Native Chromatin Immunoprecipitation

  • Alan W. Thorne
  • Fiona A. Myers
  • Tim R. Hebbes
Part of the Methods in Molecular Biology™ book series (MIMB, volume 287)

Abstract

Chromatin immunoprecipitation (ChIP) is a technique widely used for determining the genomic location of modified histones and other chromatin-associated factors. Here we describe the methodology we have used in our laboratory for the immunoprecipitation of chromatin isolated from cells in the absence of crosslinking. Chromatin released from nuclei by micrococcal nuclease digestion is centrifuged through sucrose gradients to allow selection of monoor dinucleosomes. This allows a protein or modification at a particular gene or locus to be mapped at higher resolution than in a crosslinked ChIP experiment. Two methods for the immunoprecipitation of chromatin are described: a large-scale fractionation by which it is possible to visualize the proteins of the immunoprecipitate by polyacrylamide gel electrophoresis, PAGE and a small-scale method that is more appropriate when the quantity of chromatin is limited. The sequence content of DNA extracted from the immunoprecipitated chromatin is analyzed by hybridization of Southern or slot blots, or by quantitative polymerase chain reaction. Enrichment of particular sequences in the immunoprecipitated fraction reveals the presence and extent of the modification at this location.

Key Words

Native chromatin immunoprecipitation histone acetylation active chromatin chicken β-globin human α-globin 

References

  1. 1.
    Dorbic, T. and Wittig, B. (1986) Isolation of oligonucleosomes from active chromatin using HMG17-specific monoclonal antibodies. Nucleic Acids Res. 14, 3363–3376.CrossRefPubMedGoogle Scholar
  2. 2.
    Dorbic, T. and Wittig, B. (1987) Chromatin from transcribed genes contains HMG17 only downstream from the starting point of transcription. EMBO J. 6, 2393–2399.PubMedGoogle Scholar
  3. 3.
    Hebbes, T. R., Thorne, A. W., and Crane-Robinson, C. (1988) A direct link between core histone acetylation and transcriptionally active chromatin. EMBO J. 7, 1395–1402.PubMedGoogle Scholar
  4. 4.
    Hebbes, T. R., Thorne, A. W., Clayton, A. L., and Crane-Robinson, C. (1992) Histone acetylation and globin gene switching. Nucleic Acids Res. 20, 1017–1022.CrossRefPubMedGoogle Scholar
  5. 5.
    Hebbes, T. R., Clayton, A. L., Thorne, A. W., and Crane-Robinson, C. (1994) Core histone hyperacetylation co-maps with generalized DNase I sensitivity in the chicken beta-globin chromosomal domain. EMBO J. 13, 1823–1830.PubMedGoogle Scholar
  6. 6.
    Myers, F. A., Evans, D. R., Clayton, A. L., Thorne, A. W., and Crane-Robinson, C. (2001) Targeted and extended acetylation of histones H4 and H3 at active and inactive genes in chicken embryo erythrocytes. J. Biol. Chem. 276, 20,197–20,205.CrossRefPubMedGoogle Scholar
  7. 7.
    Litt, M. D., Simpson, M., Recillas-Targa, F., Prioleau, M. N., and Felsenfeld, G. (2001). Transitions in histone acetylation reveal boundaries of three separately regulated neighboring loci. EMBO J. 20, 2224–2235.CrossRefPubMedGoogle Scholar
  8. 8.
    Litt, M. D., Simpson, M., Gaszner, M., Allis, C. D., and Felsenfeld, G. (2001). Correlation between histone lysine methylation and developmental changes at the chicken beta-globin locus. Science 293, 2453–2455.CrossRefPubMedGoogle Scholar
  9. 9.
    Madisen, L., Krumm, A., Hebbes, T. R., and Groudine, M. (1998). The immunoglobulin heavy chain locus control region increases histone acetylation along linked c-myc genes. Mol. Cell Biol. 18, 6281–6292.PubMedGoogle Scholar
  10. 10.
    Clayton, A. L., Hebbes, T. R., Thorne, A. W., and Crane-Robinson, C. (1993). Histone acetylation and gene induction in human cells. FEBS Lett. 336, 23–26.CrossRefPubMedGoogle Scholar
  11. 11.
    Pelling, A. L., Thorne, A. W., and Crane-Robinson, C. (2000). A human genomic library enriched in transcriptionally active sequences (aDNA library). Genome Res. 10, 874–886.CrossRefPubMedGoogle Scholar
  12. 12.
    O’Neill, L. P., Keohane, A. M., Lavender, J. S., et al. (1999). A developmental switch in H4 acetylation upstream of Xist plays a role in X chromosome inactivation. EMBO J. 18, 2897–2907.CrossRefPubMedGoogle Scholar
  13. 13.
    Elefant, F., Cooke, N. E., and Liebhaber, S. A. (2000). Targeted recruitment of histone acetyltransferase activity to a locus control region. J. Biol. Chem. 275, 13,827–13,834.CrossRefPubMedGoogle Scholar
  14. 14.
    Elefant, F., Su, Y., Liebhaber, S. A., and Cooke, N. E. (2000). Patterns of histone acetylation suggest dual pathways for gene activation by a bifunctional locus control region. EMBO J. 19, 6814–6822.CrossRefPubMedGoogle Scholar
  15. 15.
    Hewish, D. R. and Burgoyne, L. A. (1973). Chromatin sub-structure. The digestion of chromatin DNA at regularly spaced sites by a nuclear deoxyribonuclease. Biochem. Biophys. Res. Commun. 52, 504–510.CrossRefPubMedGoogle Scholar
  16. 16.
    Ridsdale, J. A. and Davie, J. R. (1987). Chicken erythrocyte polynucleosomes which are soluble at physiological ionic strength and contain linker histones are highly enriched in beta-globin gene sequences. Nucleic Acids Res. 15, 1081–1096.CrossRefPubMedGoogle Scholar
  17. 17.
    Ridsdale, J. A. and Davie, J. R. (1987). Selective solubilization of beta-globin oligonucleosomes at low ionic strength. Biochemistry 26, 290–295.CrossRefPubMedGoogle Scholar
  18. 18.
    Sung, M. T. and Dixon, G. H. (1970). Modification of histones during spermiogenesis in trout: a molecular mechanism for altering histone binding to DNA. Proc. Natl. Acad. Sci. USA 67, 1616–1623.CrossRefPubMedGoogle Scholar
  19. 19.
    Wood, W. I. and Felsenfeld, G. (1982). Chromatin structure of the chicken beta-globin gene region. Sensitivity to DNase I, micrococcal nuclease, and DNase II. J. Biol. Chem. 257, 7730–7736.PubMedGoogle Scholar
  20. 20.
    Stratling, W. H., Dolle, A., and Sippel, A. E. (1986). Chromatin structure of the chicken lysozyme gene domain as determined by chromatin fractionation and micrococcal nuclease digestion. Biochemistry 25, 495–502.CrossRefPubMedGoogle Scholar
  21. 21.
    Leuba, S. H., Zlatanova, J., and van Holde, K. (1994). On the location of linker DNA in the chromatin fiber. Studies with immobilized and soluble micrococcal nuclease. J. Mol. Biol. 235, 871–880.CrossRefPubMedGoogle Scholar
  22. 22.
    Hebbes, T. R., Turner, C. H., Thorne, A. W., and Crane-Robinson, C. (1989). A “minimal epitope” anti-protein antibody that recognises a single modified amino acid. Mol. Immunol. 26, 865–873.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2004

Authors and Affiliations

  • Alan W. Thorne
    • 1
  • Fiona A. Myers
    • 1
  • Tim R. Hebbes
    • 1
  1. 1.Institute of Biomedical and Biomolecular Sciences, School of Biological SciencesUniversity of PortsmouthPortsmouthUK

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