Skip to main content
SpringerLink
Log in

Preparation and Analysis of Uniquely Positioned Mononucleosomes

  • Protocol
  • First Online:
Chromatin Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 523))

Abstract

Short DNA fragments containing single, uniquely positioned nucleosome cores have been extensively employed as simple model experimental systems for analysis of many intranuclear processes, including binding of proteins to nucleosomes, transcription, DNA repair and ATP-dependent chromatin remodeling. In many cases such simple model templates faithfully recapitulate numerous important aspects of these processes. Here we describe several recently developed procedures for obtaining and analysis of mononucleosomes that are uniquely positioned on 150–600 bp DNA fragments.

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 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
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

Notes

  1. 1.

    Gaykalova and Kulaeva equally contributed to this work.

References

  1. Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., and Richmond, T. J. (1997) Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389, 251–260.

    Article  PubMed  CAS  Google Scholar 

  2. Utley, R. T., Owen-Hughes, T. A., Juan, L. J., Cote, J., Adams, C. C., and Workman, J. L. (1996) In vitro analysis of transcription factor binding to nucleosomes and nucleosome disruption/displacement. Meth. Enzymol. 274, 276–291.

    Article  PubMed  CAS  Google Scholar 

  3. Cirillo, L. A., and Zaret, K. S. (2004) Preparation of defined mononucleosomes, dinucleosomes, and nucleosome arrays in vitro and analysis of transcription factor binding. Meth. Enzymol. 375, 131–158.

    Article  PubMed  CAS  Google Scholar 

  4. Studitsky, V. M., Clark, D. J., and Felsenfeld, G. (1996) Preparation of nucleosomal templates for transcription in vitro.Meth. Enzymol. 274, 246–256.

    Article  PubMed  CAS  Google Scholar 

  5. Walter, W., and Studitsky, V. M. (2004) Construction, analysis, and transcription of model nucleosomal templates. Methods 33, 18–24.

    Article  PubMed  CAS  Google Scholar 

  6. Walter, W., Kashlev, M., and Studitsky, V. M. (2004) Transcription through the nucleosome by mRNA-producing RNA polymerases. Meth. Enzymol. 377, 445–460.

    Article  PubMed  CAS  Google Scholar 

  7. Walter, W., Kireeva, M. L., Tchernajenko, V., Kashlev, M., and Studitsky, V. M. (2003) Assay of the fate of the nucleosome during transcription by RNA polymerase II. Meth. Enzymol. 371, 564–577.

    Article  PubMed  CAS  Google Scholar 

  8. Beard, B. C., and Smerdon, M. J. (2004) Analysis of DNA repair on nucleosome templates. Meth. Enzymol. 377, 499–507.

    Article  PubMed  CAS  Google Scholar 

  9. Wittmeyer, J., Saha, A., and Cairns, B. (2004) DNA translocation and nucleosome remodeling assays by the RSC chromatin remodeling complex. Meth. Enzymol. 377, 322–343.

    Article  PubMed  CAS  Google Scholar 

  10. Lorch, Y., and Kornberg, R. D. (2004) Isolation and assay of the RSC chromatin-remodeling complex from Saccharomyces cerevisiae.Meth. Enzymol. 377, 316–322.

    Article  PubMed  CAS  Google Scholar 

  11. Eberharter, A., Langst, G., and Becker, P. B. (2004) A nucleosome sliding assay for chromatin remodeling factors. Meth. Enzymol. 377, 344–353.

    Article  PubMed  CAS  Google Scholar 

  12. Kassabov, S. R., and Bartholomew, B. (2004) Site-directed histone-DNA contact mapping for analysis of nucleosome dynamics.Meth. Enzymol. 375, 193–210.

    Article  PubMed  CAS  Google Scholar 

  13. Mizuguchi, G., Shen, X., Landry, J., Wu, W. H., Sen, S., and Wu, C. (2004) ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex. Science 303, 343–348. .

    Article  PubMed  CAS  Google Scholar 

  14. Dyer, P. N., Edayathumangalam, R. S., White, C. L., Bao, Y., Chakravarthy, S., Muthurajan, U. M., and Luger, K. (2004) Reconstitution of nucleosome core particles from recombinant histones and DNA. Meth. Enzymol. 375, 23–44.

    Article  PubMed  CAS  Google Scholar 

  15. Luger, K., Rechsteiner, T. J., and Richmond, T. J. (1999) Preparation of nucleosome core particle from recombinant histones. Meth. Enzymol. 304, 3–19.

    Article  PubMed  CAS  Google Scholar 

  16. Hanson, B. L., Alexander, C., Harp, J. M., and Bunick, G. J. (2004) Preparation and crystallization of nucleosome core particle. Meth. Enzymol. 375, 44–62.

    Article  PubMed  CAS  Google Scholar 

  17. Kireeva, M. L., Walter, W., Tchernajenko, V., Bondarenko, V., Kashlev, M., and Studitsky, V. M. (2002) Nucleosome remodeling induced by RNA polymerase II. Loss of the H2A/H2B dimer during transcription. Mol. Cell 9, 541–552.

    Article  PubMed  CAS  Google Scholar 

  18. Pennings, S., Meersseman, G., and Bradbury, E. M. (1991) Mobility of positioned nucleosomes on 5 S rDNA. J. Mol. Biol. 220, 101–110.

    Article  PubMed  CAS  Google Scholar 

  19. Meersseman, G., Pennings, S., and Bradbury, E. M. (1992) Mobile nucleosomes – a general behavior. EMBO J. 11, 2951–2959.

    PubMed  CAS  Google Scholar 

  20. Studitsky, V. M., Clark, D. J., and Felsenfeld, G. (1994). Mechanism of nucleosome displacement by a transcribing polymerase. In Structural Biology: the state of art (Adenine Press), pp. 125–131.

    Google Scholar 

  21. Widom, J. (2001) Role of DNA sequence in nucleosome stability and dynamics. Q. Rev. Biophys. 34, 269–324.

    Article  PubMed  CAS  Google Scholar 

  22. Bondarenko, V. A., Steele, L. M., Ujvari, A., Gaykalova, D., Kulaeva, O. I., Polykanov, Y. S., Luse, D. S., and Studitsky, V. M. (2006) Nucleosomes Can Form a Polar Barrier to Transcript Elongation by RNA Polymerase II. Molecular Cell, 24, 469–479.

    Google Scholar 

  23. Studitsky, V. M. (1999) Preparation and analysis of positioned nucleosomes. Meth. Mol. Biol. 119, 17–26.

    CAS  Google Scholar 

  24. Studitsky, V. M., Clark, D. J., and Felsenfeld, G. (1995) Overcoming a nucleosomal barrier to transcription. Cell 83, 19–27.

    Article  PubMed  CAS  Google Scholar 

  25. Duband-Goulet, I., Carot, V., Ulyanov, A. V., Douc-Rasy, S., and Prunell, A. (1992) Chromatin reconstitution on small DNA rings. IV. DNA supercoiling and nucleosome sequence preference. J. Mol. Biol. 224, 981–1001.

    Article  PubMed  CAS  Google Scholar 

  26. Lowary, P. T., and 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  PubMed  CAS  Google Scholar 

  27. Thastrom, A., Lowary, P. T., Widlund, H. R., Cao, H., Kubista, M., and Widom, J. (1999) Sequence motifs and free energies of selected natural and non-natural nucleosome positioning DNA sequences. J. Mol. Biol. 288, 213–229.

    Article  PubMed  CAS  Google Scholar 

  28. Wang, J. P., and Widom, J. (2005) Improved alignment of nucleosome DNA sequences using a mixture model. Nucleic Acids Res. 33, 6743–6755.

    Article  PubMed  CAS  Google Scholar 

  29. Thastrom, A., Bingham, L. M., and Widom, J. (2004) Nucleosomal locations of dominant DNA sequence motifs for histone–DNA interactions and nucleosome positioning. J. Mol. Biol. 338, 695–709.

    Article  PubMed  CAS  Google Scholar 

  30. Dorigo, B., Schalch, T., Kulangara, A., 1Duda, S., Schroeder, R. R., and Richmond, T. J. (2004) Nucleosome arrays reveal the two-start organization of the chromatin fiber. Science 306, 1571–1573.

    Article  PubMed  CAS  Google Scholar 

  31. Rhodes, D., and Laskey, R. A. (1989) Assembly of nucleosomes and chromatin in vitro. Meth. Enzymol. 170, 575–585.

    Article  PubMed  CAS  Google Scholar 

  32. Simon, R. H., and Felsenfeld, G. (1979) A new procedure for purifying histone pairs H2A + H2B and H3 + H4 from chromatin using hydroxylapatite. Nucleic Acids Res. 6, 689–696.

    Article  PubMed  CAS  Google Scholar 

  33. Owen-Hughes, T., Utley, R. T., Steger, D. J., West, J. M., John, S., Cote, J., Havas, K. M., and Workman, J. L. (1999) Analysis of nucleosome disruption by ATP-driven chromatin remodeling complexes. Meth. Mol. Biol. 119, 319–331.

    CAS  Google Scholar 

  34. Bondarenko, V. A., Steele, L. M., Ujvari, A., Gaykalova, D. A., Kulaeva, O. I., Polikanov, Y. S., Luse, D. S., and Studitsky, V. M. (2006) Nucleosomes can form a polar barrier to transcript elongation by RNA polymerase II. Mol. Cell 24, 469–479.

    Article  PubMed  CAS  Google Scholar 

  35. Schickor, P., and Heumann, H. (1994) Hydroxyl radical footprinting. Meth. Mol. Biol. 30, 21–32.

    CAS  Google Scholar 

  36. Studitsky, V. M., Clark, D. J., and Felsenfeld, G. (1994) A histone octamer can step around a transcribing polymerase without leaving the template. Cell 76, 371–382.

    Article  PubMed  CAS  Google Scholar 

  37. Morse, R. H. (1989) Nucleosomes inhibit both transcriptional initiation and elongation by RNA polymerase III in vitro. EMBO J. 8, 2343–2351.

    PubMed  CAS  Google Scholar 

  38. von Holt, C., Brandt, W. F., Greyling, H. J., Lindsey, G. G., Retief, J. D., Rodrigues, J. D., Schwager, S., and Sewell, B. T. (1989) Isolation and characterization of histones. Meth. Enzymol. 170, 431–523.

    Article  Google Scholar 

  39. Ausio, J., Dong, F., and van Holde, K. E. (1989) Use of selectively trypsinized nucleosome core particles to analyze the role of the histone "tails" in the stabilization of the nucleosome. J. Mol. Biol. 206, 451–463.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank John Widom for plasmids containing the nucleosome-positioning sequences. This work was supported by NIH grant GM58650 to V.M.S.

Author information

Authors and Affiliations

  1. Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ, USA

    Daria A. Gaykalova, Olga I. Kulaeva, Vladimir A. Bondarenko & Vasily M. Studitsky

Authors
  1. Daria A. Gaykalova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Olga I. Kulaeva
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vladimir A. Bondarenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Vasily M. Studitsky
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Research Institute, H. Lee Moffitt Cancer Center &, Magnolia Drive 12902, Tampa, 33612, U.S.A.

    Srikumar P. Chellappan

Rights and permissions

Reprints and permissions

Copyright information

© 2009 Humana Press, a part of Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Gaykalova, D.A., Kulaeva, O.I., Bondarenko, V.A., Studitsky, V.M. (2009). Preparation and Analysis of Uniquely Positioned Mononucleosomes. In: Chellappan, S. (eds) Chromatin Protocols. Methods in Molecular Biology, vol 523. Humana Press. https://doi.org/10.1007/978-1-59745-190-1_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-190-1_8

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-873-7

  • Online ISBN: 978-1-59745-190-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation