Article

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

ABSTRACT

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
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    • "To elucidate the molecular mechanisms underlying genome functions such as transcription and epigenetic regulation, it is necessary to identify molecules interacting with genome regions of interest in vivo. To this end, we developed locus-specific chromatin immunoprecipitation (locus-specific ChIP) technologies , consisting of insertional ChIP (iChIP) and engineered DNA-binding molecule-mediated ChIP (enChIP) (Hoshino & Fujii 2009; Fujita & Fujii 2011, 2013a,b, 2014a,b, 2015b ). In enChIP, an engineered "
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    ABSTRACT: The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for various biological applications, including genome editing. We developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR to isolate target genomic regions from cells for their biochemical characterization. In this study, we developed 'in vitro enChIP' using recombinant CRISPR ribonucleoproteins (RNPs) to isolate target genomic regions. in vitro enChIP has the great advantage over conventional enChIP of not requiring expression of CRISPR complexes in cells. We first showed that in vitro enChIP using recombinant CRISPR RNPs can be used to isolate target DNA from mixtures of purified DNA in a sequence-specific manner. In addition, we showed that this technology can be used to efficiently isolate target genomic regions, while retaining their intracellular molecular interactions, with negligible contamination from irrelevant genomic regions. Thus, in vitro enChIP technology is of potential use for sequence-specific isolation of DNA, as well as for identification of molecules interacting with genomic regions of interest in vivo in combination with downstream analysis.
    Full-text · Article · Feb 2016 · Genes to Cells
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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.
    Full-text · Article · Jul 2014 · PLoS ONE
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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.
    Full-text · Article · Nov 2013 · Scientific Reports
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