Two-Step Cross-linking for Analysis of Protein–Chromatin Interactions
Authors:
Open Access
- 22 October 2011
- book chapter
- Published by Springer Nature
- Vol. 809, 105-120
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.Keywords
This publication has 10 references indexed in Scilit:
- Regulation of Hepatic Six Transmembrane Epithelial Antigen of Prostate 4 (STEAP4) Expression by STAT3 and CCAAT/Enhancer-binding Protein αJournal of Biological Chemistry, 2010
- ChIP on Chip: surprising results are often artifactsBMC Genomics, 2010
- ChIP-seq: Using high-throughput sequencing to discover protein–DNA interactionsMethods, 2009
- RelA Ser 276 Phosphorylation Is Required for Activation of a Subset of NF-κB-Dependent Genes by Recruiting Cyclin-Dependent Kinase 9/Cyclin T1 ComplexesMolecular and Cellular Biology, 2008
- The Functional Role of an Interleukin 6-inducible CDK9·STAT3 Complex in Human γ-Fibrinogen Gene ExpressionJournal of Biological Chemistry, 2007
- Genome-Wide Mapping of in Vivo Protein-DNA InteractionsScience, 2007
- In Vivo Dual Cross-Linking for Identification of Indirect DNA-Associated Proteins by Chromatin ImmunoprecipitationBioTechniques, 2006
- A hyper‐dynamic equilibrium between promoter‐bound and nucleoplasmic dimers controls NF‐κB‐dependent gene activityThe EMBO Journal, 2006
- Two-Step Cross-Linking Method for Identification of NF-κB Gene Network by Chromatin ImmunoprecipitationBioTechniques, 2005
- Twenty-Five Years of the Nucleosome, Fundamental Particle of the Eukaryote ChromosomeCell, 1999