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URL: https://dx.doi.org/DOI:10.17504/protocols.io.bizgkf3w

Authors: Rafal Donczew, Amélia Lalou, Steven Hahn

Summary: ChEC-seq and other nuclease-based methods such as Cut&Run map protein locations on DNA by targeting nuclease activity to specific transcription factors and mapping the resulting DNA cleavages (Schmid et al. 2004; Skene and Henikoff 2017; Zentner et al. 2015). For ChEC-seq, yeast cells expressing a protein-micrococcal nuclease (MNase) fusion are permeabilized, MNase is activated by the addition of calcium, and the resulting DNA fragments are mapped. Potential advantages of this approach include avoiding non-specific protein-DNA crosslinking in highly transcribed regions, efficient mapping of factors that do not directly bind DNA and, more sensitive detection of protein-DNA interactions. We optimized the original ChEC-seq protocol to minimize non-specific DNA cleavage, avoid over digestion at authentic binding sites, and efficiently assay large numbers of factors. We also created a robust data analysis pipeline that incorporates peak calling to map binding sites and quantitative analysis, based on utilization of spike-in DNA, to compare factor-DNA binding under different conditions. We used this modified approach to map genome-wide distributions of the transcription coactivators TFIID and SAGA (Donczew et al. 2020) as well as transcription factors Abf1 and Rap1 (Donczew et al, submitted to Mol Cell).ReferencesDonczew R, Warfield L, Pacheco D, Erijman A, Hahn S. 2020. Two roles for the yeast transcription coactivator SAGA and a set of genes redundantly regulated by TFIID and SAGA.eLife 9: e50109.Schmid M, Durussel T, Laemmli UK. 2004. ChIC and ChEC; genomic mapping of chromatin proteins.Mol Cell 16: 147–157.Skene PJ, Henikoff S. 2017. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites.eLife 6: e21856.Warfield L, Ramachandran S, Baptista T, Devys D, Tora L, Hahn S. 2017. Transcription of nearly all yeast RNA Polymerase II-transcribed genes is dependent on transcription factor TFIID.Mol Cell. 68:118-129Zentner GE, Kasinathan S, Xin B, Rohs R, Henikoff S. 2015. ChEC-seq kinetics discriminates transcription factor binding sites by DNA sequence and shape in vivo.Nat Commun 6: 8733.

Affiliations: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109 USA, Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109 USA, Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109 USA

Version: 3

Publication Date: 2020

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