CUT&RUN detects distinct DNA footprints of RNA polymerase II near the transcription start sites
- PDF / 5,085,050 Bytes
- 13 Pages / 547.087 x 737.008 pts Page_size
- 78 Downloads / 191 Views
ORIGINAL ARTICLE
CUT&RUN detects distinct DNA footprints of RNA polymerase II near the transcription start sites Michi Miura
&
Honglin Chen
Received: 13 August 2020 / Revised: 29 September 2020 / Accepted: 30 September 2020 # The Author(s) 2020
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. Keywords CUT&RUN . Chromatin profiling . RNA polymerase II . Transcription initiation . Promoterproximal pausing . Divergent transcription Abbreviations CUT&RUN Cleavage Under Targets and Release Using Nuclease pAGProtein A/G-fused micrococcal nuclease MNase 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 transcriptsequencing
Introduction Responsible Editor: Beth Sullivan Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10577-020-09643-0) contains supplementary material. M. Miura (*) : H. Chen State Key Laboratory for Emerging Infectious Diseases and Department of Microbiology, The University of Hong Kong, Hong Kong SAR, China e-mail: [email protected]
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
M. Miura, H. Chen
CUT&RUN over conventional chromatin immunoprecipitation (ChIP). First, CUT&RUN does not require sonication for chromatin fragmentation. Physical shearing of chromatin by sonication does not occur randomly, which can skew signal detection; and excessive sonication may attenuate the epitope of the target protein (Marx 2019). Second, CUT&RUN identifies the footprints of DN
Data Loading...
