An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites
- Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, United States
Abstract
We describe Cleavage Under Targets and Release Using Nuclease (CUT&RUN), a chromatin profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. Unlike Chromatin Immunoprecipitation (ChIP), which fragments and solubilizes total chromatin, CUT&RUN is performed in situ, allowing for both quantitative high-resolution chromatin mapping and probing of the local chromatin environment. When applied to yeast and human nuclei, CUT&RUN yielded precise transcription factor profiles while avoiding cross-linking and solubilization issues. CUT&RUN is simple to perform and is inherently robust, with extremely low backgrounds requiring only ~1/10th the sequencing depth as ChIP, making CUT&RUN especially cost-effective for transcription factor and chromatin profiling. When used in conjunction with native ChIP-seq and applied to human CTCF, CUT&RUN mapped long range contacts at high resolution. We conclude that in situ mapping of protein-DNA interactions by CUT&RUN is an attractive alternative to ChIP-seq.
Data availability
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Cut-and-Run in situ factor profiling maps DNA binding and 3D contact sites at high resolutionPublicly available at the NCBI Gene Expression Omnibus (accession no: GSE84474).
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High-resolution mapping of transcription factor binding sites on native chromatin.Publicly available at the NCBI Gene Expression Omnibus (accession no: GSE45672).
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CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for TranscriptionPublicly available at the NCBI Gene Expression Omnibus (accession no: GSE72816).
Article and author information
Author details
Funding
Howard Hughes Medical Institute (Henikoff)
- Peter J Skene
Howard Hughes Medical Institute (Henikoff)
- Steven Henikoff
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2017, Skene & Henikoff
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites
eLife 6:e21856.
https://doi.org/10.7554/eLife.21856
Further reading
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- Chromosomes and Gene Expression
A new technique called CUT&RUN can map the distribution of proteins on the genome with higher resolution and accuracy than existing approaches.
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- Chromosomes and Gene Expression
Cells evoke the DNA damage checkpoint (DDC) to inhibit mitosis in the presence of DNA double-strand breaks (DSBs) to allow more time for DNA repair. In budding yeast, a single irreparable DSB is sufficient to activate the DDC and induce cell cycle arrest prior to anaphase for about 12–15 hr, after which cells ‘adapt’ to the damage by extinguishing the DDC and resuming the cell cycle. While activation of the DNA damage-dependent cell cycle arrest is well understood, how it is maintained remains unclear. To address this, we conditionally depleted key DDC proteins after the DDC was fully activated and monitored changes in the maintenance of cell cycle arrest. Degradation of Ddc2ATRIP, Rad9, Rad24, or Rad53CHK2 results in premature resumption of the cell cycle, indicating that these DDC factors are required both to establish and maintain the arrest. Dun1 is required for the establishment, but not the maintenance, of arrest, whereas Chk1 is required for prolonged maintenance but not for initial establishment of the mitotic arrest. When the cells are challenged with two persistent DSBs, they remain permanently arrested. This permanent arrest is initially dependent on the continuous presence of Ddc2, Rad9, and Rad53; however, after 15 hr these proteins become dispensable. Instead, the continued mitotic arrest is sustained by spindle assembly checkpoint (SAC) proteins Mad1, Mad2, and Bub2 but not by Bub2’s binding partner Bfa1. These data suggest that prolonged cell cycle arrest in response to 2 DSBs is achieved by a handoff from the DDC to specific components of the SAC. Furthermore, the establishment and maintenance of DNA damage-induced cell cycle arrest require overlapping but different sets of factors.
