ABSTRACT Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. We have developed a novel method, insertional chromatin immunoprecipitatin (iChIP), to isolate specific genomic regions retaining molecular interaction in order to perform non-biased identification of interacting molecules in vivo. Here, we developed a second-generation tagged LexA DNA-binding domain, 3xFNLDD, for the iChIP analysis. 3xFNLDD consists of 3 x FLAG tags, a nuclear localization signal (NLS), the DNA-binding domain (DB) and the dimerization domain of the LexA protein. Expression of 3xFNLDD can be detected by immunoblot analysis as well as flowcytometry. We showed that iChIP using 3xFNLDD is able to consistently isolate more than 10% of input genomic DNA, several-fold more efficient compared to the first-generation tagged LexA DB. 3xFNLDD would be a useful tool to perform the iChIP analysis for locus-specific biochemical epigenetics.
Cite this paper
Fujita, T. and Fujii, H. (2012) Efficient isolation of specific genomic regions by insertional chromatin immunoprecipitation (iChIP) with a second-generation tagged LexA DNA-binding domain. Advances in Bioscience and Biotechnology, 3, 626-629. doi: 10.4236/abb.2012.35081.
 Van Driel, R., Fransz, P.F., and Verschure, P.J. (2003) The eukaryotic genome: a system regulated at different hierarchical levels. Journal of Cell Science, 116: 4067-4075.
 Hoshino, A. and Fujii, H. (2009) Insertional chromatin immunoprecipitation: a method for isolating specific genomic regions. Journal of Bioscience and Bioengineering, 108: 446-449.
 Fujita, T. and Fujii, H. (2011) Direct idenification of insulator components by insertional chromatin immunoprecipitation. PLoS One, 6: e26109.
 Bartel, P., Chien, C.T., Sternglanz, R., and Fields, S. (1993) Elimination of false positives that arise in using the two-hybrid system. BioTechniques, 14: 920-924.
 Nosaka, T., Kawashima, T., Misawa, K., Ikuta, K., Mui, A.L., and Kitamura, T. (1999) STAT5 as a molecular regulator of proliferation, differentiation and apoptosis in hematopoietic cells. EMBO Journal, 18: 4754-4765.
 Hoshino, A., Matsumura, S., Kondo, K., Hirst, J.A., and Fujii, H. (2004) Inducible translocation trap: a system for detecting inducible nuclear translocation. Molecular Cell, 15: 153-159.
 Palacios, R. and Steinmetz, M. (1985) IL3-dependent mouse clones that express B-220 surface-antigen, contain Ig genes in germ-line configuration, and generate lymphocytes-B in vivo. Cell, 41: 727-734.