Abstract
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.
Highlights
CUT&RUN (Cleavage Under Targets and Release Using Nuclease) (Skene and Henikoff 2017) is a powerful tool to map protein-DNA interactions in vivo
We tested the feasibility of performing CUT&RUN on adherent cells attached to a multi-well polystyrene cell culture plate with polymerase II (Pol II) antibodies (Fig. 1a)
We have devised “CUT&RUN on plate” (CROP) for profiling protein-DNA interactions in adherent cells maintained in a multi-well cell culture plate
Summary
CUT&RUN (Cleavage Under Targets and Release Using Nuclease) (Skene and Henikoff 2017) is a powerful tool to map protein-DNA interactions in vivo. CUT&RUN directs Protein A/G-fused micrococcal nuclease (pAG-MNase) (Meers et al 2019a) to the antibody-bound protein-DNA complex, with a subsequent release of DNA fragments cleaved in the presence of calcium ions. There are two major advantages of CUT&RUN over conventional chromatin immunoprecipitation (ChIP). CUT&RUN does not require sonication for chromatin fragmentation. CUT&RUN identifies the footprints of DNA-binding proteins on the chromatin, which revealed distinct binding configurations of transcription factors in yeast (Skene and Henikoff 2017) and mammalian cells (Meers et al 2019b)
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