Comprehensive understanding of regulation mechanisms of biological phenomena mediated by functions of genomic DNA requires identification of molecules bound to genomic regions of interest in vivo. However, nonbiased methods to identify molecules bound to specific genomic loci in vivo are limited. 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. We applied iChIP to direct identification of components of insulator complexes, which function as boundaries of chromatin domain, showing that it is feasible to directly identify proteins and RNA bound to a specific genomic region in vivo by using iChIP. In addition, recently, we succeeded in identifying proteins and genomic regions interacting with a single copy endogenous locus. In this paper, we will discuss the application of iChIP to epigenetics and chromatin research. 1. Introduction Detailed biochemical and molecular biological analysis of chromatin domains is critical for understanding mechanisms of genetic and epigenetic regulation of gene expression, hetero- and euchromatinization, X-chromosome inactivation, genomic imprinting, and other important biological phenomena [1]. However, biochemical nature of chromatin domains is poorly understood. This is mainly because methods for performing biochemical and molecular biological analysis of chromatin structure are limited [2–8]. Identification of regulatory regions of gene expression has been extensively attempted in the last several decades. Conventionally, these analyses have been performed by using artificial methods such as reporter assay [9] and in silico identification of genomic regions conserved among species [10]. More recently, enhancer-specific modifications are being used to identify enhancer regions in the genome (see review [11]). However, although these approaches have been successful for relatively easy targets such as immediate early genes, it has been shown that they could produce artifactual results in many circumstances. In fact, deletion studies of candidate regulatory endogenous genomic regions have shown that the candidate regions identified by using these conventional methods could often be dispensable for expression of the genes of interest. Furthermore, these approaches cannot be used when regulatory genomic regions are far from regulated loci, for example, on other chromosomes. In fact, long-range interaction including interchromosomal interaction has been suggested to play
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