title: High-resolution mapping of in vivo genomic transcription factor binding sites using in situ DNase I footprinting and ChIP-seq.
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.
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, Japan.
authors.forename: Onuma
authors.initials: O
authors.lastname: Chumsakul
authors.forename: Kensuke
authors.initials: K
authors.lastname: Nakamura
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Kurata
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Sakamoto
authors.forename: Jon L
authors.initials: JL
authors.lastname: Hobman
authors.forename: Naotake
authors.initials: N
authors.lastname: Ogasawara
authors.forename: Taku
authors.initials: T
authors.lastname: Oshima
authors.forename: Shu
authors.initials: S
authors.lastname: Ishikawa
mesh_terms: D001412:Bacillus subtilis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D047369:Chromatin Immunoprecipitation; D017124:Conserved Sequence; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing; D008969:Molecular Sequence Data; D059372:Nucleotide Motifs; D011485:Protein Binding; D017422:Sequence Analysis, DNA / Q000379:methods*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C060762:AbrB protein, Bacillus subtilis; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D003850:Deoxyribonuclease I
keywords: AbrB; Bacillus subtilis; ChIP-seq; GeF-seq