Papers

Peer-reviewed Last author Corresponding author
Jul, 2014

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

PLOS ONE
  • Toshitsugu Fujita
  • ,
  • Hodaka Fujii

Volume
9
Number
7
Language
English
Publishing type
Research paper (scientific journal)
DOI
10.1371/journal.pone.0103084
Publisher
PUBLIC LIBRARY SCIENCE

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 IFN gamma stimulation. The list of the associated proteins contained many novel proteins in the context of IFN gamma-induced gene expression as well as proteins related to histone deacetylase complexes whose involvement has been suggested in IFN gamma-mediated gene expression. Finally, we confirmed IFN gamma-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.

Link information
DOI
https://doi.org/10.1371/journal.pone.0103084
PubMed
https://www.ncbi.nlm.nih.gov/pubmed/25051498
PubMed Central
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106880
Web of Science
https://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=JSTA_CEL&SrcApp=J_Gate_JST&DestLinkType=FullRecord&KeyUT=WOS:000339551100086&DestApp=WOS_CPL
URL
http://europepmc.org/abstract/med/25051498
URL
http://orcid.org/0000-0003-1296-4256
ID information
  • DOI : 10.1371/journal.pone.0103084
  • ISSN : 1932-6203
  • ORCID - Put Code : 49884989
  • Pubmed ID : 25051498
  • Pubmed Central ID : PMC4106880
  • Web of Science ID : WOS:000339551100086

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