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

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

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.

Key words

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

Notes

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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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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