Genome-wide Mapping of Protein-DNA Interactions by Chromatin Immunoprecipitation and DNA Microarray Hybridization

  • Jason D. Lieb
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 224)

Abstract

A critical part of understanding the mechanism and logic of cellular regulatory networks is understanding where enzymes and their regulatory proteins interact with the genome in vivo. From this, we can determine the genomic features that specify protein binding and simultaneously identify genes or other chromosomal elements whose function is affected by the binding. Recently, methods that combine well-established protocols for chromatin immunoprecipitations (1, 2, 3, 4, 5, 6) with the surveying power of DNA microarrays have allowed researchers to create high-resolution, genomewide maps of the interaction between DNA-associated proteins and DNA (7, 8, 9). Many variations of the method have been published, but all contain the same basic steps (10): growth of cells, fixation, extract preparation, immunoprecipitation, fixation reversal, DNA purification, DNA amplification, microarray hybridization, and data analysis. The purpose here is to detail a single experimental method in yeast from start to finish, rather than to review all of the different protocols that have been used. The method described in this chapter worked for a particular set of DNA-associated proteins (Rap1p, Sir2p, Sir3p, and Sir4p), and their corresponding antibody-antigen interactions (8). Since the strength, specificity, and mechanism of antibody-antigen and protein-DNA association vary widely, this protocol should be viewed as a starting point, rather than an absolute procedure.

References

  1. 1.
    Braunstein, M., Rose, A. B., Holmes, S. G., Allis, C. D., and Broach, J. R. (1993) Transcriptional silencing in yeast is associated with reduced nucleosome acetylation. Genes Dev. 7(4), 592–604.PubMedCrossRefGoogle Scholar
  2. 2.
    Solomon, M. J., Larsen, P. L., and Varshavsky, A. (1988) Mapping protein-DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene. Cell 53(6), 937–947.PubMedCrossRefGoogle Scholar
  3. 3.
    Strahl-Bolsinger, S., Hecht, A., Luo, K., and Grunstein, M. (1997) SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast. Genes Devel. 11(1), 83–93.PubMedCrossRefGoogle Scholar
  4. 4.
    Orlando, V. and Paro, R. (1993) Mapping polycomb-repressed domains in the bithorax complex using in vivo formaldehyde cross-linked chromatin. Cell 75(6), 1187–1198.PubMedCrossRefGoogle Scholar
  5. 5.
    Dedon, P. C., Soults, J. A., Allis, C. D., and Gorovsky, M. A. (1991) A simplified formaldehyde fixation and immunoprecipitation technique for studying protein-DNA interactions. Anal. Biochem. 197(1), 83–90.PubMedCrossRefGoogle Scholar
  6. 6.
    Carr, A. and Biggin, M. D. (1999) A comparison of in vivo and in vitro DNA-binding specificities suggests a new model for homeoprotein DNA binding in Drosophila embryos. EMBO J. 18(6), 1598–1608.PubMedCrossRefGoogle Scholar
  7. 7.
    Ren, B., Robert, F. Wyrick, J. J., et al. (2000) Genome-wide location and function of DNA binding proteins. Science 290(5500), 2306–2309.PubMedCrossRefGoogle Scholar
  8. 8.
    Lieb, J. D., Liu, X., Botstein, D., and Brown, P. O. (2001) Promoter-specific binding of Rap1 revealed by genome-wide maps of protein-DNA association. Nat. Genet. 28(4), 327–334.PubMedCrossRefGoogle Scholar
  9. 9.
    Iyer, V. A., Horak, C. A., Scafe, C. S., Botstein, D., Snyder, M., and Brown, P. O. (2001) Genomic binding distribution of the yeast cell-cycle transcription factors SBF and MBF. Nature 409, 533–538.PubMedCrossRefGoogle Scholar
  10. 10.
    Orlando, V. (2000) Mapping chromosomal proteins in vivo by formaldehyde-crosslinked chromatin immunoprecipitation. Trends Biochem. Sci. 25(3), 99–104.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2003

Authors and Affiliations

  • Jason D. Lieb
    • 1
  1. 1.Department of Biology and Carolina Center for Genome SciencesThe University of North Carolina at Chapel HillChapel Hill

Personalised recommendations