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.
Highlights
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 [1,2,3,4,5] and engineered DNAbinding molecule-mediated ChIP [6,7] for purification of specific genomic regions to identify their associated molecules
To generate cells stably expressing the components of engineered DNAbinding molecule-mediated ChIP (enChIP) using clustered regularly interspaced short palindromic repeats (CRISPR) more and quickly, we developed a retroviral system to express 3xFLAG-dCas9 (dCas9 tagged with the 3xFLAG tag and fused with a nuclear localization signal (NLS)) [6] and guide RNA (gRNA)
Summary
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 showed that the clustered regularly interspaced short palindromic repeats (CRISPR) system [8,9,10,11,12,13,14,15,16,17,18,19,20,21] combined with enChIP efficiently isolates specific genomic regions for identification of their associated proteins [6] In this form of enChIP, specific genomic regions are immunoprecipitated with an Ab against a tag(s), which is fused to a catalytically inactive form of Cas (dCas9) plus guide RNA (gRNA) interacting with an endogenous DNA sequence in the genomic regions (Figure S1). Stable expression of the CRISPR components would be necessary
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