Identification of proteins associated with an IFNγ-responsive promoter by a retroviral expression system for enChIP using CRISPR

PLoS One. 2014 Jul 22;9(7):e103084. doi: 10.1371/journal.pone.0103084. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Cell Line
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Epigenesis, Genetic
  • Humans
  • Interferon Regulatory Factor-1 / genetics*
  • Interferon-gamma / metabolism*
  • Promoter Regions, Genetic*

Substances

  • Interferon Regulatory Factor-1
  • Interferon-gamma

Grants and 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.