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

Chumsakul, Onuma; Nakamura, Kensuke; Kurata, Tetsuya; Hobman, Jon L.; Ogasawara, Naotake; Oshima, Taku; Ishikawa, Shu

Authors

Onuma Chumsakul

Kensuke Nakamura

Tetsuya Kurata

Jon L. Hobman

Naotake Ogasawara

Taku Oshima

Shu Ishikawa



Abstract

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.

Journal Article Type Article
Publication Date Apr 11, 2013
Journal DNA Research
Print ISSN 1340-2838
Electronic ISSN 1756-1663
Publisher Oxford University Press (OUP)
Peer Reviewed Peer Reviewed
Volume 20
Issue 4
Institution Citation Chumsakul, O., Nakamura, K., Kurata, T., Hobman, J. L., Ogasawara, N., Oshima, T., & Ishikawa, S. (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), doi:10.1093/dnares/dst013
DOI https://doi.org/10.1093/dnares/dst013
Keywords GeF-seq; ChIP-seq; AbrB; Bacillus subtilis
Publisher URL http://dnaresearch.oxfordjournals.org/content/20/4/325
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by-nc/4.0

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Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by-nc/4.0





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