Efficient isolation of specific genomic regions by insertional chromatin immunoprecipitation (iChIP) with a second-generation tagged LexA DNA-binding domain

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 in vivo molecular interaction in order to perform non-biased identification of interacting molecules. Here, we developed a second-generation tagged LexA DNA-binding domain, 3×FNLDD, for the iChIP analysis. 3×FNLDD consists of 3 × FLAG tags, a nuclear localization signal (NLS), the DNA-binding domain (DB) and the dimerization domain of the LexA protein. Expression of 3×FNLDD can be detected by immunoblot analysis as well as flowcytometry. We showed that iChIP using 3×FNLDD is able to consistently isolate more than 10% of input genomic DNA, several-fold more efficient compared to the first-generation tagged LexA DB. 3×FNLDD would be a useful tool to perform the iChIP analysis for locus-specific biochemical epigenetics.