M.J.B. and D.S. conceived the study. M.J.B., S.G., S.K., G.E.Z. and D.S. designed the experiments.
M.J.B. and S.K. performed the experiments. S.G. performed the ChEC-Seq experiments. M.J.B, S.K., S.G. and G.E.Z. carried out the bioinformatics analysis. M.J.B., S.G. and D.S. wrote the manuscript with inputs from all of the authors.
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Acknowledgments
We thank members of the Shore lab for useful discussion, Steven Hahn for comments on the manuscript, Uli Laemmli for help with the anchor-away strains and method, Mylene Docquier and the iGE3 genomics platform of the University of Geneva (http://www.ige3.unige.ch/genomics-platform.php) for high throughput DNA sequencing, Nicolas Roggli for expert artwork, Jacques Rougemont, Jean-Luc Falcone and Srinivas Ramachandran for discussion on spike controls, and Thomas Schalch for the use of his local Galaxy server. M. J. B. was supported in part by an iGE3 Ph.D.
student fellowship; S.G. was supported by NIH grant GM075114. D.S. acknowledges support from the Swiss National Fund and from the Republic and Canton of Geneva.
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Figure Legends
Figure 1. (A) Genome Browser tracks showing a region of Chromosome XII for H4ac and RNAPII ChIP-Seq in Esa1 nuclear depleted and non-depleted cells and for H3K9ac and RNAPII ChIP-ChIP-Seq in Gcn5 nuclear depleted and non-depleted cells. Gene annotations are shown below the tracks. (B) Scatterplots showing H4ac (left panel) and RNAPII (right panel) ChIP-Seq in Esa1 nuclear depleted cells (y-axis, Esa1-) versus non-depleted cells (x-axis, Esa1+). Each dot represents a gene (5038 genes in total); the color of the dots represents the density of the points (from red more dense to blue less dense). The signal for H4ac has been normalized to H3 signal. The average signal for H4ac and H3 ChIP has been quantified in a window of 500bp centered on TSS. For RNAPII the average signal has been quantified from TSS to TTS. Blue and red lines on the plots represent the threshold of 1.5 fold change. The number next to the blue and red arrows indicates the number of genes above the threshold (blue arrow for negatively affected genes, red arrow for positively affected ones). The scale for both the x- and the y-axis is Log10. (C) Scatterplot comparing the change in H4ac (normalized to H3, x-axis) and the change in RNAPII (y-axis) calculated as Log2 ratio between the signal in Esa1 nuclear depleted cells and non-depleted cells (Esa1-/Esa1+). Pearson correlation coefficient and p-value are shown. (D) Scatterplots showing H3K9ac (left panel) and RNAPII (right panel) ChIP-Seq in Gcn5 nuclear depleted cells versus non-depleted cells calculated and plotted as in (B). (E) Box plots showing RNAPII change in Esa1 (left) and Gcn5 (right) nuclear depleted cells (calculated as Log2 ratio of nuclear depleted versus non-depleted cells) for 4682 genes categorized in TFIID-dominated and SAGA-dominated according to (Huisinga and Pugh 2004).
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Figure 2. (A) Experimental outline. Yeast cells were treated with rapamycin (1 µg/mL) to induce Esa1 or Gcn5 nuclear depletion. After 60 minutes of rapamycin (or vehicle control) treatment, diamide (1.5 mM) was added to the cultures and cells were harvested for cross-linking and RNAPII ChIP-Seq at the indicated time points. (B) Plot showing average RNAPII signal (log2) normalized to time 0 (time 0 equal 0) separately for Esa1 nuclear depleted (Esa1-) and non-depleted (Esa1+) cells, for the diamide up-regulated genes (calculated as up-regulated genes at 20 minutes following diamide addition in Esa1-FRB tagged strain non-treated with rapamycin as in Figure S2A) in Esa1 nuclear depleted (Esa1-, dashed line) and non-depleted (Esa1+, continuous line) cells. (C) Heatmap representing the result of a k-means clustering analysis on the change in RNAPII occupancy (average signal from TSS to TTS) on the 1346 diamide up-regulated genes calculated as log2 ratio of the RNAPII signal in Esa1 nuclear depleted (Esa1-) versus non-depleted (Esa1+) cells. (D) Plots showing average RNAPII signal for the gene groups identified in (C) and plotted as in (B). (E) Bar plots showing the percentage of genes defined as SAGA-dominated (light brown) and TFIID-dominated (dark brown) as reported in (Huisinga and Pugh 2004) for each of the five groups identified in (C). (F) Plots showing average RNAPII signal (log2) for the diamide down-regulated genes (calculated as down-regulated genes at 20 minutes following diamide addition in Esa1-FRB tagged strain non-treated with rapamycin) plotted as in (B). (G) Plots showing average RNAPII signal (log2) for the diamide up-regulated genes (calculated as up-regulated genes at 20 minutes following diamide addition in Gcn5-FRB tagged strain non-treated with rapamycin) in Gcn5 nuclear depleted (Gcn5-, dashed line) and non-depleted (Gcn5+, continuous line) cells, plotted as in (B). (H) Same as in (G) for the diamide down-regulated genes (calculated as down-regulated genes at 20 minutes following diamide addition in Gcn5-FRB tagged strain non-treated with rapamycin).
Figure 3. (A) Scatterplots showing H3K9ac (left), H4ac (middle) and RNAPII (right) ChIP-Seq in Tra1 nuclear depleted versus non-depleted cells calculated and plotted as in Figure 1B. (B) Venn diagram depicting the overlap between genes affected at least 1.5 fold in RNAPII occupancy by Tra1 nuclear depletion and Gcn5 nuclear depletion (top) and by Tra1 nuclear depletion and Esa1 nuclear depletion (bottom). (C) Heatmaps showing 5037 genes (rows) sorted according to RNAPII change in Tra1 nuclear depleted vs non-depleted cells. RiBi genes are in blue, RPGs in orange. (D) Scatterplots
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showing H3 (top left), H3K9ac (top right), H4ac (bottom left) and RNAPII (bottom right) ChIP-Seq in Tra1 and Gcn5 nuclear depleted cells calculated and plotted as in Figure 1B. (E) Box plots showing RNAPII change in Gcn5, Tra1, and Gcn5 and Tra1 nuclear depleted cells (calculated as Log2 ratio of nuclear depleted versus non-depleted cells) for 4783 genes divided according to the effect of Gcn5 and Tra1 double depletion in “additive effect” (41 genes, top panel) and “synergistic effect” (4742 genes, bottom panel).
Figure 4. (A) Scatterplots showing RNAPII ChIP-Seq in Med17 nuclear depleted cells calculated and plotted as in Figure 1B. (B) Box plots showing RNAPII change in Med17 nuclear depleted cells for 3992 genes calculated and plotted as in Figure 1E and categorized according to the presence or absence of the TATA box and the promoter binding of TAF1 as reported in (Rhee and Pugh 2012) (TATA+ TAF1- group= 390, TATA+ TAF1+ group=307 genes, TATA- TAF1- group= 503, TATA- TAF1+
group=2790). (C) Heatmaps showing TBP occupancy (Kubik and Shore, unpublished data) (right) and Med8 ChEC signal (left) of 5036 yeast genes aligned to the TSS and sorted according to the change in RNAPII occupancy in Med17 nuclear depleted cells. (D) Genome Browser tracks showing a region of chromosome II for Med8 ChEC-Seq in Esa1 and Gcn5 nuclear depleted and non-depleted cells. (E) Scatterplots showing Med8 occupancy in Esa1 (left) or Gcn5 (right) nuclear depleted cells (y-axis) and non-depleted cells (x-axis) for the 2000 genes having the strongest Med8 ChEC signal in their UAS.
Figure 5. (A) Heatmap representing the result of a k-means clustering analysis on the change in RNAPII occupancy measured in Esa1, Gcn5, Tra1 and Med17 nuclear depleted cells. (B) Box plots showing the transcription rate measured by Net-Seq (Churchman and Weissman 2011) for the genes in the five clusters described in (A). (C-D) Bar plots showing the number of Ribi genes (C) and RPGs (D) in each of the five clusters. (E) Box plot showing TBP promoter occupancy for the genes in the five clusters (Kubik and Shore, unpublished data). (F) Bar plots showing the percentage of genes having a well-defined TATA box in their promoter (green) and significant TAF1 binding measured by ChIP (purple) as reported in (Rhee and Pugh 2012) for each of the five clusters.
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Figure 6. Schematic representation of three promoter configurations associated with moderate and high transcription. Intensity of colors (Mediator, NuA4 and Esa1) reflects importance of the co-activator in transcription. Size of the arrow next to +1 nucleosome reveals transcription level.