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. 2004;5(9):R62.
doi: 10.1186/gb-2004-5-9-r62. Epub 2004 Aug 20.

Global nucleosome occupancy in yeast

Affiliations

Global nucleosome occupancy in yeast

Bradley E Bernstein et al. Genome Biol. 2004.

Abstract

Background: Although eukaryotic genomes are generally thought to be entirely chromatin-associated, the activated PHO5 promoter in yeast is largely devoid of nucleosomes. We systematically evaluated nucleosome occupancy in yeast promoters by immunoprecipitating nucleosomal DNA and quantifying enrichment by microarrays.

Results: Nucleosome depletion is observed in promoters that regulate active genes and/or contain multiple evolutionarily conserved motifs that recruit transcription factors. The Rap1 consensus was the only binding motif identified in a completely unbiased search of nucleosome-depleted promoters. Nucleosome depletion in the vicinity of Rap1 consensus sites in ribosomal protein gene promoters was also observed by real-time PCR and micrococcal nuclease digestion. Nucleosome occupancy in these regions was increased by the small molecule rapamycin or, in the case of the RPS11B promoter, by removing the Rap1 consensus sites.

Conclusions: The presence of transcription factor-binding motifs is an important determinant of nucleosome depletion. Most motifs are associated with marked depletion only when they appear in combination, consistent with a model in which transcription factors act collaboratively to exclude nucleosomes and gain access to target sites in the DNA. In contrast, Rap1-binding sites cause marked depletion under steady-state conditions. We speculate that nucleosome depletion enables Rap1 to define chromatin domains and alter them in response to environmental cues.

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Figures

Figure 1
Figure 1
Correlation between H3 and FLAG-H2B ChIP datasets. DNA associated with histones in vivo was enriched in ChIP assays using antibodies against histone H3 or FLAG-H2B, and quantified by microarrays. (a) Relative enrichment of promoters and other non-coding regions in the H3 and H2B ChIP assays is shown. (b) Histogram showing distributions of enrichment for promoter regions in the H3 and H2B ChIP assays. (c) Overlap between regions depleted in the H3 and FLAG-H2B assays is shown. Overall, there is an 0.83 correlation between these ChIP datasets. (d) Overlap between regions depleted in the H3 ChIP assay and regions enriched by aqueous extraction is shown [62].
Figure 2
Figure 2
Inverse association between nucleosome occupancy and promoter strength. (a) Relative enrichment of promoter regions in the H3 and FLAG-H2B ChIP assays plotted against transcription rate of downstream genes (moving average, window 50). (b) Overlap between promoters upstream of active genes and the set of nucleosome-depleted promoters defined on the basis of depletion across the replicate H3 and FLAG-H2B experiments.
Figure 3
Figure 3
Sequence motifs over-represented in nucleosome-depleted promoters. (a) An unbiased search for sequences up to 10 bp in length over-represented in nucleosome-depleted promoters (relative to promoters overall) identified the poly(dA.dT) sequence element and variants of the Rap1 consensus motif ACACCCATACAT [21]. (b) Overlap between nucleosome-depleted promoters and promoters that contain multiple conserved motifs is shown [21]. (c) Histogram showing average numbers of motifs in 1,000 randomly generated promoter sets. Nucleosome-depleted promoters contain an average of 6.1 conserved motifs, significantly higher than in these randomly generated sets.
Figure 4
Figure 4
Nucleosome depletion in the vicinity of Rap1-binding sites. (a) Overlap between the 308 most nucleosome-depleted promoters and promoters found to recruit Rap1 in a global ChIP study [1]. (b) Nucleosome depletion in the vicinity of Rap1-binding sites in ribosomal gene promoters evaluated by ChIP. Fold-enrichment was determined by real-time PCR using primers that span Rap1-binding motifs in the RPS22A, RPS15, RPS11B and RPL23A promoters. (c) Southern blots showing DNA from yeast spheroplasts digested with increasing concentrations of micrococcal nuclease probed with labeled PCR products spanning the TUB2 promoter and the Rap1 sites in the RPS11B and RPS15 promoters. (d) Nucleosome occupancy for a mutant RPS11B promoter lacking Rap1 consensus sites was determined by H3 ChIP and real-time PCR. The mutant promoter is enriched 2.1-fold relative to wild type. (e) Nucleosome occupancy at Rap1-binding sites in ribosomal protein gene promoters after treatment with rapamycin evaluated by H3 ChIP and real-time PCR.

References

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