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. 2011 Aug;39(15):6369-79.
doi: 10.1093/nar/gkr255. Epub 2011 Apr 22.

Gcn5 facilitates Pol II progression, rather than recruitment to nucleosome-depleted stress promoters, in Schizosaccharomyces pombe

Miriam Sansó  1 , Itzel Vargas-Pérez, Luis Quintales, Francisco Antequera, José Ayté, Elena Hidalgo
Affiliations

Gcn5 facilitates Pol II progression, rather than recruitment to nucleosome-depleted stress promoters, in Schizosaccharomyces pombe

Miriam Sansó et al. Nucleic Acids Res. 2011 Aug.

Abstract

In the fission yeast, the MAP kinase Sty1 and the transcription factor Atf1 regulate up to 400 genes in response to environmental signals, and both proteins have been shown to bind to their promoters in a stress-dependent manner. In a genetic search, we have isolated the histone H3 acetyltransferase Gcn5, a component of the SAGA complex, as being essential for oxidative stress survival and activation of those genes. Upon stress, Gcn5 is recruited to promoters and coding sequences of stress genes in a Sty1- and Atf1-dependent manner, causing both an enhanced acetylation of histone H3 and nucleosome eviction. Unexpectedly, recruitment of RNA polymerase II (Pol II) is not impaired in Δgcn5 cells. We show here that stress genes display a 400-bp long nucleosome depleted region upstream of the transcription start site even prior to activation. Stress treatment does not alter promoter nucleosome architecture, but induces eviction of the downstream nucleosomes at stress genes, which is not observed in Δgcn5 cells. We conclude that, while Pol II is recruited to nucleosome-free stress promoters in a transcription factor dependent manner, Gcn5 mediates eviction of nucleosomes positioned downstream of promoters, allowing efficient Pol II progression along the genes.

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Figures

Figure 1.
Figure 1.
Gcn5 is essential for the activation of the Sty1-, Atf1-dependent stress response. (A) Δgcn5 strain and other mutants of the SAGA complex are sensitive to oxidative stress. Serial dilutions from cultures of strains 972 (WT), AV18 (Δsty1), MS161 (Δgcn5), MS183 (Δada2), MS184 (Δspt8), Δhfi1, Δspt20 and Δspt7 were spotted onto rich plates with (YE + 3 mM H2O2; YE + 5 mM H2O2) or without (YE) the indicated concentrations of H2O2. (B) Total levels of histone H3 acetylation at lysines 9 and 14 depend on Gcn5. Protein extracts from strains 972 (WT) and MS112 (Δgcn5) were analyzed by western blot with antibodies against acetylated H3K9/14 or total H3, as a loading control. (C) Stress-dependent transcriptional analysis of wild-type and Δgcn5 cells. Cultures of strains 972 (WT), AV18 (Δsty1) and MS112 (Δgcn5) were treated with 1 mM H2O2 for the indicated times. Total RNA was analyzed by northern blot with probes for gpd1, ctt1, hsp9, or srx1. rRNA is shown as a loading control.
Figure 2.
Figure 2.
Gcn5 is recruited to stress genes and promotes histone H3 acetylation and nucleosome eviction. (A) Gcn5 physically binds to stress promoters in an Atf1-dependent manner. Cultures of strains HMP47 (gcn5-HA WT) and MS176 (gcn5-HA Δatf1) were treated (+) or not (−) with 1 mM H2O2 for 15 min. ChIP experiments using anti-HA antibodies, coupled to quantification by real-time PCR, were performed using primers covering only promoter regions (gpd1, ctt1, hsp9 and srx1 promoters; left panel), or promoter (prom), coding (ORF) and termination (term) sequences of the ctt1 gene (right panel). Primers of an intergenic region were used as a negative control (control). Error bars (SEM) for all ChIP experiments were calculated from biological triplicates. Significant difference between Gcn5-HA binding to ORF before and after stress in wild-type cells was determined by the Student’s t-test (*P < 0.05). (B) Gcn5 promotes histone H3 acetylation upon stress imposition in promoters and coding region of stress-dependent genes. Cultures of strains 972 (WT) and MS112 (Δgcn5) were treated (+) or not (−) with 1 mM H2O2 for 15 min. ChIP assays were performed using antibodies specific for acetylated lysines 9 and 14 of histone H3 (H3Ac) or against unmodified C-terminal domain of H3 (H3), as described in A. The percentage of immunoprecipitation of acetylated H3 versus total H3 is indicated (% IP H3Ac/H3). Significant difference between wild-type versus Δgcn5 cells at ORF upon stress was determined by the Student’s t-test (*P < 0.05). (C) Gcn5 promotes a decrease in H3 levels as an indicator of nucleosome eviction at stress genes. The same experiment as in Figure 2B is represented here as the percentage of immunoprecipitation of total H3 (% IP total H3).
Figure 3.
Figure 3.
Lack of Gcn5 barely affects pre-initiation complex formation at stress promoters, but severely impairs Pol II progression. (A) Pol II recruitment to promoters is not defective in cells lacking Gcn5. Strains 393 (tbp1-HA WT) and MS194 (tbp1-HA Δgcn5), expressing Tbp1-HA, were cultured and treated (+) or not (−) with 1 mM H2O2 for 15 min. ChIP experiments were performed as in Figure 2A using primers of promoter regions. (B) Pol II recruitment to coding sequences is significantly affected in cells lacking Gcn5. Strains CN011 (rpb1-HA WT) and MS52 (rpb1-HA Δgcn5) were cultured and treated (+) or not (−) with 1 mM H2O2 for 15 min. ChIP experiments were perfomed as described in Figure 2A. Significant differences between wild-type versus Δgcn5 cells upon stress at ORF and termination (term) sites were determined from six biological replicates by the Student’s t-test (*P < 0.05). (C) Phosphorylation at Ser5 of the CTD of the large Pol II subunit Rpb1. Strains 972 (WT) and MS161 (Δgcn5) were cultured and treated (+) or not (−) with 1 mM H2O2 for 15 min. ChIP experiments with specific antibodies for phosphorylated Ser5 (Ser5-CTD) were performed as described in Figure 2A. (D) Phosphorylation at Ser2 of the CTD of the large Pol II subunit Rpb1. Strains 972 (WT) and MS112 (Δgcn5) were cultured and treated (+) or not (−) with 1 mM H2O2 for 15 min. ChIP experiments with specific antibodies for phosphorylated Ser2 (Ser2-CTD) were performed as described in Figure 2A. Significant difference between wild-type versus Δgcn5 cells at terminator (term) sites upon stress was determined by the Student’s t-test (*P < 0.05). (E) Phosphorylation at Ser5 of the CTD of the large Pol II subunit Rpb1. Strains and conditions as in (C), but analysing Ser5 occupancy at promoters (left panel). Right panel: The same experiment as in left panel is represented here as the percentage of immuno-precipitation of phosphorylated Rpb1-HA at Ser5 versus total Rpb1-HA (% IP Ser5-CTD/Pol II).
Figure 4.
Figure 4.
Short-time kinetics of Pol II progression along the ctt1 (A) and gpd1 (B) genes: Gcn5 mediates promoter escape. Strains CN011 (rpb1-HA) and MS52 (rpb1-HA Δgcn5), expressing the large Pol II subunit Rpb1-HA, were cultured and treated with 1 mM H2O2 for the time points in seconds indicated in the figure. ChIP experiments with anti-HA antibodies were performed as described in Figure 2A.
Figure 5.
Figure 5.
Promoter nucleosome signature at CESR genes. (A) Large nucleosome depleted regions in five S. pombe CESR promoters prior to gene activation. Nucleosome localization across genomic regions encompassing CESR genes gpd1, ctt1, hsp9, srx1 and atf1. Stripped and white rectangles represent untranslated and ORFs, respectively. Transcription start sites (black arrows) and the size of NDRs (thick bars) are indicated. Nucleosomes are represented by gray ovals. Data have been taken from a genome-wide nucleosome positioning analysis (40), and from published transcription start sites (50). (B) Overlay of nucleosome occupancy profiles for 35 out of the 50 genes showing the highest overexpression under oxidative stress (Supplementary Table S1). Nucleosome profiles for individual genes were determined in the absence of H2O2. Tiling microarray signals along the genomic region −500 to +500 bp relative to the TSS for each gene (40) were interpolated prior to averaging each position of the 35 genes. Nucleosome occupancy indicates the log2 ratio of mononucleosome to naked genomic DNA signal.
Figure 6.
Figure 6.
Nucleosome-scanning analysis of CESR genes shows Gcn5 dependent +1 nucleosome eviction after oxidative stress. Mononucleosomes were isolated from cultures of strains 972 (wild type, left panel) and HU799 (Δgcn5, right panel) before (closed circles, continuous lines) and after (open circles, dashed lines) 2 min with 1 mM H2O2. qPCR was performed using 15 overlapping primer pairs along 1.15 or 1.0 kb, covering the promoter, the TSS (black arrow) and coding region (white rectangle) of the ctt1 (A) or gpd1 (B) genes, respectively. Error bars (SEM) were calculated from biological triplicates (A) or duplicates (B). Nucleosomes are represented as gray ovals, where colour intensities are proportional to occupancy levels.
Figure 7.
Figure 7.
Scheme depicting the role of Gcn5 in transcription promotion in both yeasts. In S. cerevisiae, Gcn5 is required to promote signal-dependent nucleosome eviction at the PHO84 promoter (40). SAGA is also recruited to the CTT1 promoter upon heat shock, and changes in its nucleosomal architecture also occur (51–53). On the contrary, the S. pombe Gcn5-dependent CESR genes (such as ctt1) are devoid of nucleosomes under basal conditions, and therefore, only eviction of downstream nucleosomes is required upon signal activation.
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References

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