Two-Step Cross-linking for Analysis of ProteinChromatin Interactions

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.