Abstract
Competition chromatin immunoprecipitation (competition ChIP) enables experimenters to measure protein-DNA dynamics at a single locus or across the entire genome, depending on the detection method. Competition ChIP relies on a cell containing two copies of a single DNA-associated factor, with each copy of the factor differentially epitope tagged. One of the copies is expressed constitutively and the second is induced as a competitor. The ratio of isoforms associated with discrete genomic locations is detected by ChIP-on-chip (ChIP-chip) or ChIP-sequencing (ChIP-seq). The rate at which the resident isoform of the protein is replaced by the competitor at each binding location enables the calculation of residence time for that factor at each site of interaction genome wide. Here we provide a detailed protocol for designing and performing competition ChIP experiments in Saccharomyces cerevisiae, which takes ∼5 d to complete (not including strain production and characterizations, which may take as long as 6 months). Included in this protocol are guidelines for downstream bioinformatic analysis to extract residence times throughout the genome.
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Acknowledgements
We thank L.S. Kovacs and T. Jeffers for critically reading the manuscript, K. Ikegami for suggesting alternative cross-linkers and promoter systems and D. Mckay for suggesting inducible expression systems in Drosophila. J.D.L. and C.R.L. were supported by National Institutes of Health grant no. R01 GM072518. F.M. has been supported by the Région Ile-de-France under C'Nano IdF (the Center of Competences in NanoSciences for the Paris region) and the Fondation pour la Recherche Médicale en France (FRM).
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C.R.L. and F.M. performed the analysis and refined the competition ChIP protocol with input from J.D.L. C.R.L., F.M. and J.D.L. wrote the manuscript.
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Lickwar, C., Mueller, F. & Lieb, J. Genome-wide measurement of protein-DNA binding dynamics using competition ChIP. Nat Protoc 8, 1337–1353 (2013). https://doi.org/10.1038/nprot.2013.077
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DOI: https://doi.org/10.1038/nprot.2013.077
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