Cargando…
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...
Autores principales: | , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2013
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3738160/ https://www.ncbi.nlm.nih.gov/pubmed/23580539 http://dx.doi.org/10.1093/dnares/dst013 |
_version_ | 1782476815444475904 |
---|---|
author | Chumsakul, Onuma Nakamura, Kensuke Kurata, Tetsuya Sakamoto, Tomoaki Hobman, Jon L. Ogasawara, Naotake Oshima, Taku Ishikawa, Shu |
author_facet | Chumsakul, Onuma Nakamura, Kensuke Kurata, Tetsuya Sakamoto, Tomoaki Hobman, Jon L. Ogasawara, Naotake Oshima, Taku Ishikawa, Shu |
author_sort | Chumsakul, Onuma |
collection | PubMed |
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. |
format | Online Article Text |
id | pubmed-3738160 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-37381602013-08-08 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 Sakamoto, Tomoaki Hobman, Jon L. Ogasawara, Naotake Oshima, Taku Ishikawa, Shu DNA Res Full Papers 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 2013-08 2013-04-11 /pmc/articles/PMC3738160/ /pubmed/23580539 http://dx.doi.org/10.1093/dnares/dst013 Text en © The Author 2013. Published by Oxford University Press on behalf of Kazusa DNA Research Institute http://creativecommons.org/licenses/by-nc/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com. |
spellingShingle | Full Papers Chumsakul, Onuma Nakamura, Kensuke Kurata, Tetsuya Sakamoto, Tomoaki 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 | Full Papers |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3738160/ https://www.ncbi.nlm.nih.gov/pubmed/23580539 http://dx.doi.org/10.1093/dnares/dst013 |
work_keys_str_mv | AT chumsakulonuma highresolutionmappingofinvivogenomictranscriptionfactorbindingsitesusinginsitudnaseifootprintingandchipseq AT nakamurakensuke highresolutionmappingofinvivogenomictranscriptionfactorbindingsitesusinginsitudnaseifootprintingandchipseq AT kuratatetsuya highresolutionmappingofinvivogenomictranscriptionfactorbindingsitesusinginsitudnaseifootprintingandchipseq AT sakamototomoaki highresolutionmappingofinvivogenomictranscriptionfactorbindingsitesusinginsitudnaseifootprintingandchipseq AT hobmanjonl highresolutionmappingofinvivogenomictranscriptionfactorbindingsitesusinginsitudnaseifootprintingandchipseq AT ogasawaranaotake highresolutionmappingofinvivogenomictranscriptionfactorbindingsitesusinginsitudnaseifootprintingandchipseq AT oshimataku highresolutionmappingofinvivogenomictranscriptionfactorbindingsitesusinginsitudnaseifootprintingandchipseq AT ishikawashu highresolutionmappingofinvivogenomictranscriptionfactorbindingsitesusinginsitudnaseifootprintingandchipseq |