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Dynamic remodeling of individual nucleosomes across a eukaryotic genome in response to transcriptional perturbation.

Shivaswamy S, Bhinge A, Zhao Y, Jones S, Hirst M, Iyer VR - PLoS Biol. (2008)

Bottom Line: Chromatin remodeling in response to physiological perturbation was typically associated with the eviction, appearance, or repositioning of one or two nucleosomes in the promoter, rather than broader region-wide changes.However, specific nucleosomal rearrangements were also evident at promoters even when there was no apparent transcriptional change, indicating that there is no simple, globally applicable relationship between chromatin remodeling and transcriptional activity.Our study provides a detailed, high-resolution, dynamic map of single-nucleosome remodeling across the yeast genome and its relation to global transcriptional changes.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cellular and Molecular Biology, Center for Systems and Synthetic Biology, and Section of Molecular Genetics and Microbiology, University of Texas at Austin,Austin, Texas, United States of America.

ABSTRACT
The eukaryotic genome is packaged as chromatin with nucleosomes comprising its basic structural unit, but the detailed structure of chromatin and its dynamic remodeling in terms of individual nucleosome positions has not been completely defined experimentally for any genome. We used ultra-high-throughput sequencing to map the remodeling of individual nucleosomes throughout the yeast genome before and after a physiological perturbation that causes genome-wide transcriptional changes. Nearly 80% of the genome is covered by positioned nucleosomes occurring in a limited number of stereotypical patterns in relation to transcribed regions and transcription factor binding sites. Chromatin remodeling in response to physiological perturbation was typically associated with the eviction, appearance, or repositioning of one or two nucleosomes in the promoter, rather than broader region-wide changes. Dynamic nucleosome remodeling tends to increase the accessibility of binding sites for transcription factors that mediate transcriptional changes. However, specific nucleosomal rearrangements were also evident at promoters even when there was no apparent transcriptional change, indicating that there is no simple, globally applicable relationship between chromatin remodeling and transcriptional activity. Our study provides a detailed, high-resolution, dynamic map of single-nucleosome remodeling across the yeast genome and its relation to global transcriptional changes.

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Ultra-High–Throughput Sequencing Recapitulates In Vivo Nucleosome Positions(A) Detailed view of the PHO5 locus showing the raw sequence reads (brown and red profiles). The nucleosome positions calculated using our analysis algorithm are shown as ovals, shaded according to their nucleosome score as indicated. The positions of the amplicons used for qPCR analysis are marked as red (peaks) and green (troughs) lines below. The black arrows indicate the positions of genes in that region.(B) qPCR verification of the three nucleosome peaks and three troughs identified by sequencing confirm that their positions remain the same before and after heat shock.(C) The heat-shock–induced SSA4 gene and flanking regions, showing that nucleosomes are displaced specifically at the SSA4 promoter and coding region after heat shock (thick purple arrow).(D) The heat-shock–repressed ribosomal protein gene RPL17B and flanking regions, showing that a single positioned nucleosome appears after heat shock specifically at the RPL17B promoter (thin purple arrow). The nucleosome positions calculated using our analysis algorithm are indicated as in (A).
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pbio-0060065-g001: Ultra-High–Throughput Sequencing Recapitulates In Vivo Nucleosome Positions(A) Detailed view of the PHO5 locus showing the raw sequence reads (brown and red profiles). The nucleosome positions calculated using our analysis algorithm are shown as ovals, shaded according to their nucleosome score as indicated. The positions of the amplicons used for qPCR analysis are marked as red (peaks) and green (troughs) lines below. The black arrows indicate the positions of genes in that region.(B) qPCR verification of the three nucleosome peaks and three troughs identified by sequencing confirm that their positions remain the same before and after heat shock.(C) The heat-shock–induced SSA4 gene and flanking regions, showing that nucleosomes are displaced specifically at the SSA4 promoter and coding region after heat shock (thick purple arrow).(D) The heat-shock–repressed ribosomal protein gene RPL17B and flanking regions, showing that a single positioned nucleosome appears after heat shock specifically at the RPL17B promoter (thin purple arrow). The nucleosome positions calculated using our analysis algorithm are indicated as in (A).

Mentions: We assessed the quality and accuracy of our nucleosome sequencing data by examining the nucleosomes known to be positioned at the PHO5 promoter. The yeast PHO5 promoter is repressed during growth in rich media by specifically positioned nucleosomes flanking a short, hypersensitive region containing a binding site for the transcription factor Pho4 [12]. These nucleosomes were evident in the alignment of our raw sequence reads, and their precise positions calculated by our analysis algorithm corresponded to the known positions of these nucleosomes. The positions of these three nucleosomes did not vary in the two independent biological samples before and after heat shock, as this perturbation does not affect the PHO5 promoter (Figure 1A). Quantitative real-time PCR (qPCR) for the three nucleosome peaks and three troughs (linker regions) identified by sequencing provided independent experimental verification of these nucleosome positions and the fact that their positions did not change in the two samples (Figure 1B). At individual promoters where transcription is activated by heat shock, the raw data traces and our inferred nucleosome peaks showed that nucleosomes were displaced at the promoter after the perturbation (Figure 1C). Conversely, at promoters that are repressed, positioned nucleosomes appeared after the perturbation (Figure 1D). The genome-wide nucleosome positions we identified experimentally correspond well with individual nucleosomes mapped on chromosome III as well as nucleosome-bound sequences isolated in previous studies [2,4] (see Figure S1 and Table S1). While this manuscript was in preparation, a catalog of nucleosome positions in yeast was published [13]. Our mapped nucleosome positions also agree well with this recent study (Figure S1 and Table S1). Thus, our mononucleosome preparations and the high-throughput sequencing assay recapitulated bona fide in vivo nucleosome positions and rearrangements.


Dynamic remodeling of individual nucleosomes across a eukaryotic genome in response to transcriptional perturbation.

Shivaswamy S, Bhinge A, Zhao Y, Jones S, Hirst M, Iyer VR - PLoS Biol. (2008)

Ultra-High–Throughput Sequencing Recapitulates In Vivo Nucleosome Positions(A) Detailed view of the PHO5 locus showing the raw sequence reads (brown and red profiles). The nucleosome positions calculated using our analysis algorithm are shown as ovals, shaded according to their nucleosome score as indicated. The positions of the amplicons used for qPCR analysis are marked as red (peaks) and green (troughs) lines below. The black arrows indicate the positions of genes in that region.(B) qPCR verification of the three nucleosome peaks and three troughs identified by sequencing confirm that their positions remain the same before and after heat shock.(C) The heat-shock–induced SSA4 gene and flanking regions, showing that nucleosomes are displaced specifically at the SSA4 promoter and coding region after heat shock (thick purple arrow).(D) The heat-shock–repressed ribosomal protein gene RPL17B and flanking regions, showing that a single positioned nucleosome appears after heat shock specifically at the RPL17B promoter (thin purple arrow). The nucleosome positions calculated using our analysis algorithm are indicated as in (A).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2267817&req=5

pbio-0060065-g001: Ultra-High–Throughput Sequencing Recapitulates In Vivo Nucleosome Positions(A) Detailed view of the PHO5 locus showing the raw sequence reads (brown and red profiles). The nucleosome positions calculated using our analysis algorithm are shown as ovals, shaded according to their nucleosome score as indicated. The positions of the amplicons used for qPCR analysis are marked as red (peaks) and green (troughs) lines below. The black arrows indicate the positions of genes in that region.(B) qPCR verification of the three nucleosome peaks and three troughs identified by sequencing confirm that their positions remain the same before and after heat shock.(C) The heat-shock–induced SSA4 gene and flanking regions, showing that nucleosomes are displaced specifically at the SSA4 promoter and coding region after heat shock (thick purple arrow).(D) The heat-shock–repressed ribosomal protein gene RPL17B and flanking regions, showing that a single positioned nucleosome appears after heat shock specifically at the RPL17B promoter (thin purple arrow). The nucleosome positions calculated using our analysis algorithm are indicated as in (A).
Mentions: We assessed the quality and accuracy of our nucleosome sequencing data by examining the nucleosomes known to be positioned at the PHO5 promoter. The yeast PHO5 promoter is repressed during growth in rich media by specifically positioned nucleosomes flanking a short, hypersensitive region containing a binding site for the transcription factor Pho4 [12]. These nucleosomes were evident in the alignment of our raw sequence reads, and their precise positions calculated by our analysis algorithm corresponded to the known positions of these nucleosomes. The positions of these three nucleosomes did not vary in the two independent biological samples before and after heat shock, as this perturbation does not affect the PHO5 promoter (Figure 1A). Quantitative real-time PCR (qPCR) for the three nucleosome peaks and three troughs (linker regions) identified by sequencing provided independent experimental verification of these nucleosome positions and the fact that their positions did not change in the two samples (Figure 1B). At individual promoters where transcription is activated by heat shock, the raw data traces and our inferred nucleosome peaks showed that nucleosomes were displaced at the promoter after the perturbation (Figure 1C). Conversely, at promoters that are repressed, positioned nucleosomes appeared after the perturbation (Figure 1D). The genome-wide nucleosome positions we identified experimentally correspond well with individual nucleosomes mapped on chromosome III as well as nucleosome-bound sequences isolated in previous studies [2,4] (see Figure S1 and Table S1). While this manuscript was in preparation, a catalog of nucleosome positions in yeast was published [13]. Our mapped nucleosome positions also agree well with this recent study (Figure S1 and Table S1). Thus, our mononucleosome preparations and the high-throughput sequencing assay recapitulated bona fide in vivo nucleosome positions and rearrangements.

Bottom Line: Chromatin remodeling in response to physiological perturbation was typically associated with the eviction, appearance, or repositioning of one or two nucleosomes in the promoter, rather than broader region-wide changes.However, specific nucleosomal rearrangements were also evident at promoters even when there was no apparent transcriptional change, indicating that there is no simple, globally applicable relationship between chromatin remodeling and transcriptional activity.Our study provides a detailed, high-resolution, dynamic map of single-nucleosome remodeling across the yeast genome and its relation to global transcriptional changes.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cellular and Molecular Biology, Center for Systems and Synthetic Biology, and Section of Molecular Genetics and Microbiology, University of Texas at Austin,Austin, Texas, United States of America.

ABSTRACT
The eukaryotic genome is packaged as chromatin with nucleosomes comprising its basic structural unit, but the detailed structure of chromatin and its dynamic remodeling in terms of individual nucleosome positions has not been completely defined experimentally for any genome. We used ultra-high-throughput sequencing to map the remodeling of individual nucleosomes throughout the yeast genome before and after a physiological perturbation that causes genome-wide transcriptional changes. Nearly 80% of the genome is covered by positioned nucleosomes occurring in a limited number of stereotypical patterns in relation to transcribed regions and transcription factor binding sites. Chromatin remodeling in response to physiological perturbation was typically associated with the eviction, appearance, or repositioning of one or two nucleosomes in the promoter, rather than broader region-wide changes. Dynamic nucleosome remodeling tends to increase the accessibility of binding sites for transcription factors that mediate transcriptional changes. However, specific nucleosomal rearrangements were also evident at promoters even when there was no apparent transcriptional change, indicating that there is no simple, globally applicable relationship between chromatin remodeling and transcriptional activity. Our study provides a detailed, high-resolution, dynamic map of single-nucleosome remodeling across the yeast genome and its relation to global transcriptional changes.

Show MeSH
Related in: MedlinePlus