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Preparation and Analysis of Uniquely Positioned Mononucleosomes

  • Daria A. Gaykalova
  • Olga I. Kulaeva
  • Vladimir A. Bondarenko
  • Vasily M. Studitsky
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Positioned nucleosome cores chromatin reconstitution 

Notes

Acknowledgments

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

References

  1. 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.PubMedCrossRefGoogle Scholar
  2. 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.PubMedCrossRefGoogle Scholar
  3. 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.PubMedCrossRefGoogle Scholar
  4. 4.
    Studitsky, V. M., Clark, D. J., and Felsenfeld, G. (1996) Preparation of nucleosomal templates for transcription in vitro.Meth. Enzymol. 274, 246–256.PubMedCrossRefGoogle Scholar
  5. 5.
    Walter, W., and Studitsky, V. M. (2004) Construction, analysis, and transcription of model nucleosomal templates. Methods 33, 18–24.PubMedCrossRefGoogle Scholar
  6. 6.
    Walter, W., Kashlev, M., and Studitsky, V. M. (2004) Transcription through the nucleosome by mRNA-producing RNA polymerases. Meth. Enzymol. 377, 445–460.PubMedCrossRefGoogle Scholar
  7. 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.PubMedCrossRefGoogle Scholar
  8. 8.
    Beard, B. C., and Smerdon, M. J. (2004) Analysis of DNA repair on nucleosome templates. Meth. Enzymol. 377, 499–507.PubMedCrossRefGoogle Scholar
  9. 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.PubMedCrossRefGoogle Scholar
  10. 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.PubMedCrossRefGoogle Scholar
  11. 11.
    Eberharter, A., Langst, G., and Becker, P. B. (2004) A nucleosome sliding assay for chromatin remodeling factors. Meth. Enzymol. 377, 344–353.PubMedCrossRefGoogle Scholar
  12. 12.
    Kassabov, S. R., and Bartholomew, B. (2004) Site-directed histone-DNA contact mapping for analysis of nucleosome dynamics.Meth. Enzymol. 375, 193–210.PubMedCrossRefGoogle Scholar
  13. 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. .PubMedCrossRefGoogle Scholar
  14. 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.PubMedCrossRefGoogle Scholar
  15. 15.
    Luger, K., Rechsteiner, T. J., and Richmond, T. J. (1999) Preparation of nucleosome core particle from recombinant histones. Meth. Enzymol. 304, 3–19.PubMedCrossRefGoogle Scholar
  16. 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.PubMedCrossRefGoogle Scholar
  17. 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.PubMedCrossRefGoogle Scholar
  18. 18.
    Pennings, S., Meersseman, G., and Bradbury, E. M. (1991) Mobility of positioned nucleosomes on 5 S rDNA. J. Mol. Biol. 220, 101–110.PubMedCrossRefGoogle Scholar
  19. 19.
    Meersseman, G., Pennings, S., and Bradbury, E. M. (1992) Mobile nucleosomes – a general behavior. EMBO J. 11, 2951–2959.PubMedGoogle Scholar
  20. 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. 21.
    Widom, J. (2001) Role of DNA sequence in nucleosome stability and dynamics. Q. Rev. Biophys. 34, 269–324.PubMedCrossRefGoogle Scholar
  22. 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. 23.
    Studitsky, V. M. (1999) Preparation and analysis of positioned nucleosomes. Meth. Mol. Biol. 119, 17–26.Google Scholar
  24. 24.
    Studitsky, V. M., Clark, D. J., and Felsenfeld, G. (1995) Overcoming a nucleosomal barrier to transcription. Cell 83, 19–27.PubMedCrossRefGoogle Scholar
  25. 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.PubMedCrossRefGoogle Scholar
  26. 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.PubMedCrossRefGoogle Scholar
  27. 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.PubMedCrossRefGoogle Scholar
  28. 28.
    Wang, J. P., and Widom, J. (2005) Improved alignment of nucleosome DNA sequences using a mixture model. Nucleic Acids Res. 33, 6743–6755.PubMedCrossRefGoogle Scholar
  29. 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.PubMedCrossRefGoogle Scholar
  30. 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.PubMedCrossRefGoogle Scholar
  31. 31.
    Rhodes, D., and Laskey, R. A. (1989) Assembly of nucleosomes and chromatin in vitro. Meth. Enzymol. 170, 575–585.PubMedCrossRefGoogle Scholar
  32. 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.PubMedCrossRefGoogle Scholar
  33. 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.Google Scholar
  34. 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.PubMedCrossRefGoogle Scholar
  35. 35.
    Schickor, P., and Heumann, H. (1994) Hydroxyl radical footprinting. Meth. Mol. Biol. 30, 21–32.Google Scholar
  36. 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.PubMedCrossRefGoogle Scholar
  37. 37.
    Morse, R. H. (1989) Nucleosomes inhibit both transcriptional initiation and elongation by RNA polymerase III in vitro. EMBO J. 8, 2343–2351.PubMedGoogle Scholar
  38. 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.CrossRefGoogle Scholar
  39. 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.PubMedCrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Daria A. Gaykalova
    • 1
  • Olga I. Kulaeva
    • 1
  • Vladimir A. Bondarenko
    • 1
  • Vasily M. Studitsky
    • 1
  1. 1.Department of PharmacologyRobert Wood Johnson Medical School, University of Medicine and Dentistry of New JerseyPiscatawayUSA

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