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: 2.75). 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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    • "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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    • "e . Nowak et al , 2005 [ 21 ] and references therein ) . However , this feature has never been systematically evaluated or discussed . "
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    • "The CCNG2 promoter region was scanned for the presence of N-Myc, Sp1 and HDAC2 binding using the dual crosslinking ChIP assay (Nowak et al., 2005). A pre-immune serum was used as a negative control to determine the baseline of the non-specific background. "
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