High-resolution mapping of in vivo genomic transcription factor binding sites using in situ DNase I footprinting and ChIP-seq

Chumsakul, Onuma and Nakamura, Kensuke and Kurata, Tetsuya and Hobman, Jon L. and Ogasawara, Naotake and Oshima, Taku and Ishikawa, Shu (2013) High-resolution mapping of in vivo genomic transcription factor binding sites using in situ DNase I footprinting and ChIP-seq. DNA Research, 20 (4). pp. 325-338. ISSN 1756-1663

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Accurate identification of the DNA-binding sites of transcription factors and other DNA-binding proteins on the genome is crucial to understanding their molecular interactions with DNA. Here, we describe a new method: Genome Footprinting by high-throughput sequencing (GeF-seq), which combines in vivo DNase I digestion of genomic DNA with ChIP coupled with high-throughput sequencing. We have determined the in vivo binding sites of a Bacillus subtilis global regulator, AbrB, using GeF-seq. This method shows that exact DNA-binding sequences, which were protected from in vivo DNase I digestion, were resolved at a comparable resolution to that achieved by in vitro DNase I footprinting, and this was simply attained without the necessity of prediction by peak-calling programs. Moreover, DNase I digestion of the bacterial nucleoid resolved the closely positioned AbrB-binding sites, which had previously appeared as one peak in ChAP-chip and ChAP-seq experiments. The high-resolution determination of AbrB-binding sites using GeF-seq enabled us to identify bipartite TGGNA motifs in 96% of the AbrB-binding sites. Interestingly, in a thousand binding sites with very low-binding intensities, single TGGNA motifs were also identified. Thus, GeF-seq is a powerful method to elucidate the molecular mechanism of target protein binding to its cognate DNA sequences.

Item Type: Article
Keywords: GeF-seq; ChIP-seq; AbrB; Bacillus subtilis
Schools/Departments: University of Nottingham UK Campus > Faculty of Science > School of Biosciences > Division of Food Sciences
Identification Number: https://doi.org/10.1093/dnares/dst013
Depositing User: Hobman, Jon
Date Deposited: 11 Feb 2016 08:45
Last Modified: 14 Sep 2016 19:43
URI: http://eprints.nottingham.ac.uk/id/eprint/31617

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