Genomic footprinting


The advent of DNA footprinting with DNase I more than 35 years ago enabled the systematic analysis of protein-DNA interactions, and the technique has been instrumental in the decoding of cis-regulatory elements and the identification and characterization of transcription factors and other DNA-binding proteins. The ability to analyze millions of individual genomic cleavage events via massively parallel sequencing has enabled in vivo DNase I footprinting on a genomic scale, offering the potential for global analysis of transcription factor occupancy in a single experiment. Genomic footprinting has opened unique vistas on the organization, function and evolution of regulatory DNA; however, the technology is still nascent. Here we discuss both prospects and challenges of genomic footprinting, as well as considerations for its application to complex genomes.

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Figure 1: Principles of a DNase I footprinting experiment.
Figure 2: Resolving cis-regulatory architecture at nucleotide resolution in individual regulatory regions.
Figure 3: An illustrative example of interpretation of DNase I cleavage to determine TF occupancy.
Figure 4: De novo versus TF recognition site–directed analysis of TF occupancy.
Figure 5: Modeling variation in DNase I cleavages rates due to primary DNA structure.


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This work was supported by the US National Human Genome Research Institute (grant U54HG007010 to J.A.S.).

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Correspondence to Jeff Vierstra or John A Stamatoyannopoulos.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Aggregated DNase I cleavage patterns for TF recognition sequences reflecting diverse DNA-binding domains.

(a) Heatmaps of per-nucleotide DNase I cleavages and discovered footprints surrounding NRF1 recognition sequences. Left, observed cleavages. Right, the ratio of the observed cleavages to expected cleavages computed by reassigning tags to a hexamer model DNase I cleavage bias. Blue ticks indicate that the recognition sequence has an associated DNase I footprint. Line plots show the aggregate profile of mean per-nucleotide DNase I cleavages at the 20% most (left column) and 20% least (right column) accessible NRF1 recognition sequences. Top row, observed cleavages. Middle, expected cleavages computed using the hexamer model. Bottom, the log2 ratio of observed to expected. (b-g) The same as (a) for the recognition sequences for (b) SP1, (c) ELK1, (d) USF1, (e) RFX3, (f) NFIB, and (g) CTCF within accessible chromatin. In each case the cleavage patterns at occupied templates (coinciding with de novo TF footprint calls) parallel known structural features of the respective DNA binding domains.

Supplementary Figure 2 General features of DNase I sequence preference.

(a) Relative cleavage preference of all 4,096 hexamers with respect to the median hexamer as determined by deep sequencing (~100 million tags) of a DNase I digestion of deproteinized DNA from human IMR90 cells (data from ref.). (b) Biased hexamers contribute disproportionately to total DNase I cleavages for both naked DNA and chromatin (regulatory T cells cleavages mapping within DHS) when compared to the 36 bp mappable genome. Shown is the cumulative fraction all mappable positions or sequencing tags within respect to their hexamer context. Hexamers are ranked by decreasing cleavage preference as in a.

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Supplementary Figures 1 and 2, Supplementary Box 1 and Supplementary Table 1 (PDF 1055 kb)

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Vierstra, J., Stamatoyannopoulos, J. Genomic footprinting. Nat Methods 13, 213–221 (2016).

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