Skip to main content

Reconstitution of Modified Chromatin Templates for In Vitro Functional Assays

  • Protocol
  • First Online:
Book cover Chromatin Remodeling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 833))

Abstract

To study the functions of histone modifications in the context of chromatin, it is necessary to be able to prepare nucleosomal templates that carry specific posttranslational modifications in a defined biochemical system. Here, we describe two sets of protocols for reconstituting designer nucleosomes that contain specifically modified histones. The resulting nucleosomes are suitable for electromobility shift assays, chromatin remodeling assays, and other functional and structural studies.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

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 159.00
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

References

  1. Kouzarides, T. (2007) Chromatin modifications and their function. Cell 128, 693705

    Article  PubMed  CAS  Google Scholar 

  2. Li, B., Carey, M., Workman, J. L. (2007) The role of chromatin during transcription. Cell 128, 707719

    Article  PubMed  CAS  Google Scholar 

  3. Henikoff, S., McKittrick, E., Ahmad, K. (2004) Epigenetics, histone H3 variants, and the inheritance of chromatin states. Cold Spring Harb Symp Quant Biol 69, 235243

    Article  PubMed  CAS  Google Scholar 

  4. Jenuwein, T., Allis, C. D. (2001) Translating the histone code. Science 293, 10741080

    Article  PubMed  CAS  Google Scholar 

  5. Simon, M. D., Chu, F., Racki, L. R., de la Cruz, C. C., Burlingame, A. L., Panning, B., Narlikar, G. J., Shokat, K. M. (2007) The site-specific installation of methyl-lysine analogs into recombinant histones. Cell 128, 10031012

    Article  PubMed  CAS  Google Scholar 

  6. Neumann, H., Hancock, S. M., Buning, R., Routh, A., Chapman, L., Somers, J., Owen-Hughes, T., van Noort, J., Rhodes, D., Chin, J. W. (2009) A method for genetically installing site-specific acetylation in recombinant histones defines the effects of H3 K56 acetylation. Mol Cell 36, 153163

    Article  PubMed  CAS  Google Scholar 

  7. Shogren-Knaak, M., Ishii, H., Sun, J. M., Pazin, M. J., Davie, J. R., Peterson, C. L. (2006) Histone H4-K16 acetylation controls chromatin structure and protein interactions. Science 311, 844847

    Article  PubMed  CAS  Google Scholar 

  8. Li, B., Gogol, M., Carey, M., Lee, D., Seidel, C., Workman, J. L. (2007) Combined action of PHD and chromo domains directs the Rpd3S HDAC to transcribed chromatin. Science 316, 10501054

    Article  PubMed  CAS  Google Scholar 

  9. Carrozza, M. J., Florens, L., Swanson, S. K., Shia, W. J., Anderson, S., Yates, J., Washburn, M. P., Workman, J. L. (2005) Stable incorporation of sequence specific repressors Ash1 and Ume6 into the Rpd3L complex. Biochim Biophys Acta 1731, 7787; discussion 75–76

    Google Scholar 

  10. Dyer, P. N., Edayathumangalam, R. S., White, C. L., Bao, Y., Chakravarthy, S., Muthurajan, U. M., Luger, K. (2004) Reconstitution of nucleosome core particles from recombinant histones and DNA. Methods Enzymol 375, 2344

    Article  PubMed  CAS  Google Scholar 

  11. Owen-Hughes, T., Utley, R. T., Steger, D. J., West, J. M., John, S., Cote, J., Havas, K. M., Workman, J. L. (1999) Analysis of nucleosome disruption by ATP-driven chromatin remodeling complexes. Methods Mol Biol 119, 319331

    PubMed  CAS  Google Scholar 

  12. Grant, P. A., Berger, S. L., Workman, J. L. (1999) Identification and analysis of native nucleosomal histone acetyltransferase complexes. Methods Mol Biol 119, 311317

    PubMed  CAS  Google Scholar 

  13. Puig, O., Caspary, F., Rigaut, G., Rutz, B., Bouveret, E., Bragado-Nilsson, E., Wilm, M., Seraphin, B. (2001) The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, 218229

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Bing Li .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Yun, M., Ruan, C., Huh, JW., Li, B. (2012). Reconstitution of Modified Chromatin Templates for In Vitro Functional Assays. In: Morse, R. (eds) Chromatin Remodeling. Methods in Molecular Biology, vol 833. Humana Press. https://doi.org/10.1007/978-1-61779-477-3_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-477-3_15

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-476-6

  • Online ISBN: 978-1-61779-477-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics