Title:
Locus-specific biochemical epigenetics / chromatin biochemistry by insertional chromatin immunoprecipitation (iChIP)
Hodaka Fujii, M.D., Ph.D.
Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University
Abstract:
Comprehensive understanding of mechanisms of genome functions such as transcription and epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo . To perform biochemical and molecular biological analysis of specific genomic regions, we developed the insertional chromatin immunoprecipitation (iChIP) technology to purify the genomic regions of interest. Specific genomic regions tagged with the recognition sequences of an exogenous DNA-binding protein such as LexA are subjected to affinity purification in iChIP. iChIP is a comprehensive approach to purify specific genomic regions of interest to identify interacting molecules including genomic DNA, proteins, RNA, and others, with an emphasis on non-biased search using next-generation sequencing (NGS), microarrays, mass spectrometry (MS), and other methods. In addition, this approach is not restricted to cultured cell lines but easily extended to organisms in vivo .
We applied iChIP to direct identification of components of insulator complexes, which function as boundaries of chromatin domain. We combined iChIP with MS and RT-PCR (iChIP-MS and iChIP-RT-PCR) to find that the chicken beta-globin HS4 (cHS4) insulator complex contains an RNA helicase protein, p68/DDX5; an RNA species, steroid receptor RNA activator 1; and a nuclear matrix protein, Matrin-3, in vivo . Binding of p68 and Matrin-3 to the cHS4 insulator core sequence was mediated by CCCTC-binding factor (CTCF). Thus, our results showed that it is feasible to directly identify proteins and RNA bound to a specific genomic region in vivo by using iChIP.
In addition, we applied iChIP combined with SILAC (iChIP-SILAC) to direct identification of proteins interacting with the promoter region of the single-copy chicken Pax5 gene, which is the master transcription factor in B cell development. By comparing B cells with a macrophage-like cells trans-differentiated by ectopic expression of C/EBP b , iChIP-SILAC identified proteins interacting with the Pax5 promoter in a B cell-specific manner. Loss-of-function of the identified proteins induced decrease in Pax5 expression. Thus, our analysis revealed that the identified proteins are functionally required for B cell-specific expression of Pax5. Furthermore, these results showed that iChIP-SILAC would be a useful tool for identification of proteins interacting with even a single-copy locus in cells of multicellular higher eukaryotes in vivo . In addition, we used iChIP-Seq to identify genome-wide intra- and intergenomic interactions with the Pax5promoter. The significance of these interactions in Pax5 expression will be discussed.
References:
1 Hoshino A, and Fujii H : Insertional chromatin immunoprecipitation: a method for isolating specific genomic regions. J. Biosci. Bioeng. (2009) 108, 446-449.
2 Fujita T, and Fujii H : Direct identification of insulator components by insertional chromatin immunoprecipitation. PLoS One (2011) 6, e26109.
3 Fujita T, and Fujii H : Efficient isolation of specific genomic regions by insertional chromatin immunoprecipitation (iChIP) with a second-generation tagged LexA DNA-binding domain. Adv. Biosci. Biotechnol. (2012) 3, 626-629.
4 Fujita T, Fujii H : Locus-specific biochemical epigenetics / chromatin biochemistry by insertional chromatin immunoprecipitation. ISRN Biochem. (2013) 2013, Article ID 913273.
5 Fujii H , Fujita. US patent 8415098.
