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
- Genetics and Genomics
The preservation of genome integrity during sperm and egg development is vital for reproductive success. During meiosis, the tumor suppressor BRCA1/BRC-1 and structural maintenance of chromosomes 5/6 (SMC-5/6) complex genetically interact to promote high fidelity DNA double strand break (DSB) repair, but the specific DSB repair outcomes these proteins regulate remain unknown. Using genetic and cytological methods to monitor resolution of DSBs with different repair partners in Caenorhabditis elegans, we demonstrate that both BRC-1 and SMC-5 repress intersister crossover recombination events. Sequencing analysis of conversion tracts from homolog-independent DSB repair events further indicates that BRC-1 regulates intersister/intrachromatid noncrossover conversion tract length. Moreover, we find that BRC-1 specifically inhibits error prone repair of DSBs induced at mid-pachytene. Finally, we reveal functional interactions of BRC-1 and SMC-5/6 in regulating repair pathway engagement: BRC-1 is required for localization of recombinase proteins to DSBs in smc-5 mutants and enhances DSB repair defects in smc-5 mutants by repressing theta-mediated end joining (TMEJ). These results are consistent with a model in which some functions of BRC-1 act upstream of SMC-5/6 to promote recombination and inhibit error-prone DSB repair, while SMC-5/6 acts downstream of BRC-1 to regulate the formation or resolution of recombination intermediates. Taken together, our study illuminates the coordinated interplay of BRC-1 and SMC-5/6 to regulate DSB repair outcomes in the germline.
