CUT&RUN is a powerful tool to study protein-DNA interactions in vivo. DNA fragments cleaved by the targeted micrococcal nuclease identify the footprints of DNA-binding proteins on the chromatin. We performed CUT&RUN on human lung carcinoma cell line A549 maintained in a multi-well cell culture plate to profile RNA polymerase II. Long (> 270 bp) DNA fragments released by CUT&RUN corresponded to the bimodal peak around the transcription start sites, as previously seen with chromatin immunoprecipitation. However, we found that short (< 120 bp) fragments identify a well-defined peak localised at the transcription start sites. This distinct DNA footprint of short fragments, which constituted only about 5% of the total reads, suggests the transient positioning of RNA polymerase II before promoter-proximal pausing, which has not been detected in the physiological settings by standard chromatin immunoprecipitation. We showed that the positioning of the large-size-class DNA footprints around the short-fragment peak was associated with the directionality of transcription, demonstrating the biological significance of distinct CUT&RUN footprints of RNA polymerase II.

CUT&RUN 是研究体内蛋白质-DNA 相互作用的强大工具。被靶向微球菌核酸酶切割的 DNA 片段可识别 DNA 结合蛋白在染色质上的足迹。我们对保存在多孔细胞培养板中的人肺癌细胞系 A549 进行了 CUT&RUN 以分析 RNA 聚合酶 II。CUT&RUN 释放的长 (> 270 bp) DNA 片段对应于转录起始位点周围的双峰峰，如先前在染色质免疫沉淀中所见。然而，我们发现短 (< 120 bp) 片段识别了位于转录起始位点的明确定义的峰。这种独特的短片段 DNA 足迹，仅占总读数的 5% 左右，表明 RNA 聚合酶 II 在启动子近端暂停之前的瞬时定位，标准染色质免疫沉淀未在生理环境中检测到。我们表明，短片段峰周围大尺寸 DNA 足迹的定位与转录的方向性相关，证明了 RNA 聚合酶 II 不同 CUT&RUN 足迹的生物学意义。