• Home
  • Search Results
  • CUT&RUN detects distinct DNA footprints of RNA polymerase II near the transcription start sites.

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

Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology (2020-10-19)
Michi Miura, Honglin Chen
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Ethylenediaminetetraacetic acid disodium salt dihydrate, suitable for electrophoresis, for molecular biology, 99.0-101.0% (titration)
Sigma-Aldrich
Spermidine, ≥99% (GC)
Sigma-Aldrich
meso-Tetraphenylporphyrin, BioReagent, suitable for fluorescence, ≥99.0% (HPLC)
Sigma-Aldrich
Anti-RNA polymerase II Antibody, clone CTD4H8, clone CTD4H8, Upstate®, from mouse