1] Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. [2] Medical Scientist Training Program, University of Washington School of Medicine, Seattle, Washington, USA. [3] Molecular & Cellular Biology Graduate Program, University of Washington, Seattle, Washington, USA.
1] Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA. [2] Centre National de la Recherche Scientifique UMR 218 and Institut Curie, Centre de Recherche, Paris, France.
Nat Methods. 2014 Feb;11(2):203-9. doi: 10.1038/nmeth.2766. Epub 2013 Dec 15.
Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. TF profiling is commonly carried out by formaldehyde cross-linking and sonication followed by chromatin immunoprecipitation (X-ChIP). We describe a method to profile TF binding at high resolution without cross-linking. We begin with micrococcal nuclease-digested non-cross-linked chromatin and then perform affinity purification of TFs and paired-end sequencing. The resulting occupied regions of genomes from affinity-purified naturally isolated chromatin (ORGANIC) profiles of Saccharomyces cerevisiae Abf1 and Reb1 provide high-resolution maps that are accurate, as defined by the presence of known TF consensus motifs in identified binding sites, that are not biased toward accessible chromatin and that do not require input normalization. We profiled Drosophila melanogaster GAGA factor and Pipsqueak to test ORGANIC performance on larger genomes. Our results suggest that ORGANIC profiling is a widely applicable high-resolution method for sensitive and specific profiling of direct protein-DNA interactions.
包括转录因子（TFs）在内的序列特异性 DNA 结合蛋白是基因调控和染色质结构的关键决定因素。TF 分析通常通过甲醛交联和超声处理，然后进行染色质免疫沉淀（X-ChIP）来进行。我们描述了一种无需交联即可高分辨率分析 TF 结合的方法。我们从微球菌核酸酶消化的非交联染色质开始，然后进行 TF 的亲和纯化和配对末端测序。来自亲和纯化的自然分离染色质（ORGANIC）的 Saccharomyces cerevisiae Abf1 和 Reb1 的 TF 结合的基因组的占据区域提供了高分辨率图谱，这些图谱是准确的，因为在鉴定的结合位点中存在已知的 TF 共有基序，这些图谱没有偏向于可及染色质，也不需要输入归一化。我们对果蝇 GAGA 因子和 Pipsqueak 进行了分析，以测试 ORGANIC 在更大基因组上的性能。我们的结果表明，ORGANIC 分析是一种广泛适用的高分辨率方法，可用于灵敏和特异性地分析直接的蛋白质-DNA 相互作用。
