Insertional chromatin immunoprecipitation: A method for isolating specific genomic regions

Department of Pathology, New York University School of Medicine, New York, NY 10016, USA.
Journal of Bioscience and Bioengineering (Impact Factor: 1.88). 11/2009; 108(5):446-9. DOI: 10.1016/j.jbiosc.2009.05.005
Source: PubMed


We established a novel method, insertional chromatin immunoprecipitation (iChIP), for isolation of specific genomic regions. In iChIP, specific genomic domains are immunoprecipitated with antibody against a tag, which is fused to the DNA-binding domain of an exogenous DNA-binding protein, whose recognition sequence is inserted into the genomic domains of interest. The iChIP method will be a useful tool for dissecting chromatin structure of genomic region of interest.

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Available from: Hodaka Fujii, Oct 04, 2015
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    • "A comprehensive understanding of the mechanisms behind genome functions such as transcription and epigenetic regulation requires the identification of the molecules that bind to the genomic regions of interest in vivo. We previously developed the locus-specific chromatin immunoprecipitation (ChIP) technologies consisting of insertional ChIP (iChIP) [1]–[5] and engineered DNA-binding molecule-mediated ChIP (enChIP) [6], [7] for purification of specific genomic regions to identify their associated molecules. In enChIP, a tagged engineered DNA-binding molecule is expressed into the cell to be analyzed so that it recogizes an endogenous target DNA sequence. "
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    ABSTRACT: Isolation of specific genomic regions retaining molecular interactions is essential for comprehensive identification of molecules associated with the genomic regions. Recently, we developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology for purification of specific genomic regions. Here, we developed a retroviral expression system for enChIP using CRISPR. We showed that the target genomic locus can be purified with high efficiency by using this system. We also showed that contamination of potential off-target sites is negligible by using this system if the guide RNA (gRNA) for the target site has a sufficiently long unique sequence in its seed sequence. enChIP combined with stable isotope labeling using amino acids in cell culture (SILAC) analysis identified proteins whose association with the interferon (IFN) regulatory factor-1 (IRF-1) promoter region increases in response to IFNγ stimulation. The list of the associated proteins contained many novel proteins in the context of IFNγ-induced gene expression as well as proteins related to histone deacetylase complexes whose involvement has been suggested in IFNγ-mediated gene expression. Finally, we confirmed IFNγ-induced increased association of the identified proteins with the IRF-1 promoter by ChIP. Thus, our results showed that the retroviral enChIP system using CRISPR would be useful for biochemical analysis of genome functions including transcription and epigenetic regulation.
    PLoS ONE 07/2014; 9(7):e103084. DOI:10.1371/journal.pone.0103084 · 3.23 Impact Factor
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    • "enChIP is a technology related to iChIP we developed recently24. In contrast to iChIP, enChIP does not require insertion of recognition sequences of exogenous DNA-binding proteins such as LexA. "
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    ABSTRACT: Biochemical analysis of molecular interactions in specific genomic regions requires their isolation while retaining molecular interactions in vivo. Here, we report isolation of telomeres by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using a transcription activator-like (TAL) protein recognizing telomere repeats. Telomeres recognized by the tagged TAL protein were immunoprecipitated with an antibody against the tag and subjected to identification of telomere-binding molecules. enChIP-mass spectrometry (enChIP-MS) targeting telomeres identified known and novel telomere-binding proteins. The data have been deposited to the ProteomeXchange with identifier PXD000461. In addition, we showed that RNA associated with telomeres could be isolated by enChIP. Identified telomere-binding molecules may play important roles in telomere biology. enChIP using TAL proteins would be a useful tool for biochemical analysis of specific genomic regions of interest.
    Scientific Reports 11/2013; 3:3171. DOI:10.1038/srep03171 · 5.58 Impact Factor
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    • "A more comprehensive and unbiased approach would be the biochemical isolation of a specific native genomic locus for proteomic identification of proteins-associated and histone PTMs. Similar approaches have been performed for large structures like telomeres, engineered plasmids or engineered loci (8–13); however, the proteomic analysis of a small native genomic region without genomic engineering for specifically associated proteins and histone PTMs has yet to be demonstrated. To work toward proteomic studies of native chromatin regions (i.e. "
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    ABSTRACT: Here, we describe an approach to isolate native chromatin sections without genomic engineering for label-free proteomic identification of associated proteins and histone post-translational modifications. A transcription activator-like (TAL) protein A fusion protein was designed to recognize a unique site in the yeast GAL1 promoter. The TAL-PrA fusion enabled chromatin affinity purification (ChAP) of a small section of native chromatin upstream from the GAL1 locus, permitting mass spectrometric (MS) identification of proteins and histone post-translational modifications regulating galactose-induced transcription. This TAL-ChAP-MS approach allows the biochemical isolation of a specific native genomic locus for proteomic studies and will provide for unprecedented objective insight into protein and epigenetic mechanisms regulating site-specific chromosome metabolism.
    Nucleic Acids Research 09/2013; 41(20). DOI:10.1093/nar/gkt822 · 9.11 Impact Factor
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