Article

Two-step cross-linking method for Identification of NF-k B gene network by chromatin immunoprecipitation

The University of Texas Medical Branch, Galveston 77555-1060, USA.
BioTechniques (Impact Factor: 2.95). 12/2005; 39(5):715-25.
Source: PubMed

ABSTRACT

The chromatin immunoprecipitation (ChIP) assay has recently been exploited as a powerful and versatile technique for probing protein-DNA interactions within the chromatin environment. In this method, intact cells are fixed with a reversible DNA-protein cross-linking agent (formaldehyde), and associated DNA is enriched by immunoprecipitating a target DNA binding protein. The bound DNA in the immune complexes is then used to identify that specific DNA binding protein's endogenous genomic targets. Nuclear factor kappaB (NF-kappaB) is a highly inducible transcription factor that controls genetic networks important for pathogen- or cytokine-induced inflammation, immune response, and cellular survival. In our studies of the genetic network under control of the inducible NF-kappaB transcription factor, we found that the conventional ChIP technique using a single formaldehyde cross-linking step did not reproducibly cross-link it to DNA. As a result, we have developed a novel ChIP assay using a two-step cross-linking procedure, incorporating N-hydroxysuccinimide (NHS)-ester-mediated protein-protein cross-linking prior to conventional DNA-protein cross-linking. We demonstrate that this technique is highly efficient, cross-linking virtually all NF-kappaB/Rel A into covalent complexes, resulting in quantitative and robust identification of inducible NF-kappaB family binding to a variety of validated NF-kappaB-dependent genomic targets. To demonstrate the general utility of this two-step cross-linking procedure, we performed enhanced capture of cytokine-inducible signal transducer and activator of transcription-3 (STAT3) binding to one of its known target genes. Our method represents a significant improvement in the efficiency of ChIP analysis in the study of endogenous targets for rare transcription factors.

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Available from: Bing Tian, Mar 27, 2014
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    • "ChIP assay was performed in HuH7 cells treated with vehicle or 40 ng/ml TNFa for 6 h. The same anti-p65 antibody was used for ChIP analysis following a two-step cross-linking method as described [19]. See Supplementary Table for the EMSA and ChIP primer sequences. "
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    • "However, formaldehyde has a short crosslinking spacer arm and is not efficient to examine the proteins indirectly associated with DNA, such as PERs and CRYs. Dual cross-linking using a protein–protein cross-linker and formaldehyde works better in these cases (Koike et al., 2012; Nowak, Tian, & Brasier, 2005; Zeng, Vakoc, Chen, Blobel, & Berger, 2006). "
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    • "Two-step cross-link chromatin immunoprecipitation (ChIP) was performed as described previously (17). Briefly, A549 cells were washed twice with PBS and incubated with disuccinimidyl glutarate (2 mM for 45 min) for protein–protein cross-linking. "
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