The yeast Snt2 protein coordinates the transcriptional response to hydrogen peroxide-mediated oxidative stress

Abstract

Regulation of gene expression is a vital part of the cellular stress response, yet the full set of proteins that orchestrate this regulation remains unknown. Snt2 is a Saccharomyces cerevisiae protein whose function has not been well characterized that was recently shown to associate with Ecm5 and the Rpd3 deacetylase. Here, we confirm that Snt2, Ecm5, and Rpd3 physically associate. We then demonstrate that cells lacking Rpd3 or Snt2 are resistant to hydrogen peroxide (H2O2)-mediated oxidative stress and use chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to show that Snt2 and Ecm5 recruit Rpd3 to a small number of promoters and in response to H2O2, colocalize independently of Rpd3 to the promoters of stress response genes. By integrating ChIP-seq and expression analyses, we identify target genes that require Snt2 for proper expression after H2O2. Finally, we show that cells lacking Snt2 are also resistant to nutrient stress imparted by the TOR (target of rapamycin) pathway inhibitor rapamycin and identify a common set of genes targeted by Snt2 and Ecm5 in response to both H2O2 and rapamycin. Our results establish a function for Snt2 in regulating transcription in response to oxidative stress and suggest Snt2 may also function in multiple stress pathways.

Fig 1

Snt2 associates with Ecm5 and the Rpd3 deacetylase. (A) Domain structures of Snt2, Ecm5, and Rpd3. BAH, bromo-adjacent homology; PHD, plant homeodomain finger; SANT, Spt3–Ada3–N-CoR–TFIIS (N-CoR stands for nuclear receptor corepressor 1, and TFIIS stands for transcription elongation factor IIS); ARID, AT-rich interaction domain; JmjC, Jumonji C; HDAC, histone deacetylase. (B) Silver-stained SDS-polyacrylamide gels of eluents from Snt2-PrA and control (no tag) affinity purifications. Proteins identified by LC-MS are listed next to bands of the appropriate size. Rpd3 comigrated on the gel with IgG. Numbers to the left are molecular mass in kilodaltons. (C) Coomassie blue-stained gel analysis of Ecm5-PrA and control purifications. Proteins identified by LC-MS are listed next to the corresponding bands. (D) Lysates of untagged, Ecm5-Myc, Snt2-GFP, or Ecm5-Myc Snt2-GFP strains were immunoprecipitated with anti-Myc antibody, and inputs and immunoprecipitates (IPs) were immunoblotted (IB) with anti-Myc (to detect Ecm5), anti-GFP (to detect Snt2), anti-Rpd3, or anti-Sin3.

Fig 2

Cells lacking Snt2 or Rpd3 are resistant to H₂O₂. (A) Fivefold serial dilutions of the wild-type (WT) strain or indicated knockout strains were spotted onto YPD plates that were untreated or supplemented with 2.3 mM H₂O₂. Plates were imaged after 2 days. (B) Log-phase cultures of the wild-type strain or indicated knockout strains were treated with 0.4 mM H₂O₂ for 4 h. Percent survival was determined by CFU assay. Data are means ± standard errors of the means (SEMs) (error bars) from 3 biological replicates. Values that are statistically significantly different (P < 0.01) are indicated by the bar and two asterisks. (C and D) Plate spotting assays with the indicated knockout strains were performed as described above for panel A.

Fig 3

H₂O₂ treatment does not affect the levels of Snt2 and Rpd3 associating with Ecm5 or the levels of Ecm5 and Snt2 associating with Rpd3. (A) Immunoblot analysis of Myc immunoprecipitations from ECM5-MYC SNT2-GFP (EM SG) or SNT2-GFP (SG) strains that were untreated (−) or treated with H₂O₂ (+). For Ecm5-Myc and Snt2-GFP immunoblots, input is 15% of IP; for Rpd3 immunoblot, input is 1% of IP. (B) Immunoblot analysis of Rpd3 immunoprecipitation from the ECM5-MYC SNT2-GFP strain treated as described above for panel A. Inputs are 15% of IPs.

Fig 4

Snt2 and Ecm5 are highly colocalized and associate with additional promoters after H₂O₂ stress. (A) ChIP-seq tracks showing Snt2 and Ecm5 ChIP enrichment in a representative region before and after H₂O₂ treatment. The coverage values for each track are scaled by 1,000,000/number of reads, and the scale of the y axes for all tracks is shown in brackets. The locations of genes and peaks and the chromosomal coordinates are shown under the tracks. chrII, chromosome II. (B) Venn diagrams show that the majority of Snt2 and Ecm5 ChIP peaks overlap. (C) Correlations between Snt2 and Ecm5 enrichment before and after H₂O₂ treatment (Pearson's correlation coefficients are indicated in the bottom right-hand corners of the graphs). (D) Genomic distributions of shared Snt2/Ecm5 peaks before and after H₂O₂ stress. (E) Average number of Snt2 or Ecm5 ChIP-seq reads per 50-bp window around transcription start sites (TSSs) for all yeast genes, scaled by 1,000,000/total reads. (F) Overlaps of Snt2 or Ecm5 peaks before and after H₂O₂ treatment. (G) Box-and-whisker plots showing the distributions of Snt2 and Ecm5 enrichment at all peak regions before and after H₂O₂ treatment in the treated and untreated data sets. The bottoms, middles, and tops of the boxes depict the 25th, 50th, and 75th percentiles, respectively, and the top and bottom whiskers depict the 10th and 90th percentiles, respectively, of Snt2 or Ecm5 enrichment levels. The P values were determined by Wilcoxon rank sum test. (H) Snt2 or Ecm5 enrichment levels at peaks after H₂O₂ treatment relative to the levels before treatment. Peaks where Snt2/Ecm5 enrichment was >1.5-fold increased, >1.5-fold decreased, or unchanged after treatment are colored purple, orange, or gray, respectively. (I) Motif analysis using the 20 most-enriched Snt2/Ecm5 peaks (left) or all peaks where Snt2/Ecm5 levels increased after treatment (right). (J) Categories of genes significantly enriched by GO analysis of Snt2/Ecm5 peaks that increased, decreased, or did not change enrichment after treatment. ox.stress response, oxidative stress response; a.a. metabolism, amino acid metabolism.

Fig 5

After H₂O₂ treatment, Snt2 and Ecm5 localize to stress and metabolism genes. (A) Examples of ESR genes whose promoters are enriched for Snt2 and Ecm5 after H₂O₂ treatment. For bidirectional promoters, the gene associated with the category above the panel is shown in a larger font. (B) ChIP-seq results were confirmed by ChIP-qPCR. Relative enrichment was determined by normalizing percent inputs at the target locus to percent inputs at a control region on the right arm of telomere 6. The means ± SEMs of 3 biological replicates are shown. Samples in which the ChIP enrichment after treatment differs significantly from enrichment before treatment are indicated by asterisks as follows: ∗, P < 0.05; ∗∗, P < 0.01. Enrichment in the ACT1 ORF is shown as a negative control.

Fig 6

Snt2 and Ecm5 are required for Rpd3 recruitment to superenriched promoters but not for recruitment of the Rpd3(L) complex member Sds3. (A) Promoters superenriched for Snt2 and Ecm5 (note the ranges in brackets for ChIP-seq tracks) before and after H₂O₂ treatment. (B) ChIP-qPCR confirmation of high levels of Snt2 and Ecm5 at superenriched promoters shown in panel A. Data were normalized as described in the legend to Fig. 5B. (C) Control immunoblots showing that deletion of SNT2 or ECM5 did not affect Rpd3-Myc levels and that deletion of SNT2 did not affect Sds3-Myc levels before or after H₂O₂ treatment. Actin blots serve as a loading controls. (D) Rpd3-Myc ChIP-qPCR performed on untagged, RPD3-MYC, RPD3-MYC snt2Δ, or RPD3-MYC ecm5Δ strains that were treated with H₂O₂. Data were normalized as described in the legend to Fig. 5B. ACT1 ORF is the negative control. (E) Sds3-Myc ChIP-qPCR performed on untagged, SDS3-MYC, or SDS3-MYC snt2Δ strains that were treated with H₂O₂. Values are means ± standard deviations of 3 independent measurements and are representative of 2 experiments.

Fig 7

Snt2 is required for proper expression of ChIP target genes after H₂O₂ treatment. (A) Numbers of genes significantly up- or downregulated from wild-type (WT) levels in the snt2Δ or ecm5Δ strains before and after H₂O₂ treatment. (B) Two cell wall genes misexpressed in opposite ways in the snt2Δ and ecm5Δ strains. The graph shows log₂ ratios of expression in the mutant (knockout [KO]) strains relative to the WT levels before treatment. (C) Categories of genes significantly up- or downregulated in the snt2Δ strain relative to wild-type levels after H₂O₂ treatment. (D) Overlap between Snt2/Ecm5 H₂O₂-enriched target genes that were up- or downregulated relative to WT levels in the snt2Δ strain after treatment and target genes that changed expression in the WT strain after treatment. (E) Target genes that function in either the oxidative stress response or the cytochrome c pathway that were up- or downregulated in the snt2Δ strain after treatment. The graph shows log₂ ratios of expression levels in the snt2Δ strain after treatment relative to WT levels after treatment. (F) Heat map showing expression levels of the 309 genes described above for panel D. Four clusters of genes with similar expression patterns are noted at the top of the heat map. (G) Scatterplots of the 309 genes described above for panel D, comparing the log₂ expression ratios in snt2Δ cells after treatment relative to WT levels after treatment (y axis) to the log₂ expression ratios in WT cells after treatment relative to WT levels before treatment (x axis). In the three right scatterplots, genes in the categories above the plots are colored red. (H) Overlap between the 309 genes described above for panel D and Ste12 or Rap1 target genes. TF targets, transcription factor targets.

Fig 8

Rapamycin treatment recapitulates a subset of the Snt2 and Ecm5 localization changes seen after H₂O₂ treatment. (A) Serial dilutions of the indicated WT or knockout strains were spotted onto SD CSM plates supplemented with DMSO alone or rapamycin dissolved in DMSO (50 nM final concentration). (B) Snt2/Ecm5 ChIP-seq enrichment in control and rapamycin (RAP)-treated cells at a representative region on chromosome VII. Arrows indicate regions where Snt2 and Ecm5 levels were enriched after rapamycin treatment. (C) Overlap between peaks where the levels of Snt2 and Ecm5 were at least 1.5-fold higher after rapamycin treatment and peaks where Snt2/Ecm5 levels were higher after H₂O₂ treatment. (D) Promoters enriched for Snt2 and Ecm5 after H₂O₂ or rapamycin treatment.