Identification of proteins associated with an IFNγ-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 purificatio...

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Autores principales:	Toshitsugu Fujita, Hodaka Fujii
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/814c1abda91041eb9963c81cf82b7a25
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spelling	oai:doaj.org-article:814c1abda91041eb9963c81cf82b7a252021-11-25T06:07:32ZIdentification of proteins associated with an IFNγ-responsive promoter by a retroviral expression system for enChIP using CRISPR.1932-620310.1371/journal.pone.0103084https://doaj.org/article/814c1abda91041eb9963c81cf82b7a252014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/25051498/?tool=EBIhttps://doaj.org/toc/1932-6203Isolation 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.Toshitsugu FujitaHodaka FujiiPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 7, p e103084 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Toshitsugu Fujita Hodaka Fujii Identification of proteins associated with an IFNγ-responsive promoter by a retroviral expression system for enChIP using CRISPR.
description	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.
format	article
author	Toshitsugu Fujita Hodaka Fujii
author_facet	Toshitsugu Fujita Hodaka Fujii
author_sort	Toshitsugu Fujita
title	Identification of proteins associated with an IFNγ-responsive promoter by a retroviral expression system for enChIP using CRISPR.
title_short	Identification of proteins associated with an IFNγ-responsive promoter by a retroviral expression system for enChIP using CRISPR.
title_full	Identification of proteins associated with an IFNγ-responsive promoter by a retroviral expression system for enChIP using CRISPR.
title_fullStr	Identification of proteins associated with an IFNγ-responsive promoter by a retroviral expression system for enChIP using CRISPR.
title_full_unstemmed	Identification of proteins associated with an IFNγ-responsive promoter by a retroviral expression system for enChIP using CRISPR.
title_sort	identification of proteins associated with an ifnγ-responsive promoter by a retroviral expression system for enchip using crispr.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/814c1abda91041eb9963c81cf82b7a25
work_keys_str_mv	AT toshitsugufujita identificationofproteinsassociatedwithanifngresponsivepromoterbyaretroviralexpressionsystemforenchipusingcrispr AT hodakafujii identificationofproteinsassociatedwithanifngresponsivepromoterbyaretroviralexpressionsystemforenchipusingcrispr
_version_	1718414172917596160

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

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