Abstract
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.
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References
Kornberg, R.D., and Lorch, Y. (1999) Twenty five years of the nucleosome, fundamental particle of the eukaryote chromosome. Cell 98:285–294.
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.
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.
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.
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.
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.
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.
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.
Waldminhaus, T., and Skarstad, K. (2010) ChIP on Chip: surprising results are often artifacts. BMC Genomics 11:414.
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.
Acknowledgments
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).
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Tian, B., Yang, J., Brasier, A.R. (2012). Two-Step Cross-linking for Analysis of Protein–Chromatin Interactions. In: Vancura, A. (eds) Transcriptional Regulation. Methods in Molecular Biology, vol 809. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-376-9_7
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DOI: https://doi.org/10.1007/978-1-61779-376-9_7
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