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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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Individual yeast regulatory regions and factor binding sites(a) Rap1 binds to two adjacent sites also predicted from ChIP experiments upstream of RPS6A (chr16:378,775-378,874). (b) Reb1 binds to a canonical site upstream of TUF1 (chr15:683,707-683,806) but a non-canonical site upstream is only inferred from ChIP data (c) Mcm1 site upstream of MFA1 (chr4:1,384,893-1,384,993) exhibits hypersensitive nucleotides illustrated in Fig. 3a. (d) Hsf1 site identified by ChIP in BTN2 promoter (chr7:772,068-772,167) is identified as a footprint. (e) Two Reb1 binding sites in the REB1 promoter (chr2:336,885-337,084) are identified as footprints; a Cbf1 site predicted by ChIP shows a footprint, but a Rpn4 site defined by ChIP does not. (f) Two Pdr3 sites in the PDR5 promoter (chr15:619,227-619,476) are identified as footprints, in addition to an evolutionarily conserved region further upstream. Each panel shows per nucleotide DNase I cleavage, detected footprints (red boxes), assigned motifs (pink boxes), binding sites inferred from ChIP experiments (blue boxes), and evolutionary conservation (dark blue, Phastcons, bottom).
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Figure 4: Individual yeast regulatory regions and factor binding sites(a) Rap1 binds to two adjacent sites also predicted from ChIP experiments upstream of RPS6A (chr16:378,775-378,874). (b) Reb1 binds to a canonical site upstream of TUF1 (chr15:683,707-683,806) but a non-canonical site upstream is only inferred from ChIP data (c) Mcm1 site upstream of MFA1 (chr4:1,384,893-1,384,993) exhibits hypersensitive nucleotides illustrated in Fig. 3a. (d) Hsf1 site identified by ChIP in BTN2 promoter (chr7:772,068-772,167) is identified as a footprint. (e) Two Reb1 binding sites in the REB1 promoter (chr2:336,885-337,084) are identified as footprints; a Cbf1 site predicted by ChIP shows a footprint, but a Rpn4 site defined by ChIP does not. (f) Two Pdr3 sites in the PDR5 promoter (chr15:619,227-619,476) are identified as footprints, in addition to an evolutionarily conserved region further upstream. Each panel shows per nucleotide DNase I cleavage, detected footprints (red boxes), assigned motifs (pink boxes), binding sites inferred from ChIP experiments (blue boxes), and evolutionary conservation (dark blue, Phastcons, bottom).

Mentions: Digital genomic footprinting data are sufficiently dense to enable analysis of regulatory factor occupancy patterns at the level of individual regulatory regions. The examples in Fig.4 and Supplementary Fig.6 provide snapshots of a diverse population of regulators and binding site contexts. In many cases, high-confidence footprints agree with previous predictions for specific regulators (Fig.4a,b,d,e and Supplementary Fig.6a). However, we also observed numerous examples of discordance (Fig.4c,e), possibly reflecting condition-specific binding. For example, at the REB1 promoter (Fig.4e), we detected footprints at two previously-identified evolutionarily-conserved Reb1 binding sites19, neither of which were identified under conditions used in prior ChIP experiments. Conversely, ChIP data annotated a nearby Rpn4 site that does not fall within an FDR=0.05 footprint.


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)

Individual yeast regulatory regions and factor binding sites(a) Rap1 binds to two adjacent sites also predicted from ChIP experiments upstream of RPS6A (chr16:378,775-378,874). (b) Reb1 binds to a canonical site upstream of TUF1 (chr15:683,707-683,806) but a non-canonical site upstream is only inferred from ChIP data (c) Mcm1 site upstream of MFA1 (chr4:1,384,893-1,384,993) exhibits hypersensitive nucleotides illustrated in Fig. 3a. (d) Hsf1 site identified by ChIP in BTN2 promoter (chr7:772,068-772,167) is identified as a footprint. (e) Two Reb1 binding sites in the REB1 promoter (chr2:336,885-337,084) are identified as footprints; a Cbf1 site predicted by ChIP shows a footprint, but a Rpn4 site defined by ChIP does not. (f) Two Pdr3 sites in the PDR5 promoter (chr15:619,227-619,476) are identified as footprints, in addition to an evolutionarily conserved region further upstream. Each panel shows per nucleotide DNase I cleavage, detected footprints (red boxes), assigned motifs (pink boxes), binding sites inferred from ChIP experiments (blue boxes), and evolutionary conservation (dark blue, Phastcons, bottom).
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Related In: Results  -  Collection

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Figure 4: Individual yeast regulatory regions and factor binding sites(a) Rap1 binds to two adjacent sites also predicted from ChIP experiments upstream of RPS6A (chr16:378,775-378,874). (b) Reb1 binds to a canonical site upstream of TUF1 (chr15:683,707-683,806) but a non-canonical site upstream is only inferred from ChIP data (c) Mcm1 site upstream of MFA1 (chr4:1,384,893-1,384,993) exhibits hypersensitive nucleotides illustrated in Fig. 3a. (d) Hsf1 site identified by ChIP in BTN2 promoter (chr7:772,068-772,167) is identified as a footprint. (e) Two Reb1 binding sites in the REB1 promoter (chr2:336,885-337,084) are identified as footprints; a Cbf1 site predicted by ChIP shows a footprint, but a Rpn4 site defined by ChIP does not. (f) Two Pdr3 sites in the PDR5 promoter (chr15:619,227-619,476) are identified as footprints, in addition to an evolutionarily conserved region further upstream. Each panel shows per nucleotide DNase I cleavage, detected footprints (red boxes), assigned motifs (pink boxes), binding sites inferred from ChIP experiments (blue boxes), and evolutionary conservation (dark blue, Phastcons, bottom).
Mentions: Digital genomic footprinting data are sufficiently dense to enable analysis of regulatory factor occupancy patterns at the level of individual regulatory regions. The examples in Fig.4 and Supplementary Fig.6 provide snapshots of a diverse population of regulators and binding site contexts. In many cases, high-confidence footprints agree with previous predictions for specific regulators (Fig.4a,b,d,e and Supplementary Fig.6a). However, we also observed numerous examples of discordance (Fig.4c,e), possibly reflecting condition-specific binding. For example, at the REB1 promoter (Fig.4e), we detected footprints at two previously-identified evolutionarily-conserved Reb1 binding sites19, neither of which were identified under conditions used in prior ChIP experiments. Conversely, ChIP data annotated a nearby Rpn4 site that does not fall within an FDR=0.05 footprint.

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