Abstract
Comprehensive understanding of genome functions requires identification of molecules (proteins, RNAs, genomic regions, etc.) bound to specific genomic regions of interest in vivo. To perform biochemical and molecular biological analysis of specific genomic regions, we developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) to purify genomic regions of interest. In enChIP, specific genomic regions are tagged for biochemical purification using engineered DNA-binding molecules, such as transcription activator-like (TAL) proteins and a catalytically inactive form of the clustered regularly interspaced short palindromic repeats (CRISPR) system. enChIP is a comprehensive approach that emphasizes non-biased search using next-generation sequencing (NGS), microarrays, mass spectrometry (MS), and other methods. Moreover, this approach is not restricted to cultured cell lines and can be easily extended to organisms. In this review, we discuss applications of enChIP to elucidating the molecular mechanisms underlying genome functions.
Highlights
Genome functions, such as transcription and epigenetic regulation, play essential roles in biological activities
We developed the insertional chromatin immunoprecipitation technology [2,3,4,5,6,7]
(viii) Identification of proteins associated with the isolated locus using mass spectrometry, RNAs using RNA sequencing, or interacting genomic regions using next-generation sequencing (NGS)
Summary
Genome functions, such as transcription and epigenetic regulation, play essential roles in biological activities. These approaches often neglect the physiological localization of these molecules, leading to detection of interactions that do not occur in the nucleus In this regard, for the purpose of biochemical identification of chromatin-associated molecules, it would be straightforward to purify specific genomic regions that retain their molecular interactions. For the purpose of biochemical identification of chromatin-associated molecules, it would be straightforward to purify specific genomic regions that retain their molecular interactions To this end, we developed the insertional chromatin immunoprecipitation (iChIP) technology [2,3,4,5,6,7]. We developed “in vitro iChIP” [6], in which fragmented chromatin from cells harboring a recognition sequence is incubated with a synthetic or purified form of the cognate DNA-binding molecule, e.g., the recombinant LexA protein, and the target genomic region is isolated by affinity purification. The Principle and Applications of Engineered DNA-Binding Molecule-Mediated Chromatin
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