High-resolution, genome-wide mapping of positive supercoiling in chromosomes [yChIP]

Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to E. coli and S. cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and enriched between convergently-oriented genes, consistent with the ?twin-domain? model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin binding sites, replication-transcription encounters, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach that can be applied in any organism to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods. Sequencing data from E. coli MG1655 and S. cerevisiae. (i) E. coli GapR and GapR1-76 ChIP-seq, (ii) E. coli RNA-seq +/- GapR-3xFLAG expression, (iii) S. cerevisiae GapR ChIP-seq in raffinose +/- alpha-factor and in glycerol, (iv) S. cerevisiae RNA-seq +/- GapR, and (v) S. cerevisiae RNA-seq in raffinose +/- alpha-factor and in glycerol after GapR induction

DNA超螺旋（DNA supercoiling）可影响DNA复制、转录、蛋白质与DNA的结合以及染色体的三维组织结构。然而，目前尚无能够直接检测或绘制正超螺旋的方法，因此其在基因组中的分布仍不明确。本研究报道了一种基于GapR染色质免疫沉淀（chromatin immunoprecipitation, ChIP）的方法：GapR是一种优先识别过度缠绕DNA的细菌蛋白，可用于生成高分辨率的正超螺旋图谱。我们将该方法应用于大肠杆菌（Escherichia coli, E. coli）和酿酒酵母（Saccharomyces cerevisiae, S. cerevisiae），发现正超螺旋广泛存在，与转录过程相关，并富集于相向转录的基因之间，这与超螺旋的"双结构域"模型相符。在酿酒酵母中，我们还发现正超螺旋与着丝粒、黏连蛋白（cohesin）结合位点、复制-转录碰撞位点以及R环（DNA-RNA杂交双链）的边界相关。研究结果表明，GapR-seq是一种强大的研究手段，可应用于任何生物体，用于探究Hi-C技术及其他现有方法无法解析的染色体结构与组织特征。本研究的测序数据来源于大肠杆菌MG1655（E. coli MG1655）和酿酒酵母，具体包括：(i) 大肠杆菌GapR及GapR1-76的染色质免疫沉淀测序（ChIP-seq）数据；(ii) 表达与不表达GapR-3xFLAG的大肠杆菌转录组测序（RNA-seq）数据；(iii) 酿酒酵母在棉子糖培养基（添加/不添加α因子）及甘油培养基中的GapR ChIP-seq数据；(iv) 表达与不表达GapR的酿酒酵母转录组测序（RNA-seq）数据；(v) GapR诱导后，酿酒酵母在棉子糖培养基（添加/不添加α因子）及甘油培养基中的转录组测序（RNA-seq）数据。