Two-step cross-linking for analysis of protein-chromatin interactions

Methods Mol Biol. 2012;809:105-20. doi: 10.1007/978-1-61779-376-9_7.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell Line
  • Chromatin Immunoprecipitation / methods*
  • Histones / metabolism
  • Humans
  • Models, Biological
  • NF-kappa B / metabolism
  • Polymerase Chain Reaction
  • Protein Binding
  • RNA Polymerase II / metabolism
  • STAT3 Transcription Factor / metabolism

Substances

  • Histones
  • NF-kappa B
  • STAT3 Transcription Factor
  • RNA Polymerase II