High-resolution mapping of in vivo genomic transcription factor binding sites using in situ DNase I footprinting and ChIP-seq
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 viv...
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| Format: | Article |
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Oxford University Press
2013
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| Online Access: | https://eprints.nottingham.ac.uk/31617/ |
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| author | Chumsakul, Onuma Nakamura, Kensuke Kurata, Tetsuya Hobman, Jon L. Ogasawara, Naotake Oshima, Taku Ishikawa, Shu |
| author_facet | Chumsakul, Onuma Nakamura, Kensuke Kurata, Tetsuya Hobman, Jon L. Ogasawara, Naotake Oshima, Taku Ishikawa, Shu |
| author_sort | Chumsakul, Onuma |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | 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. |
| first_indexed | 2025-11-14T19:13:00Z |
| format | Article |
| id | nottingham-31617 |
| institution | University of Nottingham Malaysia Campus |
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| last_indexed | 2025-11-14T19:13:00Z |
| publishDate | 2013 |
| publisher | Oxford University Press |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-316172020-05-04T16:36:28Z https://eprints.nottingham.ac.uk/31617/ 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 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. Oxford University Press 2013-04-11 Article PeerReviewed Chumsakul, Onuma, Nakamura, Kensuke, Kurata, Tetsuya, Hobman, Jon L., Ogasawara, Naotake, 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 GeF-seq; ChIP-seq; AbrB; Bacillus subtilis http://dnaresearch.oxfordjournals.org/content/20/4/325 doi:10.1093/dnares/dst013 doi:10.1093/dnares/dst013 |
| spellingShingle | GeF-seq; ChIP-seq; AbrB; Bacillus subtilis Chumsakul, Onuma Nakamura, Kensuke Kurata, Tetsuya Hobman, Jon L. Ogasawara, Naotake Oshima, Taku Ishikawa, Shu High-resolution mapping of in vivo genomic transcription factor binding sites using in situ DNase I footprinting and ChIP-seq |
| title | High-resolution mapping of in vivo genomic transcription factor binding sites using in situ DNase I footprinting and ChIP-seq |
| title_full | High-resolution mapping of in vivo genomic transcription factor binding sites using in situ DNase I footprinting and ChIP-seq |
| title_fullStr | High-resolution mapping of in vivo genomic transcription factor binding sites using in situ DNase I footprinting and ChIP-seq |
| title_full_unstemmed | High-resolution mapping of in vivo genomic transcription factor binding sites using in situ DNase I footprinting and ChIP-seq |
| title_short | High-resolution mapping of in vivo genomic transcription factor binding sites using in situ DNase I footprinting and ChIP-seq |
| title_sort | high-resolution mapping of in vivo genomic transcription factor binding sites using in situ dnase i footprinting and chip-seq |
| topic | GeF-seq; ChIP-seq; AbrB; Bacillus subtilis |
| url | https://eprints.nottingham.ac.uk/31617/ https://eprints.nottingham.ac.uk/31617/ https://eprints.nottingham.ac.uk/31617/ |