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
- Structural Biology and Molecular Biophysics
Type II nuclear receptors (T2NRs) require heterodimerization with a common partner, the retinoid X receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and overexpression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single-molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged RXR and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR, increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.
