatin biology. Cell J 17:1–6

    PubMed  PubMed Central  Google Scholar 

  11. Szenker E, Ray-Gallet D, Almouzni G (2011) The double face of the histone variant H3.3. Cell Res 21:421–434

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Franklin SG, Zweidler A (1977) Non-allelic variants of histones 2a, 2b and 3 in mammals. Nature 266:273–275

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  14. Palmer DK, O’Day K, Wener MH et al (1987) A 17-kD centromere protein (CENP-A) copurifies with nucleosome core particles and with histones. J Cell Biol 104:805–815

    Article  CAS  PubMed  Google Scholar 

  15. Albig W, Ebentheuer J, Klobeck G et al (1996) A solitary human H3 histone gene on chromosome 1. Hum Genet 97:486–491

    Article  CAS  PubMed  Google Scholar 

  16. Witt O, Albig W, Doenecke D (1996) Testis-specific expression of a novel human H3 histone gene. Exp Cell Res 229:301–306

    Article  CAS  PubMed  Google Scholar 

  17. Brush D, Dodgson JB, Choi OR et al (1985) Replacement variant histone genes contain intervening sequences. Mol Cell Biol 5:1307–1317

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Schenk R, Jenke A, Zilbauer M et al (2011) H3.5 is a novel hominid-specific histone H3 variant that is specifically expressed in the seminiferous tubules of human testes. Chromosoma 120:275–285

    Article  CAS  PubMed  Google Scholar 

  19. Wiedemann SM, Mildner SN, Bönisch C et al (2010) Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y. J Cell Biol 190:777–791

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Taguchi H, Xie Y, Horikoshi N et al (2017) Crystal structure and characterization of novel human histone H3 variants, H3.6, H3.7, and H3.8. Biochemistry 56:2184–2196

    Article  CAS  PubMed  Google Scholar 

  21. West MH, Bonner WM (1980) Histone 2A, a heteromorphous family of eight protein species. Biochemistry 19:3238–3245

    Article  CAS  PubMed  Google Scholar 

  22. Iouzalen N, Moreau J, Méchali M (1996) H2A.ZI, a new variant histone expressed during Xenopus early development exhibits several distinct features from the core histone H2A. Nucleic Acids Res 24:3947–3952

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Eirín-López JM, González-Romero R, Dryhurst D et al (2009) The evolutionary differentiation of two histone H2A.Z variants in chordates (H2A.Z-1 and H2A.Z-2) is mediated by a stepwise mutation process that affects three amino acid residues. BMC Evol Biol 9:31

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Chadwick BP, Willard HF (2001) A novel chromatin protein, distantly related to histone H2A, is largely excluded from the inactive X chromosome. J Cell Biol 152:375–384

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Pehrson JR, Fried VA (1992) MacroH2A, a core histone containing a large nonhistone region. Science 257:1398–1400

    Article  CAS  PubMed  Google Scholar 

  26. Dyer PN, Edayathumangalam RS, White CL et al (2004) Reconstitution of nucleosome core particles from recombinant histones and DNA. Methods Enzymol 375:23–44

    Article  CAS  PubMed  Google Scholar 

  27. Suto RK, Clarkson MJ, Tremethick DJ et al (2000) Crystal structure of a nucleosome core particle containing the variant histone H2A.Z. Nat Struct Biol 7:1121–1124

    Article  CAS  PubMed  Google Scholar 

  28. Fan JY, Gordon F, Luger K et al (2002) The essential histone variant H2A.Z regulates the equilibrium between different chromatin conformational states. Nat Struct Biol 9:172–176

    Article  CAS  PubMed  Google Scholar 

  29. Park YJ, Dyer PN, Tremethick DJ et al (2004) A new fluorescence resonance energy transfer approach demonstrates that the histone variant H2AZ stabilizes the histone octamer within the nucleosome. J Biol Chem 279:24274–24282

    Article  CAS  PubMed  Google Scholar 

  30. Horikoshi N, Sato K, Shimada K et al (2013) Structural polymorphism in the L1 loop regions of human H2A.Z.1 and H2A.Z.2. Acta Crystallogr D Biol Crystallogr 69:2431–2439

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Sugiyama M, Horikoshi N, Suzuki Y et al (2015) Solution structure of variant H2A.Z.1 nucleosome investigated by small-angle X-ray and neutron scatterings. Biochem Biophys Rep 4:28–32

    PubMed  PubMed Central  Google Scholar 

  32. Horikoshi N, Arimura Y, Taguchi H et al (2016) Crystal structures of heterotypic nucleosomes containing histones H2A.Z and H2A. Open Biol 6:160127

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Bao Y, Konesky K, Park YJ et al (2004) Nucleosomes containing the histone variant H2A.Bbd organize only 118 base pairs of DNA. EMBO J 23:3314–3324

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Gautier T, Abbott DW, Molla A et al (2004) Histone variant H2ABbd confers lower stability to the nucleosome. EMBO Rep 5:715–720

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Angelov D, Verdel A, An W et al (2004) SWI/SNF remodeling and p300-dependent transcription of histone variant H2ABbd nucleosomal arrays. EMBO J 23:3815–3824

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Sugiyama M, Arimura Y, Shirayama K et al (2014) Distinct features of the histone core structure in nucleosomes containing the histone H2A.B variant. Biophys J 106:2206–2213

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Arimura Y, Kimura H, Oda T et al (2013) Structural basis of a nucleosome containing histone H2A.B/H2A.Bbd that transiently associates with reorganized chromatin. Sci Rep 3:3510

    Article  PubMed  PubMed Central  Google Scholar 

  38. Chakravarthy S, Gundimella SK, Caron C et al (2005) Structural characterization of the histone variant macroH2A. Mol Cell Biol 25:7616–7624

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Tachiwana H, Osakabe A, Shiga T et al (2011) Structures of human nucleosomes containing major histone H3 variants. Acta Crystallogr D Biol Crystallogr 67:578–583

    Article  CAS  PubMed  Google Scholar 

  40. Tachiwana H, Kagawa W, Osakabe A et al (2010) Structural basis of instability of the nucleosome containing a testis-specific histone variant, human H3T. Proc Natl Acad Sci U S A 107:10454–10459

    Article  PubMed  PubMed Central  Google Scholar 

  41. Urahama T, Harada A, Maehara K et al (2016) Histone H3.5 forms an unstable nucleosome and accumulates around transcription start sites in human testis. Epigenetics Chromatin 9:2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kujirai T, Horikoshi N, Sato K et al (2016) Structure and function of human histone H3.Y nucleosome. Nucleic Acids Res 44:6127–6141

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Tachiwana H, Kagawa W, Shiga T et al (2011) Crystal structure of the human centromeric nucleosome containing CENP-A. Nature 476:232–235

    Article  CAS  PubMed  Google Scholar 

  44. Arimura Y, Shirayama K, Horikoshi N et al (2014) Crystal structure and stable property of the cancer-associated heterotypic nucleosome containing CENP-A and H3.3. Sci Rep 4:7115

    Article  PubMed  PubMed Central  Google Scholar 

  45. Tanaka Y, Tawaramoto-Sasanuma M, Kawaguchi S et al (2004) Expression and purification of recombinant human histones. Methods 33:3–11

    Article  CAS  PubMed  Google Scholar 

  46. Lowary PT, Widom J (1998) New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning. J Mol Biol 276:19–42

    Article  CAS  PubMed  Google Scholar 

  47. Viens A, Mechold U, Brouillard F et al (2006) Analysis of human histone H2AZ deposition in vivo argues against its direct role in epigenetic templating mechanisms. Mol Cell Biol 26:5325–5335

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Nekrasov M, Amrichova J, Parker BJ et al (2012) Histone H2A.Z inheritance during the cell cycle and its impact on promoter organization and dynamics. Nat Struct Mol Biol 19:1076–1083

    Article  CAS  PubMed  Google Scholar 

  49. Lacoste N, Woolfe A, Tachiwana H et al (2014) Mislocalization of the centromeric histone variant CenH3/CENP-A in human cells depends on the chaperone DAXX. Mol Cell 53:631–644

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Ms. Yukari Iikura (Waseda University) for her assistance. This work was supported in part by JSPS KAKENHI Grant Numbers JP25116002 and JP17H01408, by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP18am0101076, and by JST CREST Grant Number PMJCR16G1, Japan [to H.K.]. H.K. and N.H. were supported by the Waseda Research Institute for Science and Engineering. T.K. and R.F. were supported by Research Fellowships from JSPS for Young Scientists [JP15J06807 and JP16J10043, respectively]. Y.A. and S.M. were supported in part by JSPS KAKENHI Grant Numbers JP17K15043 [to Y.A.] and JP16K18473 [to S.M.].

Author information

Authors and Affiliations

  1. Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, 113-0032, Tokyo, Japan

    Tomoya Kujirai, Yasuhiro Arimura, Risa Fujita & Hitoshi Kurumizaka

  2. Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, 162-8480, Tokyo, Japan

    Tomoya Kujirai, Yasuhiro Arimura, Risa Fujita, Naoki Horikoshi, Shinichi Machida & Hitoshi Kurumizaka

Authors
  1. Tomoya Kujirai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yasuhiro Arimura
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Risa Fujita
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Naoki Horikoshi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shinichi Machida
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hitoshi Kurumizaka
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hitoshi Kurumizaka .

Editor information

Editors and Affiliations

  1. Institut Curie, PSL Research University CNRS, UMR3664, Equipe Labellisée Ligue contre le Cancer, Paris, France

    Guillermo A. Orsi

  2. Institut Curie, PSL Research University CNRS, UMR3664, Equipe Labellisée Ligue contre le Cancer, Paris, France

    Geneviève Almouzni

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Kujirai, T., Arimura, Y., Fujita, R., Horikoshi, N., Machida, S., Kurumizaka, H. (2018). Methods for Preparing Nucleosomes Containing Histone Variants. In: Orsi, G., Almouzni, G. (eds) Histone Variants. Methods in Molecular Biology, vol 1832. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8663-7_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8663-7_1

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8662-0

  • Online ISBN: 978-1-4939-8663-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation