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.

Download full-text


Available from: Bing Tian, Mar 27, 2014
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Genome-wide analyses have revolutionized our ability to study the transcriptional regulation of circadian rhythms. The advent of next-generation sequencing methods has facilitated the use of two such technologies, ChIP-seq and RNA-seq. In this chapter, we describe detailed methods and protocols for these two techniques, with emphasis on their usage in circadian rhythm experiments in the mouse liver, a major target organ of the circadian clock system. Critical factors for these methods are highlighted and issues arising with time series samples for ChIP-seq and RNA-seq are discussed. Finally, detailed protocols for library preparation suitable for Illumina sequencing platforms are presented. © 2015 Elsevier Inc. All rights reserved.
  • Source
    • "e . Nowak et al , 2005 [ 21 ] and references therein ) . However , this feature has never been systematically evaluated or discussed . "
    [Show abstract] [Hide abstract]
    ABSTRACT: NF-κB is a transcription factor that upon activation undergoes cycles of cytoplasmic-to-nuclear and nuclear-to-cytoplasmic transport, giving rise to so called "oscillations". In turn, oscillations tune the transcriptional output. Since a detailed understanding of oscillations requires a systems biology approach, we developed a method to acquire and analyze large volumes of data on NF-κB dynamics in single cells. We measured the time evolution of the nuclear to total ratio of GFP-p65 in knock-in mouse embryonic fibroblasts using time-lapse imaging. We automatically produced a precise segmentation of nucleus and cytoplasm based on an accurate estimation of the signal and image background. Finally, we defined a set of quantifiers that describe the oscillatory dynamics, which are internally normalized and can be used to compare data recorded by different labs. Using our method, we analyzed NF-κB dynamics in over 2000 cells exposed to different concentrations of TNF- α α. We reproduced known features of the NF-κB system, such as the heterogeneity of the response in the cell population upon stimulation and we confirmed that a fraction of the responding cells does not oscillate. We also unveiled important features: the second and third oscillatory peaks were often comparable to the first one, a basal amount of nuclear NF-κB could be detected in unstimulated cells, and at any time a small fraction of unstimulated cells showed spontaneous random activation of the NF-κB system. Our work lays the ground for systematic, high-throughput, and unbiased analysis of the dynamics of transcription factors that can shuttle between the nucleus and other cell compartments.
    PLoS ONE 03/2014; 9(3):e90104. DOI:10.1371/journal.pone.0090104 · 3.53 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Myc oncoproteins and histone deacetylases (HDACs) modulate gene transcription and enhance cancer cell proliferation, and HDAC inhibitors are among the most promising new classes of anticancer drugs. Here, we show that N-Myc and c-Myc upregulated HDAC2 gene expression in neuroblastoma and pancreatic cancer cells, respectively, which contributed to N-Myc- and c-Myc-induced cell proliferation. Cyclin G2 (CCNG2) was commonly repressed by N-Myc and HDAC2 in neuroblastoma cells and by c-Myc and HDAC2 in pancreatic cancer cells, and could be reactivated by HDAC inhibitors. 5-bromo-2'-deoxyuridine incorporation assays showed that transcriptional repression of CCNG2 was, in part, responsible for N-Myc-, c-Myc- and HDAC2-induced cell proliferation. Dual crosslinking chromatin immunoprecipitation assay demonstrated that N-Myc acted as a transrepressor by recruiting the HDAC2 protein to Sp1-binding sites at the CCNG2 gene core promoter. Moreover, HDAC2 was upregulated, and CCNG2 downregulated, in pre-cancerous and neuroblastoma tissues from N-Myc transgenic mice, and c-Myc overexpression correlated with upregulation of HDAC2 and repression of CCNG2 in tumour tissues from pancreatic cancer patients. Taken together, our data indicate the critical roles of upregulation of HDAC2 and suppression of CCNG2 in Myc-induced oncogenesis, and have significant implications for the application of HDAC inhibitors in the prevention and treatment of Myc-driven cancers.
    Oncogene 11/2010; 29(44):5957-68. DOI:10.1038/onc.2010.332 · 8.56 Impact Factor
Show more