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Global mapping of protein-DNA interactions in vivo by digital genomic footprinting

Nature Methods volume 6, pages 283–289 (2009)Cite this article

Abstract

The orchestrated binding of transcriptional activators and repressors to specific DNA sequences in the context of chromatin defines the regulatory program of eukaryotic genomes. We developed a digital approach to assay regulatory protein occupancy on genomic DNA in vivo by dense mapping of individual DNase I cleavages from intact nuclei using massively parallel DNA sequencing. Analysis of >23 million cleavages across the Saccharomyces cerevisiae genome revealed thousands of protected regulatory protein footprints, enabling de novo derivation of factor binding motifs and the identification of hundreds of new binding sites for major regulators. We observed striking correspondence between single-nucleotide resolution DNase I cleavage patterns and protein-DNA interactions determined by crystallography. The data also yielded a detailed view of larger chromatin features including positioned nucleosomes flanking factor binding regions. Digital genomic footprinting should be a powerful approach to delineate the cis-regulatory framework of any organism with an available genome sequence.

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Figure 1: Digital DNase I analysis of yeast chromatin structure from chromosomal to nucleotide resolution.
Figure 2: Detection of footprints and corresponding sequence motifs.
Figure 3: Mean nucleotide-level accessibility parallels protein-DNA interactions.
Figure 4: Individual yeast regulatory regions and factor binding sites.
Figure 5: Higher-order patterns of DNA accessibility.

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Acknowledgements

We thank the staff of the University of Washington Genome Sciences High-Throughput Genomics Unit for technical assistance with Illumina-Solexa sequencing, and members of the Stamatoyannopoulos and Fields laboratories for many helpful discussions. This work was supported by US National Institutes of Health grants R01GM071923 and U54HG004592 to J.A.S., and P41RR11823 to S.F. and W.S.N.; X.C. was supported by a fellowship from the Natural Sciences and Engineering Research Council of Canada (NSERC PGS D3).

Author information

Author notes

  1. Zhihong Zhang

    Present address: Present address: Illumina, Inc., San Diego, California, USA.,

  2. Jay R Hesselberth and Xiaoyu Chen: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Genome Sciences, University of Washington, Seattle, Washington, USA

    Jay R Hesselberth, Zhihong Zhang, Peter J Sabo, Richard Sandstrom, Alex P Reynolds, Robert E Thurman, Shane Neph, Michael S Kuehn, William S Noble, Stanley Fields & John A Stamatoyannopoulos

  2. Department of Computer Science, University of Washington, Seattle, Washington, USA

    Xiaoyu Chen & William S Noble

  3. Howard Hughes Medical Institute, University of Washington, Seattle, Washington, USA

    Zhihong Zhang & Stanley Fields

  4. Department of Medicine, University of Washington, Seattle, Washington, USA

    John A Stamatoyannopoulos

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Correspondence to John A Stamatoyannopoulos.

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Competing interests

Z.Z. is presently an employee of Illumina, Inc.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 1–4, Supplementary Methods (PDF 2438 kb)

Supplementary Software

Footprint detection software. (ZIP 9 kb)

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Hesselberth, J., Chen, X., Zhang, Z. et al. Global mapping of protein-DNA interactions in vivo by digital genomic footprinting. Nat Methods 6, 283–289 (2009). https://doi.org/10.1038/nmeth.1313

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