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

Hesselberth JR, Chen X, Zhang Z, Sabo PJ, Sandstrom R, Reynolds AP, Thurman RE, Neph S, Kuehn MS, Noble WS, Fields S, Stamatoyannopoulos JA - Nat. Methods (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genome Sciences, University of Washington, Seattle, USA.

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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Detection of footprints and corresponding sequence motifs(a) Visualization of DNase I protection (footprinting) around 907 computationally-predicted Reb1 sites in a heat map. Rows show levels of DNase I cleavage 25 bp up- and downstream of each motif instance and are sorted by the ratio of mean cleavage over flanking regions to that within the motif itself. Red ticks (at left) indicate motif instances (n = 580) that coincide with footprints (FDR = 0.05) containing de novo-derived Reb1 motifs. Blue ticks (right) indicate motif instances (n = 151) coinciding with those identified by ChIP10. All motif instances are uniquely mappable within the yeast genome. (b) Mean per nucleotide DNase I cleavage (red) and evolutionary conservation (Phastcons?; blue) calculated for footprints that match the Reb1, Abf1, Rap1 and Hsf1 motifs (subpanel vertical axes). Significance of observed conservation patterns (blue text) (Supplementary Methods), extent of consensus motifs derived from the footprinted region (green shading), motifs derived from ChIP and footprinting below. Venn diagrams depict the overlap of motifs derived from and mapping to footprints (red) vs. ChIP (blue).
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Figure 2: Detection of footprints and corresponding sequence motifs(a) Visualization of DNase I protection (footprinting) around 907 computationally-predicted Reb1 sites in a heat map. Rows show levels of DNase I cleavage 25 bp up- and downstream of each motif instance and are sorted by the ratio of mean cleavage over flanking regions to that within the motif itself. Red ticks (at left) indicate motif instances (n = 580) that coincide with footprints (FDR = 0.05) containing de novo-derived Reb1 motifs. Blue ticks (right) indicate motif instances (n = 151) coinciding with those identified by ChIP10. All motif instances are uniquely mappable within the yeast genome. (b) Mean per nucleotide DNase I cleavage (red) and evolutionary conservation (Phastcons?; blue) calculated for footprints that match the Reb1, Abf1, Rap1 and Hsf1 motifs (subpanel vertical axes). Significance of observed conservation patterns (blue text) (Supplementary Methods), extent of consensus motifs derived from the footprinted region (green shading), motifs derived from ChIP and footprinting below. Venn diagrams depict the overlap of motifs derived from and mapping to footprints (red) vs. ChIP (blue).

Mentions: On close inspection, we observed that DNase I cleavage patterns upstream of transcriptional start sites (TSSs) were punctuated by short stretches of protected nucleotides consistent with the footprints of DNA-binding proteins, and that in many cases individual footprints could be matched to known DNA-binding motifs (Fig.1d). We also examined the degree to which computationally predicted factor binding sites within yeast intergenic regions exhibited DNase I protection. For any given factor, computational predictions are expected to contain a mixture of true- and false-positive sites. Fig.2a shows the DNase I cleavage patterns surrounding 907 computationally-predicted9 Reb1 binding sites (+/-25 bp) within yeast intergenic regions, ranked by the ratio of DNase I cleavage flanking the motif to that within the motif. This analysis showed that a significant proportion of predicted Reb1 sites exhibited DNase I protection consistent with protein binding in vivo and, moreover, that the DNase I protection patterns were specifically localized to the motif region. We observed analogous patterns for other motifs, with considerable variation in the fraction of computationally predicted motif instances that evidenced DNase I protection (data not shown), commensurate with the expectation that many (if not most) binding sites predicted from motif scans alone are not actuated in vivo.


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

Hesselberth JR, Chen X, Zhang Z, Sabo PJ, Sandstrom R, Reynolds AP, Thurman RE, Neph S, Kuehn MS, Noble WS, Fields S, Stamatoyannopoulos JA - Nat. Methods (2009)

Detection of footprints and corresponding sequence motifs(a) Visualization of DNase I protection (footprinting) around 907 computationally-predicted Reb1 sites in a heat map. Rows show levels of DNase I cleavage 25 bp up- and downstream of each motif instance and are sorted by the ratio of mean cleavage over flanking regions to that within the motif itself. Red ticks (at left) indicate motif instances (n = 580) that coincide with footprints (FDR = 0.05) containing de novo-derived Reb1 motifs. Blue ticks (right) indicate motif instances (n = 151) coinciding with those identified by ChIP10. All motif instances are uniquely mappable within the yeast genome. (b) Mean per nucleotide DNase I cleavage (red) and evolutionary conservation (Phastcons?; blue) calculated for footprints that match the Reb1, Abf1, Rap1 and Hsf1 motifs (subpanel vertical axes). Significance of observed conservation patterns (blue text) (Supplementary Methods), extent of consensus motifs derived from the footprinted region (green shading), motifs derived from ChIP and footprinting below. Venn diagrams depict the overlap of motifs derived from and mapping to footprints (red) vs. ChIP (blue).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2668528&req=5

Figure 2: Detection of footprints and corresponding sequence motifs(a) Visualization of DNase I protection (footprinting) around 907 computationally-predicted Reb1 sites in a heat map. Rows show levels of DNase I cleavage 25 bp up- and downstream of each motif instance and are sorted by the ratio of mean cleavage over flanking regions to that within the motif itself. Red ticks (at left) indicate motif instances (n = 580) that coincide with footprints (FDR = 0.05) containing de novo-derived Reb1 motifs. Blue ticks (right) indicate motif instances (n = 151) coinciding with those identified by ChIP10. All motif instances are uniquely mappable within the yeast genome. (b) Mean per nucleotide DNase I cleavage (red) and evolutionary conservation (Phastcons?; blue) calculated for footprints that match the Reb1, Abf1, Rap1 and Hsf1 motifs (subpanel vertical axes). Significance of observed conservation patterns (blue text) (Supplementary Methods), extent of consensus motifs derived from the footprinted region (green shading), motifs derived from ChIP and footprinting below. Venn diagrams depict the overlap of motifs derived from and mapping to footprints (red) vs. ChIP (blue).
Mentions: On close inspection, we observed that DNase I cleavage patterns upstream of transcriptional start sites (TSSs) were punctuated by short stretches of protected nucleotides consistent with the footprints of DNA-binding proteins, and that in many cases individual footprints could be matched to known DNA-binding motifs (Fig.1d). We also examined the degree to which computationally predicted factor binding sites within yeast intergenic regions exhibited DNase I protection. For any given factor, computational predictions are expected to contain a mixture of true- and false-positive sites. Fig.2a shows the DNase I cleavage patterns surrounding 907 computationally-predicted9 Reb1 binding sites (+/-25 bp) within yeast intergenic regions, ranked by the ratio of DNase I cleavage flanking the motif to that within the motif. This analysis showed that a significant proportion of predicted Reb1 sites exhibited DNase I protection consistent with protein binding in vivo and, moreover, that the DNase I protection patterns were specifically localized to the motif region. We observed analogous patterns for other motifs, with considerable variation in the fraction of computationally predicted motif instances that evidenced DNase I protection (data not shown), commensurate with the expectation that many (if not most) binding sites predicted from motif scans alone are not actuated in vivo.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genome Sciences, University of Washington, Seattle, USA.

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.

Show MeSH
Related in: MedlinePlus