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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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Higher-order patterns of DNA accessibility(a) Mapped DNase I cleavages relative to 5,006 TSSs29. Four major clusters are exposed by k-means analysis (red, blue, green and purple bars, respectively). In the red cluster, maximal DNase I cleavage occurs in a stereotypic ∼50 bp band ∼100 bp upstream of the TSS (grey arrowhead, top). In the blue, green and purple clusters, the extent and intensity of DNase I cleavage upstream of the TSS widens to the -1, -2, and -3 nucleosomes (respectively). (b) Spatial restriction of footprints near TSSs. Distribution of footprints matching Reb1, Abf1, Rap1, Mcm1, Pdr3, Cbf1 and Hsf1 relative to the TSSs (dashed black lines) and start codons of 1,260 genes sorted by the length of the 5′UTR. Enrichment within a ∼50 bp region centered ∼100 bp upstream of the TSS (dashed red lines). (c) DNase I cleavage profiles aligned relative to Reb1, Abf1, Rap1 and Mcm1 footprints. (d) mRNA abundance for genes found in each of the four clusters correlates with the accessibility of the promoters of those genes (colors as in a; median expression denoted by a black bar).
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Figure 5: Higher-order patterns of DNA accessibility(a) Mapped DNase I cleavages relative to 5,006 TSSs29. Four major clusters are exposed by k-means analysis (red, blue, green and purple bars, respectively). In the red cluster, maximal DNase I cleavage occurs in a stereotypic ∼50 bp band ∼100 bp upstream of the TSS (grey arrowhead, top). In the blue, green and purple clusters, the extent and intensity of DNase I cleavage upstream of the TSS widens to the -1, -2, and -3 nucleosomes (respectively). (b) Spatial restriction of footprints near TSSs. Distribution of footprints matching Reb1, Abf1, Rap1, Mcm1, Pdr3, Cbf1 and Hsf1 relative to the TSSs (dashed black lines) and start codons of 1,260 genes sorted by the length of the 5′UTR. Enrichment within a ∼50 bp region centered ∼100 bp upstream of the TSS (dashed red lines). (c) DNase I cleavage profiles aligned relative to Reb1, Abf1, Rap1 and Mcm1 footprints. (d) mRNA abundance for genes found in each of the four clusters correlates with the accessibility of the promoters of those genes (colors as in a; median expression denoted by a black bar).

Mentions: We next sought to visualize patterns of DNase I cleavage and protection at the level of extended promoter domains. We extracted DNase I cleavage data from -1 kb to +1 kb intervals around the TSSs of ∼5,000 yeast genes and performed hierarchical clustering (Fig.5a). This revealed that 93% of yeast genes could be organized into four distinct clusters, ranging from low (red cluster) to high (purple) mean chromatin accessibility (Fig.5a). For genes in the red cluster, chromatin accessibility was maximal over the -100 region, visualized in Fig.5a as a prominent central vertical yellow stripe. Even at this resolution, a ∼10 bp footprint centrally positioned within the -100 region could be discerned at a surprising proportion of genes (Fig.5a). A prominent feature of the DNase I cleavage patterns is the presence of regular undulations in accessibility, with a period of ∼175 bp symmetrically flanking the central high-accessibility zone (Fig.5a). This pattern is consistent with the presence of phased nucleosomes. We further observed that the periodic pattern emanated from the boundaries of the central high-accessibility region, even though this region varied in size between the four clusters. This observation suggested that phased nucleosomes were in fact distributed relative to central sites occupied by factors. To explore further the relationship between nucleosome-level features and factor occupancy, we examined the long-range distribution of DNase I cleavages surrounding footprints of individual regulators across the genome. The distribution of DNase I cleavages relative to footprints for Reb1 and Abf1 revealed periodic undulations, consistent with phased nucleosome arrays symmetrically distributed relative to the factor-binding sites. However, Rap1 and Mcm1 exhibited less prominent patterns (Fig.5c), suggesting that some factors (e.g. Reb1 and Abf1) have a more determinative role in establishing chromatin architecture at promoters20. Collectively, these data are consistent with statistical positioning of nucleosomes relative to factor binding-induced ‘barrier’ events21,22.


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)

Higher-order patterns of DNA accessibility(a) Mapped DNase I cleavages relative to 5,006 TSSs29. Four major clusters are exposed by k-means analysis (red, blue, green and purple bars, respectively). In the red cluster, maximal DNase I cleavage occurs in a stereotypic ∼50 bp band ∼100 bp upstream of the TSS (grey arrowhead, top). In the blue, green and purple clusters, the extent and intensity of DNase I cleavage upstream of the TSS widens to the -1, -2, and -3 nucleosomes (respectively). (b) Spatial restriction of footprints near TSSs. Distribution of footprints matching Reb1, Abf1, Rap1, Mcm1, Pdr3, Cbf1 and Hsf1 relative to the TSSs (dashed black lines) and start codons of 1,260 genes sorted by the length of the 5′UTR. Enrichment within a ∼50 bp region centered ∼100 bp upstream of the TSS (dashed red lines). (c) DNase I cleavage profiles aligned relative to Reb1, Abf1, Rap1 and Mcm1 footprints. (d) mRNA abundance for genes found in each of the four clusters correlates with the accessibility of the promoters of those genes (colors as in a; median expression denoted by a black bar).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2668528&req=5

Figure 5: Higher-order patterns of DNA accessibility(a) Mapped DNase I cleavages relative to 5,006 TSSs29. Four major clusters are exposed by k-means analysis (red, blue, green and purple bars, respectively). In the red cluster, maximal DNase I cleavage occurs in a stereotypic ∼50 bp band ∼100 bp upstream of the TSS (grey arrowhead, top). In the blue, green and purple clusters, the extent and intensity of DNase I cleavage upstream of the TSS widens to the -1, -2, and -3 nucleosomes (respectively). (b) Spatial restriction of footprints near TSSs. Distribution of footprints matching Reb1, Abf1, Rap1, Mcm1, Pdr3, Cbf1 and Hsf1 relative to the TSSs (dashed black lines) and start codons of 1,260 genes sorted by the length of the 5′UTR. Enrichment within a ∼50 bp region centered ∼100 bp upstream of the TSS (dashed red lines). (c) DNase I cleavage profiles aligned relative to Reb1, Abf1, Rap1 and Mcm1 footprints. (d) mRNA abundance for genes found in each of the four clusters correlates with the accessibility of the promoters of those genes (colors as in a; median expression denoted by a black bar).
Mentions: We next sought to visualize patterns of DNase I cleavage and protection at the level of extended promoter domains. We extracted DNase I cleavage data from -1 kb to +1 kb intervals around the TSSs of ∼5,000 yeast genes and performed hierarchical clustering (Fig.5a). This revealed that 93% of yeast genes could be organized into four distinct clusters, ranging from low (red cluster) to high (purple) mean chromatin accessibility (Fig.5a). For genes in the red cluster, chromatin accessibility was maximal over the -100 region, visualized in Fig.5a as a prominent central vertical yellow stripe. Even at this resolution, a ∼10 bp footprint centrally positioned within the -100 region could be discerned at a surprising proportion of genes (Fig.5a). A prominent feature of the DNase I cleavage patterns is the presence of regular undulations in accessibility, with a period of ∼175 bp symmetrically flanking the central high-accessibility zone (Fig.5a). This pattern is consistent with the presence of phased nucleosomes. We further observed that the periodic pattern emanated from the boundaries of the central high-accessibility region, even though this region varied in size between the four clusters. This observation suggested that phased nucleosomes were in fact distributed relative to central sites occupied by factors. To explore further the relationship between nucleosome-level features and factor occupancy, we examined the long-range distribution of DNase I cleavages surrounding footprints of individual regulators across the genome. The distribution of DNase I cleavages relative to footprints for Reb1 and Abf1 revealed periodic undulations, consistent with phased nucleosome arrays symmetrically distributed relative to the factor-binding sites. However, Rap1 and Mcm1 exhibited less prominent patterns (Fig.5c), suggesting that some factors (e.g. Reb1 and Abf1) have a more determinative role in establishing chromatin architecture at promoters20. Collectively, these data are consistent with statistical positioning of nucleosomes relative to factor binding-induced ‘barrier’ events21,22.

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