Identification of Proteins Associated with an IFN c-Responsive Promoter by a Retroviral Expression System for enChIP Using CRISPR

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 IFNc stimulation. The list of the associated proteins contained many novel proteins in the context of IFNc-induced gene expression as well as proteins related to histone deacetylase complexes whose involvement has been suggested in IFNc-mediated gene expression. Finally, we confirmed IFNc-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. Citation: Fujita T, Fujii H (2014) Identification of Proteins Associated with an IFNc-Responsive Promoter by a Retroviral Expression System for enChIP Using CRISPR. PLoS ONE 9(7): e103084. doi:10.1371/journal.pone. 0103084 Editor: Luwen Zhang, University of Nebraska Lincoln, United States of America Received April 13, 2014; Accepted June 27, 2014; Published July 22, 2014 Copyright: 2014 Fujita, Fujii. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files Funding: This work was supported by Takeda Science Foundation (http://www.takeda-sci.or.jp) (T.F.), the Uehara Memorial Foundation (http://www. ueharazaidan.or.jp) (H.F.), the Kurata Memorial Hitachi Science and Technology Foundation (http://www.hitachi-zaidan.org/global/kurata/) (T.F. and H.F.), Adaptable & Seamless Technology Transfer Program through Target-driven R&D (A-STEP) by the Japan Science and Technology Agency (JST) (http://www.jst.go. jp/tt/EN/univ-ip/a-step.html) (#AS251Z01861Q) (H.F.), Grant-in-Aid for Young Scientists (B) (#25830131) (T.F.), Grant-in-Aid for Scientific Research on Innovative Areas "Cell Fate" (#23118516) (T.F.), "Transcription Cycle" (#25118512) (H.F.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (http://www.mext.go.jp/english/). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Competing Interests: The authors filed a patent application on enChIP. Patent name: "Method for isolating specific genomic regions using DNA-binding molecules recognizing endogenous DNA sequences" Number: PCT/JP2013/74107. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * Email: hodaka@biken.osaka-u.ac. jp