Locus-specific biochemical epigenetics/chromatin biochemistry by insertional chromatin immunoprecipitation.

Toshitsugu Fujita,Hodaka Fujii

doi:10.1155/2013/913273

Abstract

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.

Highlights

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]
These analyses have been performed by using arti cial methods such as reporter assay [9] and in silico identi cation of genomic regions conserved among species [10]
It has been shown that such regulatory regions have physical contact with the regulated loci, forming a loop [13, 14]. is led to the idea of identi cation of regulatory genomic regions by detecting genomic regions interacting with the genomic region of interest. us, development of methods to identify intra- and interchromosomal interaction is vital for the advancement of the eld

Summary

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]. Long-range interaction including interchromosomal interaction has been suggested to play important roles in regulation of gene expression and other biological phenomena [12] In this regard, it has been shown that such regulatory regions have physical contact with the regulated loci, forming a loop [13, 14]. Molecules interacting with a speci c genomic region have been identi ed using arti cial approaches including a nity puri cation, yeast one-hybrid, ISRN Biochemistry electrophoretic mobility shi assay (EMSA), and others [15]. These approaches are successful in some cases, especially for the analyses of easier targets such as immediate early responses, they can be very problematic. We will discuss insertional chromatin immunoprecipitation (iChIP) we developed for the locus-speci c biochemical epigenetics/chromatin biochemistry and its application

Methods to Analyze Molecular Interaction

Application of iChIP

Findings

Future Application of iChIP