Distinct patterns of histone acetyltransferase and Mediator deployment at yeast protein-coding genes

Abstract

The transcriptional coactivators Mediator and two histone acetyltransferase (HAT) complexes, NuA4 and SAGA, play global roles in transcriptional activation. Here we explore the relative contributions of these factors to RNA polymerase II association at specific genes and gene classes by rapid nuclear depletion of key complex subunits. We show that the NuA4 HAT Esa1 differentially affects certain groups of genes, whereas the SAGA HAT Gcn5 has a weaker but more uniform effect. Relative dependence on Esa1 and Tra1, a shared component of NuA4 and SAGA, distinguishes two large groups of coregulated growth-promoting genes. In contrast, we show that the activity of Mediator is particularly important at a separate, small set of highly transcribed TATA-box-containing genes. Our analysis indicates that at least three distinct combinations of coactivator deployment are used to generate moderate or high transcription levels and suggests that each may be associated with distinct forms of regulation.

Keywords: Esa1; Gcn5; Mediator; Tra1; histone acetylation; transcription.

Figure 1.

(A) Genome browser tracks showing a region of chromosome XII for H4ac and RNAPII ChIP-seq in Esa1 nuclear-depleted and nondepleted cells and H3K9ac and RNAPII ChIP-seq in Gcn5 nuclear-depleted and nondepleted cells. Gene annotations are shown below the tracks. (B) Scatter plots showing H4ac (left panel) and RNAPII (right panel) ChIP-seq in Esa1 nuclear-depleted cells (Esa1⁻; Y-axis) versus nondepleted cells (Esa1⁺; X-axis). Each dot represents a gene (5038 genes in total); the color of the dots represents the density of the points (from more dense [red] to less dense [blue]). The signal for H4ac was normalized to the H3 signal. The average signal for H4ac and H3 ChIP was quantified in a window of 500 base pairs (bp) centered on the transcription start site (TSS). For RNAPII, the average signal was quantified from the TSS to the transcription termination site (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) Negatively affected genes; (red arrow) positively affected genes. The scale for both the X-axis and the Y-axis is log₁₀. (C) Scatter plot 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 nondepleted cells (Esa1⁻/Esa1⁺). Pearson correlation coefficient and P-value are shown. (D) Scatter plots showing H3K9ac (left panel) and RNAPII (right panel) ChIP-seq in Gcn5 nuclear-depleted cells versus nondepleted 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 vs. nondepleted cells) for 4682 genes categorized in TFIID-dominated and SAGA-dominated according to Huisinga and Pugh (2004).

Figure 2.

(A) Experimental outline. Yeast cells were treated with 1 µg/mL rapamycin to induce Esa1 or Gcn5 nuclear depletion. After 60 min of rapamycin (or vehicle control) treatment, 1.5 mM diamide 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 = 0) separately for Esa1 nuclear-depleted (Esa1⁻) and nondepleted (Esa1⁺) cells for the diamide-up-regulated genes (calculated as up-regulated genes at 20 min following diamide addition in the Esa1-FRB-tagged strain untreated with rapamycin, as in Supplemental Fig. S2A) in Esa1 nuclear-depleted (Esa1⁻; dashed line) and nondepleted (Esa1⁺; continuous line) cells. (C) Heat map representing the result of a k-means clustering analysis on the change in RNAPII occupancy (average signal from the TSS to the TTS) on the 1346 diamide-up-regulated genes, calculated as log₂ ratio of the RNAPII signal in Esa1 nuclear-depleted (Esa1⁻) versus nondepleted (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 min following diamide addition in the Esa1-FRB-tagged strain not 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 min following diamide addition in the Gcn5-FRB-tagged strain not treated with rapamycin) in Gcn5 nuclear-depleted (Gcn5⁻; dashed line) and nondepleted (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 min following diamide addition in the Gcn5-FRB-tagged strain not treated with rapamycin).

Figure 3.

(A) Scatter plots showing H3K9ac (left), H4ac (middle), and RNAPII (right) ChIP-seq in Tra1 nuclear-depleted versus nondepleted 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) Heat maps showing 5037 genes (rows) sorted according to RNAPII change in Tra1 nuclear-depleted versus nondepleted cells. (Blue) RiBi genes; (orange) RPGs. (D) Scatter plots 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 vs. nondepleted 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) Scatter plots 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, n = 390; TATA⁺ TAF1⁺ group, n = 307; TATA⁻ TAF1⁻ group, n = 503; TATA⁻ TAF1⁺ group, n = 2790. (C) Heat maps showing TBP occupancy (Kubik et al. 2018) (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 nondepleted cells. (E) Scatter plots showing Med8 occupancy in Esa1 (left) or Gcn5 (right) nuclear-depleted cells (Y-axis) and nondepleted cells (X-axis) for the 2000 genes having the strongest Med8 ChEC signal in their UASs.

Figure 5.

(A) Heat map 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 nascent elongating transcript sequencing (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 et al. 2018). (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.

Figure 6.

Schematic representation of three promoter configurations associated with moderate and high transcription. The intensity of the colors (Mediator, NuA4, and Esa1) reflects the importance of the coactivator in transcription. The size of the arrow next to the +1 nucleosome indicates the transcription level. The sizes and positions of the coactivators in the cartoon are illustrative. For simplicity, TFIID binding downstream from the TSS is not depicted.