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Native Accessible ChIP: A Novel Approach to Interrogate Protein‐DNA Interactions in Accessible Chromatin
Author(s) -
Wang Yan,
Meng Xiangdong,
Kong Michelle,
Okino Steven T
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.923.4
Subject(s) - chromatin immunoprecipitation , chromatin , dna , chip on chip , histone , promoter , chip sequencing , microbiology and biotechnology , nuclease , chemistry , biology , chromatin remodeling , genetics , gene , gene expression
We have developed a novel technology for isolating accessible chromatin. When cells are permeabilized and treated with nuclease in situ, small DNA fragments, corresponding to accessible chromatin can be easily isolated and characterized. We now report that the isolated accessible chromatin is still associated with its cognate DNA‐binding proteins and histones. Based on this, we developed a novel chromatin immunoprecipitation (ChIP) procedure that does not require protein‐DNA cross‐linking or DNA shearing. Using this method, termed NA‐ChIP (for native accessible ChIP), we demonstrate that RNA polymerase II (RNAPII), TBP and H3K4me3 are all associated with active promoters, but are rarely detected at silenced promoters. Comparison of NA‐ChIP and a standard ChIP assay shows that immunoprecipitation (IP) efficiency is similar, but IP specificity is dramatically different. To measure IP specificity we compared the ratio of active promoter DNA precipitated with RNAPII (signal) relative to inactive promoter DNA precipitated (noise). We find that NA‐ChIP is highly specific with a signal‐to‐noise ratio >500; standard ChIP is less specific with a signal‐to‐noise ratio of 9. We conclude that NA‐ChIP offers significant advantages over standard ChIP, including dramatically reduced background and an easier workflow.