Abstract
Comprehensive understanding of mechanisms of genome functions requires identification of molecules interacting with genomic regions of interest in vivo. We previously developed the insertional chromatin immunoprecipitation (iChIP) technology to isolate specific genomic regions retaining molecular interactions and identify their associated molecules. iChIP consists of locus-tagging and affinity purification. The recognition sequences of an exogenous DNA-binding protein such as LexA are inserted into a genomic region of interest in the cell to be analyzed. The exogenous DNA-binding protein fused with a tag(s) is expressed in the cell and the target genomic region is purified with antibody against the tag(s). In this study, we developed the iChIP system using recombinant DNA-binding proteins to make iChIP more straightforward than the conventional iChIP system using expression of the exogenous DNA-binding proteins in the cells to be analyzed. In this system, recombinant 3xFNLDD-D (r3xFNLDD-D) consisting of the 3xFLAG-tag, a nuclear localization signal (NLS), the DNA-binding domain plus the dimerization domain of the LexA protein, and the Dock-tag is used for isolation of specific genomic regions. r3xFNLDD-D was expressed using a silkworm-baculovirus expression system and purified by affinity purification. iChIP using r3xFNLDD-D could efficiently isolate the single-copy chicken Pax5 (cPax5) locus, in which LexA binding elements were inserted, with negligible contamination of other genomic regions. In addition, we could detect RNA associated with the cPax5 locus using this form of the iChIP system combined with RT-PCR. The iChIP system using r3xFNLDD-D can isolate specific genomic regions retaining molecular interactions without expression of the exogenous DNA-binding protein in the cell to be analyzed. iChIP using r3xFNLDD-D would be more straightforward and useful for analysis of specific genomic regions to elucidate their functions as compared to the previously published iChIP protocol.
Highlights
Comprehensive understanding of mechanisms of genome functions requires identification of molecules interacting with genomic regions of interest in vivo
We successfully identified proteins and RNA components of an insulator, which functions as boundaries of chromatin domains [9], by using insertional chromatin immunoprecipitation (iChIP) combined with mass spectrometry or RT-PCR [3]. iChIP has been used for identification of proteins or DNA interacting with specific genomic regions by other researchers [10,11,12,13]
We could detect RNA associated with the chicken Pax5 (cPax5) locus using this form of the iChIP system combined with RT-PCR
Summary
Comprehensive understanding of mechanisms of genome functions requires identification of molecules interacting with genomic regions of interest in vivo. We previously developed the insertional chromatin immunoprecipitation (iChIP) technology to isolate specific genomic regions retaining molecular interactions and identify their associated molecules. The recognition sequences of an exogenous DNA-binding protein such as LexA are inserted into a genomic region of interest in the cell to be analyzed. IChIP is based on locus-tagging by inserting recognition sequences of an exogenous DNA-binding protein to isolate specific genomic regions using the exogenous DNA-binding molecule. The scheme of iChIP is as follows: (i) The recognition sequences of an exogenous DNA-binding protein such as a bacterial protein, LexA, are inserted into the genomic region of interest in the cell to be analyzed. (vi) The isolated complexes which retain molecular interactions are reverse crosslinked, if necessary, and subsequent purification of DNA, RNA, proteins, or other molecules allows their identification and characterization. IChIP is a useful technology for elucidation of molecular mechanisms of genome functions
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