title: Two-step cross-linking for analysis of protein-chromatin interactions.
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.
authors: Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX, USA.
authors: Bing
authors: B
authors: Tian
authors: Jun
authors: J
authors: Yang
authors: Allan R
authors: AR
authors: Brasier
mesh_terms: D002460:Cell Line; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D006657:Histones / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012319:RNA Polymerase II / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism
chemical_list: D006657:Histones; D016328:NF-kappa B; D050796:STAT3 Transcription Factor; D012319:RNA Polymerase II