Two-Step Cross-linking for Analysis of Protein–Chromatin Interactions

  • Bing Tian
  • Jun Yang
  • Allan R. BrasierEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 809)


Eukaryotic gene regulation is controlled, in part, by inducible transcription factor-binding regulatory sequences in a tissue-specific and hormone-responsive manner. The development of methods for the analysis of transcription factor interaction within native chromatin has been a significant advance for the systematic analyses of the timing of gene regulation and studies on the effects of chromatin modifying enzymes on promoter accessibility. Chromatin immunoprecipitation (ChIP) is a specific method involving formaldehyde mediated protein–chromatin fixation to preserve the interaction for subsequent target identification. However, the conventional single-step cross-linking technique does not preserve all protein–DNA interactions, especially for transcription factors in hyper-dynamic equilibrium with chromatin or for coactivator interactions. Here, we describe a versatile, efficient “two-step” XChIP method that involves sequential protein–protein fixation followed by protein–DNA fixation. This method has been used successfully for analysis of chromatin binding for transcription factors (NF-κB, STAT3), polymerases (RNA Pol II), coactivators (CBP/p300, CDK9), and chromatin structural proteins (modified histones). Modifications of DNA extraction and sonication suitable for downstream target identification by quantitative genomic PCR and next generation sequencing are described.

Key words

Chromatin immunoprecipitation Nuclear factor-κB Polymerase chain reaction Next generation sequencing 



This work was supported, in part, by NIH grants AI062885 (A.R.B.), NHLBI contract BAA-HL-02-04 (A.R.B.), and ES06676 (to K. Elferink, UTMB).


  1. 1.
    Kornberg, R.D., and Lorch, Y. (1999) Twenty five years of the nucleosome, fundamental particle of the eukaryote chromosome. Cell 98:285–294.PubMedCrossRefGoogle Scholar
  2. 2.
    Nowak, D.E., Tian, B., and Brasier, A.R. (2005) Two-step cross-linking method for identification of NF-κB gene network by chromatin immunoprecipitation. Biotechniques 39:715–725.PubMedCrossRefGoogle Scholar
  3. 3.
    Bosisio, D., Marazzi, I., Agresti, A., Shimizu, N., Bianchi, M.E., and Natoli, G. (2006) A hyper-dynamic equilibrium between promoter-bound and nucleoplasmic dimers controls NFκB-dependent gene activity. EMBO J 25:798–810.PubMedCrossRefGoogle Scholar
  4. 4.
    Nowak, D.E., Tian, B., Jamaluddin, M., Boldogh, I., Vergara,L. A., Choudhary, S., and Brasier, A.R. (2008) RelA Ser276 phosphorylation is required for activation of a subset of NFκB-dependent genes by recruiting cyclin-dependent kinase 9/cyclin T1 complexes. Mol Cell Biol 28:3623–3638.PubMedCrossRefGoogle Scholar
  5. 5.
    Hou, T., Ray, S., and Brasier, A.R. (2007) The functional role of an IL-6 inducible CDK9-STAT3 complex in human fibrinogen gene expression. J. Biol. Chem. 282:37091–37102.PubMedCrossRefGoogle Scholar
  6. 6.
    Ramadoss, P., Chiappini, F., Bilban, M., and Hollenberg,A.N. (2010) Regulation of hepatic six transmembrane epithelial antigen of prostate 4 (STEAP4) expression by STAT3 and CCAAT/enhancer-binding protein  +  ¦. J. Biol. Chem. 285:16453–16466.PubMedCrossRefGoogle Scholar
  7. 7.
    Johnson, D.S., Mortazavi, A., Myers, R.M., and Wold,B. (2007) Genome-wide mapping of in vivo protein-DNA interactions. Science 316:1497–1502.PubMedCrossRefGoogle Scholar
  8. 8.
    Zeng, P.Y., Vakoc, C.R., Chen, Z.-C., Blobel, G.A., and Berger, S.L. (2006) In vivo dual cross-linking for identification of indirect DNA-associate proteins by chromatin Immunoprecipitation. Biotechniques 41:694698.CrossRefGoogle Scholar
  9. 9.
    Waldminhaus, T., and Skarstad, K. (2010) ChIP on Chip: surprising results are often artifacts. BMC Genomics 11:414.CrossRefGoogle Scholar
  10. 10.
    Schmidt, D., Wilson, M.D., Spyrou, C., Brown, G.D., Hadfield, J., and Odom, D.T. (2009) ChIP-seq: using high-throughput sequencing to discover protein-DNA interactions. Methods 48:240–248.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Internal MedicineUniversity of Texas Medical BranchGalvestonUSA
  2. 2.Department of Internal Medicine, Institute for Translational Sciences, and Sealy Center for Molecular MedicineUniversity of Texas Medical BranchGalvestonUSA

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