Molecular and Cell Biology Methods for Fungi pp 211-224

Part of the Methods in Molecular Biology book series (MIMB, volume 638)

A Detailed Protocol for Chromatin Immunoprecipitation in the Yeast Saccharomyces cerevisiae

  • Melanie Grably
  • David Engelberg


Critical cellular processes such as DNA replication, DNA damage repair, and transcription are mediated and regulated by DNA-binding proteins. Many efforts have been invested therefore in developing methods that monitor the dynamics of protein-DNA association. As older techniques such as DNA footprinting, and electrophoretic mobility shift assays (EMSA) could be applied mostly in vitro, the development of the chromatin immunoprecipitation (ChIP) method, which allows quantitative measurement of protein-bound DNA most accurately in vivo, revolutionized our capabilities of understanding the mechanisms underlying the aforementioned processes. Furthermore, this powerful tool could be applied at the genomic-scale providing a global picture of the protein-DNA complexes at the entire genome.

The procedure is conceptually simple; involves rapid crosslinking of proteins to DNA by the addition of formaldehyde to the culture, shearing the DNA and immunoprecipitating the protein of interest while covalently bound to its DNA targets. Following decrosslinking, DNA that was coimmunoprecipitated could be amplified by PCR or could serve as a probe of a genomic microarray to identify all DNA fragments that were bound to the protein.

Although simple in principle, the method is not trivial to implement and the results might be misleading if proper controls are not included in the experiment. In this chapter, we provide therefore a highly detailed protocol of ChIP assay as is applied successfully in our laboratory. We pay special attention to describe every small detail, in order that any investigator could readily and successfully apply this important and powerful technology.

Key words

Saccharomyces cerevisiae Chromatin immunoprecipitation DNA binding proteins Transcription Heat shock 


  1. 1.
    Bhaumik SR, Green MR (2002) Differential requirement of SAGA components for recruitment of TATA-box-binding protein to promoters in vivo. Mol Cell Biol 22:7365–7371CrossRefPubMedGoogle Scholar
  2. 2.
    Geisberg JV, Struhl K (2004) Cellular stress alters the transcriptional properties of promoter-bound Mot1-TBP complexes. Mol Cell 14:479–489CrossRefPubMedGoogle Scholar
  3. 3.
    Jasiak AJ, Hartmann H, Karakasili E, Kalocsay M, Flatley A, Kremmer E, Strasser K, Martin DE, Soding J, Cramer P (2008) Genome-associated RNA polymerase II includes the dissociable Rpb4/ 7 subcomplex. J Biol Chem 283:26423–26427CrossRefPubMedGoogle Scholar
  4. 4.
    Krogan NJ, Kim M, Ahn SH, Zhong G, Kobor MS, Cagney G, Emili A, Shilatifard A, Buratowski S, Greenblatt JF (2002) RNA polymerase II elongation factors of Saccharo-myces cerevisiae: a targeted proteomics approach. Mol Cell Biol 22:6979–6992CrossRefPubMedGoogle Scholar
  5. 5.
    Li J, Lin Q, Wang W, Wade P, Wong J (2002) Specific targeting and constitutive association of histone deacetylase complexes during transcriptional repression. Genes Dev 16:687–692CrossRefPubMedGoogle Scholar
  6. 6.
    Moqtaderi Z, Struhl K (2004) Genome-wide occupancy profile of the RNA polymerase III machinery in Saccharomyces cerevisiae reveals loci with incomplete transcription complexes. Mol Cell Biol 24:4118–4127CrossRefPubMedGoogle Scholar
  7. 7.
    Radonjic M, Andrau JC, Lijnzaad P, Kemmeren P, Kockelkorn TT, van Leenen D, van Berkum NL, Holstege FC (2005) Genome-wide analyses reveal RNA polymerase II located upstream of genes poised for rapid response upon S. cerevisiae stationary phase exit. Mol Cell 18:171–183CrossRefPubMedGoogle Scholar
  8. 8.
    Robyr D, Suka Y, Xenarios I, Kurdistani SK, Wang A, Suka N, Grunstein M (2002) Microarray deacetylation maps determine genome-wide functions for yeast histone deacetylases. Cell 109:437–446CrossRefPubMedGoogle Scholar
  9. 9.
    Simic R, Lindstrom DL, Tran HG, Roinick KL, Costa PJ, Johnson AD, Hartzog GA, Arndt KM (2003) Chromatin remodeling protein Chd1 interacts with transcription elongation factors and localizes to transcribed genes. EMBO J 22:1846–1856CrossRefPubMedGoogle Scholar
  10. 10.
    Vogelauer M, Wu J, Suka N, Grunstein M (2000) Global histone acetylation and deacetylation in yeast. Nature 408:495–498CrossRefPubMedGoogle Scholar
  11. 11.
    Wong MM, Cox LK, Chrivia JC (2007) The chromatin remodeling protein, SRCAP, is critical for deposition of the histone variant H2A.Z at promoters. J Biol Chem 282:26132–26139CrossRefPubMedGoogle Scholar
  12. 12.
    Krebs JE, Kuo MH, Allis CD, Peterson CL (1999) Cell cycle-regulated histone acetylation required for expression of the yeast HO gene. Genes Dev 13:1412–1421CrossRefPubMedGoogle Scholar
  13. 13.
    Kuras L, Borggrefe T, Kornberg RD (2003) Association of the mediator complex with enhancers of active genes. Proc Natl Acad Sci U S A 100:13887–13891CrossRefPubMedGoogle Scholar
  14. 14.
    Larschan E, Winston F (2001) The S. cerevisiae SAGA complex functions in vivo as a coactivator for transcriptional activation by Gal4. Genes Dev 15:1946–1956CrossRefPubMedGoogle Scholar
  15. 15.
    Swanson MJ, Qiu H, Sumibcay L, Krueger A, Kim SJ, Natarajan K, Yoon S, Hinnebusch AG (2003) A multiplicity of coactivators is required by Gcn4p at individual promoters in vivo. Mol Cell Biol 23:2800–2820CrossRefPubMedGoogle Scholar
  16. 16.
    Deckert J, Struhl K (2001) Histone acetylation at promoters is differentially affected by specific activators and repressors. Mol Cell Biol 21:2726–2735CrossRefPubMedGoogle Scholar
  17. 17.
    Kurdistani SK, Robyr D, Tavazoie S, Grunstein M (2002) Genome-wide binding map of the histone deacetylase Rpd3 in yeast. Nat Genet 31:248–254CrossRefPubMedGoogle Scholar
  18. 18.
    Pokholok DK, Harbison CT, Levine S, Cole M, Hannett NM, Lee TI, Bell GW, Walker K, Rolfe PA, Herbolsheimer E, Zeitlinger J, Lewitter F, Gifford DK, Young RA (2005) Genome-wide map of nucleosome acetylation and methylation in yeast. Cell 122:517–527CrossRefPubMedGoogle Scholar
  19. 19.
    Roh TY, Ngau WC, Cui K, Landsman D, Zhao K (2004) High-resolution genome-wide mapping of histone modifications. Nat Biotechnol 22:1013–1016CrossRefPubMedGoogle Scholar
  20. 20.
    Bernstein BE, Liu CL, Humphrey EL, Perlstein EO, Schreiber SL (2004) Global nucleosome occupancy in yeast. Genome Biol 5:R62CrossRefPubMedGoogle Scholar
  21. 21.
    Alepuz PM, de Nadal E, Zapater M, Ammerer G, Posas F (2003) Osmostress-induced transcription by Hot1 depends on a Hog1-mediated recruitment of the RNA Pol II. EMBO J 22:2433–2442CrossRefPubMedGoogle Scholar
  22. 22.
    Pokholok DK, Zeitlinger J, Hannett NM, Reynolds DB, Young RA (2006) Activated signal transduction kinases frequently occupy target genes. Science 313:533–536CrossRefPubMedGoogle Scholar
  23. 23.
    Aparicio O, Geisberg JV, Sekinger E, Yang A, Moqtaderi Z, Struhl K (2005) Chromatin immunoprecipitation for determining the association of proteins with specific genomic sequences in vivo. In: Ausubel FM, Brent R, Kington RE, Moore DD, Seidman JG, Smith JA, Struhl KE (eds) Current protocols in molecular biology. Wiley, New York, pp 21.3.1-21.3.33Google Scholar
  24. 24.
    Ezhkova E, Tansey WP (2004) Chromatin immunoprecipitation to study Protein-DNA Interactions in Budding Yeast, vol 313, 2nd edn, Methods in molecular biology. Humana Press Inc, Totawa, NJ, pp 225–244Google Scholar
  25. 25.
    Nelson JD, Denisenko O, Sova P, Bomsztyk K (2006) Fast chromatin immunoprecipitation assay. Nucleic Acids Res 34:e2CrossRefPubMedGoogle Scholar
  26. 26.
    Ren B, Dynlacht BD (2003) Use of chromatin immunoprecipitation assays in genome-wide location analysis of mammalian transcription factors. Methods Enzymol 376:304–315CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Melanie Grably
    • 1
  • David Engelberg
    • 1
  1. 1.The Department of Biological ChemistryThe Institute of Life Sciences, The Hebrew University of JerusalemJerusalemIsrael

Personalised recommendations