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High-resolution mapping of transcription factor binding sites on native chromatin

Abstract

Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. TF profiling is commonly carried out by formaldehyde cross-linking and sonication followed by chromatin immunoprecipitation (X-ChIP). We describe a method to profile TF binding at high resolution without cross-linking. We begin with micrococcal nuclease–digested non-cross-linked chromatin and then perform affinity purification of TFs and paired-end sequencing. The resulting occupied regions of genomes from affinity-purified naturally isolated chromatin (ORGANIC) profiles of Saccharomyces cerevisiae Abf1 and Reb1 provide high-resolution maps that are accurate, as defined by the presence of known TF consensus motifs in identified binding sites, that are not biased toward accessible chromatin and that do not require input normalization. We profiled Drosophila melanogaster GAGA factor and Pipsqueak to test ORGANIC performance on larger genomes. Our results suggest that ORGANIC profiling is a widely applicable high-resolution method for sensitive and specific profiling of direct protein-DNA interactions.

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Figure 1: Identification of Reb1 binding sites on native chromatin.
Figure 2: ORGANIC TF binding sites have characteristic motifs.
Figure 3: High sensitivity and specificity of ORGANIC profiling of TFs.
Figure 4: ORGANIC sites are stably bound in vivo and are conserved throughout Saccharomyces evolution.
Figure 5: ORGANIC profiling identifies TF binding sites in inaccessible chromatin.
Figure 6: ORGANIC profiling of Drosophila GAGA factor (GAF) and Pipsqueak (Psq) DNA-binding proteins.

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Acknowledgements

We thank J.G. Henikoff for help with data analysis, members of the S. Henikoff laboratory and L. Gabrovsek for comments on the manuscript, G. Cavalli (Institut Génétique Humaine) and C.A. Berg (University of Washington) for GAF and Psq antibodies, respectively, and the Drosophila RNAi Screening Center for cells. This work was supported by the Howard Hughes Medical Institute, grant R01 ES020116 from the US National Institutes of Health (NIH) (S.H. and K.A.), the Fred Hutchinson Cancer Research Center (FHCRC) Chromosome Metabolism and Cancer Training grant NIH 5T32 CA009657 (G.E.Z.), the European Research Council (ERC) 7th Framework Program, Marie Curie Actions IOF 300710 (G.A.O.) and the Micki & Robert Flowers ARCS Endowment from the Seattle Chapter of the ARCS Foundation (S.K.).

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Contributions

S.H. conceived of the strategy; S.K., S.H. and G.E.Z. designed the yeast experiments; K.A. and G.A.O. designed and performed the Drosophila experiments; S.K. performed the yeast experiments and yeast analysis; S.K. and G.A.O. performed the Drosophila analysis; and S.K. wrote the paper.

Corresponding author

Correspondence to Steven Henikoff.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–17 and Supplementary Results (PDF 7038 kb)

Supplementary Table 1

Peaks called for ORGANIC datasets reported in this study. Reb1: 2.5 min MNase/80mM NaCl/len50, 10 min MNase/80mM NaCl/len50, 10 min MNase/150mM NaCl/len50, 10 min MNase/600mM NaCl/len50; Abf1: 2.5 min MNase/80mM NaCl/len50, 10 min MNase/80mM NaCl/len50, 10 min MNase/600mM NaCl/len50; Psq len25 and GAF len25. Thresholds used for peak calling (see Online Methods) are indicated. (XLS 1017 kb)

Supplementary Table 2

Log-odds position-specific scoring matrices from S. cerevisiae ORGANIC experiments. For a given log-odds entry (row,column) in the matrix, row specifies the nucleotide and column the position in the motif. (XLS 27 kb)

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Kasinathan, S., Orsi, G., Zentner, G. et al. High-resolution mapping of transcription factor binding sites on native chromatin. Nat Methods 11, 203–209 (2014). https://doi.org/10.1038/nmeth.2766

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