Plant Signal Transduction pp 25-35

Part of the Methods in Molecular Biology book series (MIMB, volume 1363) | Cite as

DNA-Binding Factor Target Identification by Chromatin Immunoprecipitation (ChIP) in Plants

Abstract

Chromatin immunoprecipitation (ChIP) allows the precise identification of genomic loci that physically interact with a protein of interest, whether that protein is a transcription factor, a core polymerase, a histone, or other chromatin-associated protein. In short, tissue is first cross-linked to freeze a population of DNA-protein interactions at a stage of interest. Chromatin is then extracted, fragmented, and incubated with a specific antibody against the protein of interest. Next, the resultant DNA-protein complexes are immunoprecipitated and captured using beads that bind to the antibody constant region. Samples are finally reverse cross-linked to separate the bound fragments and the DNA is purified. This DNA is analyzed by quantitative PCR for enrichment of genomic regions expected to be bound by the protein under study. The protocol detailed in this chapter has been successfully applied in the identification of target genes for seven transcriptional regulators of diverse classes involved in Arabidopsis thaliana floral transition.

Key words

Chromatin immunoprecipitation ChIP ChIP-seq ChIP-chip Transcription factor Antibody Direct target 

References

  1. 1.
    Garner MM, Revzin A (1981) A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res 9:3047–3060PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505–510CrossRefPubMedGoogle Scholar
  3. 3.
    Solomon MJ, Larsen PL, Varshavsky A (1988) Mapping protein-DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene. Cell 53:937–947CrossRefPubMedGoogle Scholar
  4. 4.
    Thibaud-Nissen F, Wu H, Richmond T et al (2006) Development of Arabidopsis whole-genome microarrays and their application to the discovery of binding sites for the TGA2 transcription factor in salicylic acid-treated plants. Plant J 47:152–162CrossRefPubMedGoogle Scholar
  5. 5.
    Mardis ER (2007) ChIP-seq: welcome to the new frontier. Nat Methods 4:613–614CrossRefPubMedGoogle Scholar
  6. 6.
    Haring M, Offermann S, Danker T et al (2007) Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant Methods 3:11PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Morohashi K, Casas MI, Falcone Ferreyra ML et al (2012) A genome-wide regulatory framework identifies maize pericarp color1 controlled genes. Plant Cell 24:2745–2764PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    He G, Chen B, Wang X et al (2013) Conservation and divergence of transcriptomic and epigenomic variation in maize hybrids. Genome Biol 14:R57PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Ito Y, Kitagawa M, Ihashi N et al (2008) DNA-binding specificity, transcriptional activation potential, and the rin mutation effect for the tomato fruit-ripening regulator RIN. Plant J 55:212–223CrossRefPubMedGoogle Scholar
  10. 10.
    Ricardi MM, González RM, Iusem ND (2010) Protocol: fine-tuning of a Chromatin Immunoprecipitation (ChIP) protocol in tomato. Plant Methods 6:11PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Fujisawa M, Nakano T, Shima Y et al (2013) A large-scale identification of direct targets of the tomato MADS box transcription factor RIPENING INHIBITOR reveals the regulation of fruit ripening. Plant Cell 25:371–386PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Malone BM, Tan F, Bridges SM et al (2011) Comparison of four ChIP-Seq analytical algorithms using rice endosperm H3K27 trimethylation profiling data. PLoS One 6:e25260PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Oh E, Zhu J-Y, Wang Z-Y (2012) Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses. Nat Cell Biol 14:802–809PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Zhu J-Y, Sun Y, Wang Z-Y (2012) Genome-wide identification of transcription factor-binding sites in plants using chromatin immunoprecipitation followed by microarray (ChIP-chip) or sequencing (ChIP-seq). Methods Mol Biol 876:173–188CrossRefPubMedGoogle Scholar
  15. 15.
    Shamimuzzaman M, Vodkin L (2013) Genome-wide identification of binding sites for NAC and YABBY transcription factors and co-regulated genes during soybean seedling development by ChIP-Seq and RNA-Seq. BMC Genomics 14:477PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Liu L, Missirian V, Zinkgraf M et al (2014) Evaluation of experimental design and computational parameter choices affecting analyses of ChIP-seq and RNA-seq data in undomesticated poplar trees. BMC Genomics 15(Suppl 5):S3PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Yant L (2012) Genome-wide mapping of transcription factor binding reveals developmental process integration and a fresh look at evolutionary dynamics. Am J Bot 99:277–290CrossRefPubMedGoogle Scholar
  18. 18.
    Heyndrickx KS, Van de Velde J, Wang C et al (2014) A functional and evolutionary perspective on transcription factor binding in Arabidopsis thaliana. Plant Cell 26:3894. doi:10.1105/tpc.114.130591 PubMedCentralCrossRefPubMedGoogle Scholar
  19. 19.
    Mathieu J, Yant LJ, Mürdter F et al (2009) Repression of flowering by the miR172 target SMZ. PLoS Biol 7:e1000148PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Moyroud E, Minguet EG, Ott F et al (2011) Prediction of regulatory interactions from genome sequences using a biophysical model for the Arabidopsis LEAFY transcription factor. Plant Cell 23:1293–1306PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Yant L, Mathieu J, Dinh TT et al (2010) Orchestration of the floral transition and floral development in Arabidopsis by the bifunctional transcription factor APETALA2. Plant Cell 22:2156–2170PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Posé D, Verhage L, Ott F et al (2013) Temperature-dependent regulation of flowering by antagonistic FLM variants. Nature 503:414–417CrossRefPubMedGoogle Scholar
  23. 23.
    Immink RGH, Posé D, Ferrario S et al (2012) Characterization of SOC1’s central role in flowering by the identification of its upstream and downstream regulators. Plant Physiol 160:433–449PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Instituto de Hortofruticultura Subtropical y MediterráneaUniversidad de Málaga-Consejo Superior de Investigaciones CientíficasMálagaSpain
  2. 2.Department of Cell and Development BiologyJohn Innes Centre, Norwich Research parkNorwichUK

Personalised recommendations