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 as well as the identification of hundreds of novel binding sites for major regulators. We observed striking correspondence between nucleotide-level 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 provides a powerful approach to delineate the cis-regulatory framework of any organism with an available genome sequence.
DNaseI-seq cleavage counts are displayed at nucleotide resolution, along with a 'mappability' track that indicates whether tag sequences starting at that location on both the forward and the reverse strands can be uniquely mapped to the yeast genome. Finally, the set of footprints with q values <0.1 are included, where the q value is defined as the minimal false discovery rate threshold at which the given footprint is deemed significant. The name associated with each footprint is its q value.
To visualize regulatory protein occupancy across the genome of Saccharomyces cerevisiae, DNase I digestion of yeast nuclei was coupled with massively parallel DNA sequencing to create a dense whole-genome map of DNA template accessibility at the nucleotide-level.
Yeast nuclei were isolated and treated with a DNase I concentration sufficient to release short (<300 bp) DNA fragments. Small fragments were derived from two DNase I "hits" in close proximity. Each end of those fragments represents an in vivo DNase I cleavage site. The sequence and hence genomic location of these sites were then determined by DNA sequencing.
Footprints were identified using a computational algorithm that evaluates short regions (between 8 and 30 bp) over which the DNase I cleavage density was significantly reduced compared with the immediately flanking regions. FDR thresholds were assigned to each footprint by comparing p-values obtained from real and shuffled cleavage data.
Detailed methods are given in Hesselberth et al. (2009), and supplementary data and source code are available here.
This track was produced at the University of Washington by Jay R. Hesselberth, Xiaoyu Chen, Zhihong Zhang, Peter J. Sabo, Richard Sandstrom, Alex P. Reynolds, Robert E. Thurman, Shane Neph, Michael S. Kuehn, William S. Noble (william-noble@u.washington.edu), Stanley Fields (fields@u.washington.edu) and John A. Stamatoyannopoulos (jstam@stamlab.org).
Hesselberth JR, Chen X, Zhang Z, Sabo PJ, Sandstrom R, Reynolds AP, Thurman RE, Neph S, Kuehn MS, Noble WS, Fields S and Stamatoyannopoulos JA. Global mapping of protein-DNA interactions in vivo by digital genomic footprinting. Nature Methods. 2009 Mar 22;6:283-289.