High-resolution mapping of in vivo genomic transcription factor binding sites using in situ DNase I footprinting and ChIP-seq.
High-resolution mapping of in vivo genomic transcription factor binding sites using in situ DNase I footprinting and ChIP-seq.
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DOI：
10.1093/dnares/dst013
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发表时间：
2013-08
期刊：
影响因子：
--
通讯作者：
Ishikawa S
中科院分区：
文献类型：
--
作者：
Chumsakul O;Nakamura K;Kurata T;Sakamoto T;Hobman JL;Ogasawara N;Oshima T;Ishikawa S
准确鉴定基因组上转录因子和其他DNA结合蛋白的DNA结合位点对于理解它们与DNA的分子相互作用至关重要。在这里，我们描述了一种新的方法：基因组足迹通过高通量测序（GeF-seq），它结合了体内DNA酶I消化基因组DNA与ChIP加上高通量测序。我们已经使用GeF-seq确定了枯草芽孢杆菌全局调节剂AbrB的体内结合位点。这种方法表明，确切的DNA结合序列，这是保护从体内DNA酶I消化，解决了在一个相当的分辨率，实现了体外DNA酶I足迹，这是简单地实现了没有必要的预测峰调用程序。此外，DNA酶I消化细菌类核解析了紧密定位的AbrB结合位点，其先前在ChAP芯片和ChAP-seq实验中作为一个峰出现。使用GeF-seq的AbrB结合位点的高分辨率测定使我们能够在96%的AbrB结合位点中鉴定出二分TGGNA基序。有趣的是，在具有非常低的结合强度的一千个结合位点中，也鉴定出单个TGGNA基序。因此，GeF-seq是阐明靶蛋白与其同源DNA序列结合的分子机制的有力方法。
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.
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