The histone deacetylase Rpd3p is required for transient changes in genomic expression in response to stress

Abstract

Background: Yeast responding to stress activate a large gene expression program called the Environmental Stress Response that consists of approximately 600 repressed genes and approximately 300 induced genes. Numerous factors are implicated in regulating subsets of Environmental Stress Response genes; however, a complete picture of Environmental Stress Response regulation remains unclear. We investigated the role of the histone deacetylase Rpd3p, previously linked to the upstream regions of many Environmental Stress Response genes, in producing Environmental Stress Response gene expression changes in response to stress.

Results: We found that the Rpd3-Large complex is required for proper expression of both induced and repressed Environmental Stress Response genes under multiple stress conditions. Cells lacking RPD3 or the Rpd3-Large subunit PHO23 had a major defect in Environmental Stress Response initiation, particularly during the transient phase of expression immediately after stress exposure. Chromatin-immunoprecipitation showed a direct role for Rpd3-Large at representative genes; however, there were different effects on nucleosome occupancy and histone deacetylation at different promoters. Computational analysis implicated regulators that may act with Rpd3p at Environmental Stress Response genes. We provide genetic and biochemical evidence that Rpd3p is required for binding and action of the stress-activated transcription factor Msn2p, although the contribution of these factors differs for different genes.

Conclusions: Our results implicate Rpd3p as an important co-factor in the Environmental Stress Response regulatory network, and suggest the importance of histone modification in producing transient changes in gene expression triggered by stress.

Figure 1

Rpd3p is required for stress-dependent activation of the environmental stress response. Gene expression in wild-type and rpd3Δ cells responding to 25°C to 37°C heat shock (left panels), 0.4 mM H₂O₂ treatment (middle panels), or 0.75 M NaCl exposure (right panels) as described in Materials and methods. (a) The gene expression diagram represents the induced (red) or repressed (green) expression measurements of each gene (represented as rows) in the protein synthesis (PS), ribosomal protein (RP), and induced environmental stress response (iESR) gene groups for each microarray experiment (represented as columns organized temporally within each time course). The difference ('dif.') between wild type and rpd3Δ is represented to the right of each expression diagram: yellow indicates weaker repression and blue indicates weaker induction in the rpd3Δ mutant. Basal expression differences between rpd3Δ and wild type grown in the absence of stress are also shown. (b) The average log₂ expression change of genes in the PS, RP, and iESR subgroups shown in (a) plotted for wild type and rpd3Δ cells. Time points with statistically smaller changes in expression in rpd3Δ cells (P < 0.01, paired t-test) are indicated with an asterisk.

Figure 2

Rpd3p is bound upstream of several target genes after stress. Rpd3-myc binding upstream of several genes (including the positive control INO1, PS gene GAR1, RP gene RPL16A, and iESR genes UBC5 and XKS1) was assessed using ChIP before and 10 minutes after 0.4 mM H₂O₂ treatment or cold phosphate-buffered saline shock (see Materials and methods for details). The log2 enrichment of each fragment recovered from the Rpd3-myc expressing strain versus an untagged control strain is shown, for unstressed cells and cells responding to stress, according to the key on the right. Error bars represent the standard deviation of biological triplicates. The enrichment of each locus in whole-cell extracts (WCE) is shown as a control.

Figure 3

Rpd3p mediates stress-dependent changes in histone acetylation. Changes in nucleosome occupancy (NucOcc) and histone H4 acetylation (H4Ac) at specific nucleosomes (blue bars) spanning representative repressed (green) and induced (red) ESR genes shown in Figure 2 was measured in wild-type, rpd3Δ and pho23Δ cells responding to 0.4 mM H₂O₂ treatment (see Materials and methods for details). The log2 changes in (a) nucleosome occupancy and (b) fraction of nucleosomes acetylated on H4 following H₂O₂ exposure is shown for each gene. Error bars represent the range of two replicates for wild type or the standard deviation of at least three experiments for rpd3Δ and pho23Δ. H4 acetylation levels were normalized to levels of nucleosome occupancy to capture the change in the fraction of acetylated nucleosomes. (c) Expression changes of each gene as measured by microarray experiments at 10, 20, 30, 40, and 60 minutes after H₂O₂ treatment in wild-type, rpd3Δ and pho23Δ cells, according to the key shown.

Figure 4

Targets of Hsf1p and Yap1p show conditional dependence on Rpd3p. The average expression of (a) Hsf1p targets [14] or (b) Yap1p targets [1] was plotted for wild-type (dark purple) and rpd3 (light purple) cells responding to heat shock (left panels), H₂O₂ treatment (middle panels), or NaCl exposure (right panels) as described in Materials and methods. Time points with smaller expression changes in rpd3Δ cells (P < 0.01, paired t-test) are indicated with an asterisk.

Figure 5

Rpd3p is required for proper Msn2/4p action. (a-c) Gene expression measured in wild-type (WT), rpd3Δ, msn2Δ /msn4Δ, and msn2Δ /msn4Δ /rpd3Δ cells treated with 0.4 mM H₂O₂ for 30 minutes. Average log₂ expression changes of (a) 215 genes equally affected by deletion of RPD3, MSN2/MSN4, or MSN2/MSN4/RPD3, (b) 83 genes affected more by deletion of MSN2/MSN4 than RPD3, and (c) 103 genes that display additive dependence on RPD3 and MSN2/MSN4. The standard deviation of the genes' expression is shown for each gene group. (d) Msn2p binding before and 10 minutes after 0.4 mM H₂O₂ treatment in wild-type and rpd3Δ cells, according to the key for: TSA2 (from (a)), DDR2 (from (b)), YGP1 (from (c)), HOR7 (dependent on Rpd3p only), and YPS127W (dependent on Msn2/4p but not Rpd3p). Fold-change in Msn2p occupancy between stressed and unstressed cells is listed below each plot. Error bars represent the standard deviation of triplicate experiments.