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
RNA interference (RNAi) is a conserved pathway that utilizes Argonaute proteins and their associated small RNAs to exert gene regulatory function on complementary transcripts. While the majority of germline-expressed RNAi proteins reside in perinuclear germ granules, it is unknown whether and how RNAi pathways are spatially organized in other cell types. Here, we find that the small RNA biogenesis machinery is spatially and temporally organized during Caenorhabditis elegans embryogenesis. Specifically, the RNAi factor, SIMR-1, forms visible concentrates during mid-embryogenesis that contain an RNA-dependent RNA polymerase, a poly-UG polymerase, and the unloaded nuclear Argonaute protein, NRDE-3. Curiously, coincident with the appearance of the SIMR granules, the small RNAs bound to NRDE-3 switch from predominantly CSR-class 22G-RNAs to ERGO-dependent 22G-RNAs. NRDE-3 binds ERGO-dependent 22G-RNAs in the somatic cells of larvae and adults to silence ERGO-target genes; here we further demonstrate that NRDE-3-bound, CSR-class 22G-RNAs repress transcription in oocytes. Thus, our study defines two separable roles for NRDE-3, targeting germline-expressed genes during oogenesis to promote global transcriptional repression, and switching during embryogenesis to repress recently duplicated genes and retrotransposons in somatic cells, highlighting the plasticity of Argonaute proteins and the need for more precise temporal characterization of Argonaute-small RNA interactions.
