Tracking Histone Variant Nucleosomes Across the Human Cell Cycle Using Biophysical, Biochemical, and Cytological Analyses

  • Marcin P. Walkiewicz
  • Minh Bui
  • Delphine Quénet
  • Yamini Dalal
Part of the Methods in Molecular Biology book series (MIMB, volume 1170)

Abstract

Histone variants such as H3.3, macroH2A, H2A.Z, and CENP-A are important epigenetic modifiers of the chromatin state in eukaryotic genomes. The centromeric histone H3 variant CENP-A/CENH3 epigenetically marks centromeres and is required for assembly of the kinetochore complex, a region of the chromosome that is responsible for proper genome segregation during mitosis. Several diverse techniques using biochemical, cell biology, and biophysical approaches have been utilized to study the nature of the CENP-A nucleosome across the cell cycle. In this chapter, we describe methods for CENP-A nucleosome purification and separation of CENP-A from other core histones using traditional SDS-PAGE and more resolving techniques such as Triton acid urea (TAU) and two-dimensional gels. We also discuss methods for observation of CENP-A on chromatin fibers using immunofluorescence. Finally, we provide a detailed description of analysis of chromatin structures using atomic force microscopy.

Key words

CENP-A Histones Cell cycle SDS-PAGE TAU Western blotting Chromatin fiber Immunofluorescence Atomic force microscopy AFM 

Notes

Acknowledgements

We thank Dr. Rajbir Gill for expert advice on chromatin extraction, TAU gel preparation, and FISH protocols, and Dr. Emilios Dimitriadis for helpful advice on automated AFM image analysis.

References

  1. 1.
    Thomas JO, Kornberg RD (1975) An octamer of histones in chromatin and free in solution. Proc Natl Acad Sci U S A 72(7):2626–2630PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Woodcock CL, Frado LL, Rattner JB (1984) The higher-order structure of chromatin: evidence for a helical ribbon arrangement. J Cell Biol 99(1 Pt 1):42–52PubMedCrossRefGoogle Scholar
  3. 3.
    Palmer DK, O’Day K, Wener MH, Andrews BS, Margolis RL (1987) A 17-kD centromere protein (CENP-A) copurifies with nucleosome core particles and with histones. J Cell Biol 104(4):805–815PubMedCrossRefGoogle Scholar
  4. 4.
    Earnshaw WC, Migeon BR (1985) Three related centromere proteins are absent from the inactive centromere of a stable isodicentric chromosome. Chromosoma 92(4):290–296PubMedCrossRefGoogle Scholar
  5. 5.
    Bui M, Dimitriadis EK, Hoischen C, An E, Quenet D, Giebe S, Nita-Lazar A, Diekmann S, Dalal Y (2012) Cell-cycle-dependent structural transitions in the human CENP-A nucleosome in vivo. Cell 150(2):317–326PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Shechter D, Dormann HL, Allis CD, Hake SB (2007) Extraction, purification and analysis of histones. Nat Protoc 2(6):1445–1457PubMedCrossRefGoogle Scholar
  7. 7.
    Zweidler A (1978) Resolution of histones by polyacrylamide gel electrophoresis in presence of nonionic detergents. Methods Cell Biol 17:223–233PubMedCrossRefGoogle Scholar
  8. 8.
    Waterborg JH (2002) Acid-urea-triton polyacrylamide gel electrophoresis of histones. In: Walker JM (ed) The protein protocols handbook. Springer, New York, pp 113–123CrossRefGoogle Scholar
  9. 9.
    Earley KW, Shook MS, Brower-Toland B, Hicks L, Pikaard CS (2007) In vitro specificities of Arabidopsis co-activator histone acetyltransferases: implications for histone hyperacetylation in gene activation. Plant J 52(4):615–626PubMedCrossRefGoogle Scholar
  10. 10.
    Ravi M, Shibata F, Ramahi JS, Nagaki K, Chen C, Murata M, Chan SW (2011) Meiosis-specific loading of the centromere-specific histone CENH3 in Arabidopsis thaliana. PLoS Genet 7(6):e1002121PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Blower MD, Sullivan BA, Karpen GH (2002) Conserved organization of centromeric chromatin in flies and humans. Dev Cell 2(3):319–330PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Sullivan BA (2010) Optical mapping of protein-DNA complexes on chromatin fibers. Methods Mol Biol 659:99–115PubMedCrossRefGoogle Scholar
  13. 13.
    Binnig G, Quate CF, Gerber C (1986) Atomic force microscope. Phys Rev Lett 56(9):930–933PubMedCrossRefGoogle Scholar
  14. 14.
    Bui M, Walkiewicz MP, Dimitriadis EK, Dalal Y (2013) The CENP-A nucleosome: a battle between Dr. Jekyll and Mr. Hyde. Nucleus 4(1):37–42PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Dimitriadis EK, Weber C, Gill RK, Diekmann S, Dalal Y (2010) Tetrameric organization of vertebrate centromeric nucleosomes. Proc Natl Acad Sci U S A 107(47):20317–20322PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Lyubchenko YL, Gall AA, Shlyakhtenko LS (2001) Atomic force microscopy of DNA and protein-DNA complexes using functionalized mica substrates. Methods Mol Biol 148: 569–578PubMedGoogle Scholar
  17. 17.
    Shlyakhtenko LS, Gall AA, Lyubchenko YL (2013) Mica functionalization for imaging of DNA and protein-DNA complexes with atomic force microscopy. Methods Mol Biol 931:295–312PubMedCrossRefGoogle Scholar
  18. 18.
    Shlyakhtenko LS, Gall AA, Filonov A, Cerovac Z, Lushnikov A, Lyubchenko YL (2003) Silatrane-based surface chemistry for immobilization of DNA, protein-DNA complexes and other biological materials. Ultramicroscopy 97(1–4):279–287PubMedCrossRefGoogle Scholar
  19. 19.
    Juan G, Hernando E, Cordon-Cardo C (2002) Separation of live cells in different phases of the cell cycle for gene expression analysis. Cytometry 49(4):170–175PubMedCrossRefGoogle Scholar
  20. 20.
    Pretlow TG II, Pretlow TP (1979) Centrifugal elutriation (counterstreaming centrifugation) of cells. Cell Biophys 1(2):195–210PubMedCrossRefGoogle Scholar
  21. 21.
    Quenet D, Dimitriadis EK, Dalal Y (2012) Atomic force microscopy of chromatin. In: Atomic force microscopy investigations into biology—from cell to protein Rijeka, Croatia: InTech 195–218Google Scholar
  22. 22.
    Willard HF, Waye JS (1987) Hierarchical order in chromosome-specific human alpha satellite DNA. Trends Genet 3(7):192–198CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Marcin P. Walkiewicz
    • 1
  • Minh Bui
    • 1
  • Delphine Quénet
    • 1
  • Yamini Dalal
    • 1
  1. 1.Chromatin Structure and Epigenetic Mechanisms Unit, Laboratory of Receptor Biology and Gene Expression, Center for Cancer ResearchNational Cancer Institute, NIHBethesdaUSA

Personalised recommendations