CUT&RUN detects distinct DNA footprints of RNA polymerase II near the transcription start sites

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. ### Competing Interest Statement The authors have declared no competing interest. * CUT&RUN : Cleavage Under Targets and Release Using Nuclease pAG-MNase : Protein A/G-fused micrococcal nuclease ChIP : chromatin immunoprecipitation Pol II : RNA polymerase II TSS : transcription start site S5p Pol II : Serine 5-phosphorylated RNA polymerase II mNET-seq : mammalian native elongating transcript-sequencing

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