Transcription dependent dynamic supercoiling in Raji human B cells in vivo. Homo sapiens

Transcription has the capacity to modify mechanically DNA topology, DNA structure, and nucleosome arrangement. Resulting from ongoing transcription, these modifications in turn, may provide instant feedback to the transcription machinery. To substantiate the connection between transcription and DNA dynamics, we charted an ENCODE map of transcription-dependent dynamic supercoiling in human Burkitt lymphoma cells using psoralen photobinding to probe DNA topology in vivo. Dynamic supercoils spread ~1.5 kb upstream of the start sites of active genes. Low and high output promoters handle this torsional stress differently as shown using inhibitors of transcription and topoisomerases, and by chromatin immunoprecipation of RNA polymerase and topoisomerases I and II. Whereas lower outputs are managed adequately by topoisomerase I, high output promoters additionally require topoisomerase II. The genome-wide coupling between transcription and DNA topology emphasizes the importance of dynamic supercoiling for gene regulation. Overall design: Raji cells: untreated and treated with DRB, CPT and BLAP. Three biological replicates per treatment, each hybridized to new array. Total: 12 samples (4 treatments x 3 replicates).

转录能够从力学层面改变DNA拓扑结构、DNA构型以及核小体排布。这类由持续转录产生的修饰，反过来又可对转录机器提供即时反馈。为证实转录与DNA动态特性之间的联系，我们利用补骨脂素光结合（psoralen photobinding）技术在活体内探测DNA拓扑结构，绘制了人类伯基特淋巴瘤（Burkitt lymphoma）细胞中转录依赖性动态超螺旋的ENCODE（DNA元件百科全书）图谱。动态超螺旋可在活跃基因的转录起始位点上游约1.5千碱基对（kb）的区域内扩散。低、高表达量启动子对这种扭转应力的处理方式存在差异，这一结论通过转录抑制剂、拓扑异构酶抑制剂实验，以及RNA聚合酶与拓扑异构酶I、II的染色质免疫共沉淀（chromatin immunoprecipitation）实验得以验证。低表达量启动子的扭转应力可通过拓扑异构酶I得到充分调控，而高表达量启动子还需要拓扑异构酶II的参与。转录与DNA拓扑结构之间的全基因组层面耦合关系，凸显了动态超螺旋在基因调控中的重要性。实验整体设计：以Raji细胞为研究对象，设置未处理组与分别经DRB、CPT、BLAP处理的实验组；每组设置3次生物学重复，每个重复样本均使用全新芯片进行杂交；总样本量为12例（4种处理方式×3次生物学重复）。