Skip to main content

Chromatin Immunoprecipitation Assay to Identify Genomic Binding Sites of Regulatory Factors

  • Protocol
Estrogen Receptors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1366))

Abstract

DNA–protein interactions are vital to fundamental cellular events including transcription, replication, DNA repair, and recombination. Thus, their study holds the key to our understanding of mechanisms underlying normal development and homeostasis as well as disease. Transcriptional regulation is a highly complex process that involves recruitment of numerous factors resulting in formation of multi-protein complexes at gene promoters to regulate gene expression. The studied proteins can be, for example, transcription factors, epigenetic regulators, co-activators, co-repressors, or ligand-activated nuclear receptors as estrogen receptor-α (ERα) bound either directly to the DNA or indirectly by interaction with other DNA-bound factors. Chromatin immunoprecipitation (ChIP) assay is a powerful method to study interactions of proteins and a specific genomic DNA region. Recruitment of ERα to promoters of estrogen-dependent genes is a common mechanism to activate or enhance gene transcription in breast cancer thus promoting tumor progression. In this chapter, we demonstrate a stepwise protocol for ChIP assay using binding of ERα to its genomic targets after stimulation with 17β-estradiol (E2) in breast cancer cells as an example.

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 119.00
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 169.99
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

Similar content being viewed by others

References

  1. Thakurela S, Garding A, Jung J et al (2013) Gene regulation and priming by topoisomerase IIα in embryonic stem cells. Nat Commun 4:2478

    Article  Google Scholar 

  2. Tiwari VK, Stadler MB, Wirbelauer C et al (2012) A chromatin-modifying function of JNK during stem cell differentiation. Nat Genet 44:94–100

    Article  CAS  Google Scholar 

  3. Schnitt SJ (2010) Classification and prognosis of invasive breast cancer: from morphology to molecular taxonomy. Mod Pathol 23(Suppl 2):S60–S64

    Article  Google Scholar 

  4. Thomas C, Gustafsson J-Å (2011) The different roles of ER subtypes in cancer biology and therapy. Nat Rev Cancer 11:597–608

    Article  CAS  Google Scholar 

  5. Cheskis BJ, Greger JG, Nagpal S et al (2007) Signaling by estrogens. J Cell Physiol 213:610–617

    Article  CAS  Google Scholar 

  6. Hall JM, Couse JF, Korach KS (2001) The multifaceted mechanisms of estradiol and estrogen receptor signaling. J Biol Chem 276:36869–36872

    Article  CAS  Google Scholar 

  7. Safe S, Kim K, Kim K (2008) Non-classical genomic estrogen receptor (ER)/specificity protein and ER/activating protein-1 signaling pathways. J Mol Endocrinol 41:263–275

    Article  CAS  Google Scholar 

  8. Robinson-Rechavi M, Escriva Garcia H, Laudet V (2003) The nuclear receptor superfamily. J Cell Sci 116:585–586

    Article  Google Scholar 

  9. Dong J, Tsai-Morris C-H, Dufau ML (2006) A novel estradiol/estrogen receptor alpha-dependent transcriptional mechanism controls expression of the human prolactin receptor. J Biol Chem 281:18825–18836

    Article  CAS  Google Scholar 

  10. Koslowski M, Türeci O, Biesterfeld S et al (2009) Selective activation of trophoblast-specific PLAC1 in breast cancer by CCAAT/enhancer-binding protein beta (C/EBPbeta) isoform 2. J Biol Chem 284:28607–28615

    Article  CAS  Google Scholar 

  11. Wagner M, Koslowski M, Paret C et al (2013) NCOA3 is a selective co-activator of estrogen receptor α-mediated transactivation of PLAC1 in MCF-7 breast cancer cells. BMC Cancer 13:570

    Article  Google Scholar 

  12. Carey MF, Peterson CL, Smale ST (2009) Chromatin immunoprecipitation (ChIP). Cold Spring Harb Protoc pdb.prot5279

    Google Scholar 

  13. O’Neill LP, Turner BM (1996) Immunoprecipitation of chromatin. Methods Enzymol 274:189–197

    Article  Google Scholar 

  14. Barski A, Cuddapah S, Cui K et al (2007) High-resolution profiling of histone methylations in the human genome. Cell 129:823–837

    Article  CAS  Google Scholar 

  15. Robertson G, Hirst M, Bainbridge M et al (2007) Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nat Methods 4:651–657

    Article  CAS  Google Scholar 

  16. Kharchenko PV, Tolstorukov MY, Park PJ (2008) Design and analysis of ChIP-seq experiments for DNA-binding proteins. Nat Biotechnol 26:1351–1359

    Article  CAS  Google Scholar 

  17. Schmidt D, Wilson MD, Spyrou C et al (2009) ChIP-seq: using high-throughput sequencing to discover protein-DNA interactions. Methods 48:240–248

    Article  CAS  Google Scholar 

  18. Grober OMV, Mutarelli M, Giurato G et al (2011) Global analysis of estrogen receptor beta binding to breast cancer cell genome reveals an extensive interplay with estrogen receptor alpha for target gene regulation. BMC Genomics 12:36

    Article  CAS  Google Scholar 

  19. Ross-Innes CS, Stark R, Teschendorff AE et al (2012) Differential oestrogen receptor binding is associated with clinical outcome in breast cancer. Nature 481:389–393

    Article  CAS  Google Scholar 

  20. Stender JD, Kim K, Charn TH et al (2010) Genome-wide analysis of estrogen receptor alpha DNA binding and tethering mechanisms identifies Runx1 as a novel tethering factor in receptor-mediated transcriptional activation. Mol Cell Biol 30:3943–3955

    Article  CAS  Google Scholar 

  21. Zeng P-Y, Vakoc CR, Chen Z-C et al (2006) In vivo dual cross-linking for identification of indirect DNA-associated proteins by chromatin immunoprecipitation. Biotechniques 41:694, 696, 698

    Article  CAS  Google Scholar 

  22. Hebbes TR, Thorne AW, Crane-Robinson C (1988) A direct link between core histone acetylation and transcriptionally active chromatin. EMBO J 7:1395–1402

    Article  CAS  Google Scholar 

  23. O’Neill LP, Turner BM (2003) Immunoprecipitation of native chromatin: NChIP. Methods 31:76–82

    Article  Google Scholar 

  24. Levenson AS, Jordan VC (1997) MCF-7: the first hormone-responsive breast cancer cell line. Cancer Res 57:3071–3078

    CAS  PubMed  Google Scholar 

  25. Soule HD, Vazguez J, Long A et al (1973) A human cell line from a pleural effusion derived from a breast carcinoma. J Natl Cancer Inst 51:1409–1416

    Article  CAS  Google Scholar 

  26. Harlow E, Lane D (1988) Antibodies: a laboratory manual. Cold Spring Harbor Laboratory, New York

    Google Scholar 

  27. Métivier R, Penot G, Hübner MR et al (2003) Estrogen receptor-alpha directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Cell 115:751–763

    Article  Google Scholar 

Download references

Acknowledgements

Research in the laboratory of V.K.T. is supported by the Wilhelm Sander Stiftung 2012.009.2, EpiGeneSys RISE1 program, Marie Curie CIG 322210 and Deutsche Forschungsgemeinschaft (DFG) Grant TI 799/1-1.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Vijay K. Tiwari or Ugur Sahin .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Wagner, M., Jung, J., Koslowski, M., Türeci, Ö., Tiwari, V.K., Sahin, U. (2016). Chromatin Immunoprecipitation Assay to Identify Genomic Binding Sites of Regulatory Factors. In: Eyster, K.M. (eds) Estrogen Receptors. Methods in Molecular Biology, vol 1366. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3127-9_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3127-9_6

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3126-2

  • Online ISBN: 978-1-4939-3127-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics