Genome-wide Screening of Regulators of Catalase Expression: ROLE OF A TRANSCRIPTION COMPLEX AND HISTONE AND tRNA MODIFICATION COMPLEXES ON ADAPTATION TO STRESS

Abstract

In response to environmental cues, the mitogen-activated protein kinase Sty1-driven signaling cascade activates hundreds of genes to induce a robust anti-stress cellular response in fission yeast. Thus, upon stress imposition Sty1 transiently accumulates in the nucleus where it up-regulates transcription through the Atf1 transcription factor. Several regulators of transcription and translation have been identified as important to mount an integral response to oxidative stress, such as the Spt-Ada-Gcn5-acetyl transferase or Elongator complexes, respectively. With the aim of identifying new regulators of this massive gene expression program, we have used a GFP-based protein reporter and screened a fission yeast deletion collection using flow cytometry. We find that the levels of catalase fused to GFP, both before and after a threat of peroxides, are altered in hundreds of strains lacking components of chromatin modifiers, transcription complexes, and modulators of translation. Thus, the transcription elongation complex Paf1, the histone methylase Set1-COMPASS, and the translation-related Trm112 dimers are all involved in full expression of Ctt1-GFP and in wild-type tolerance to peroxides.

Keywords: Schizosaccharomyces pombe; catalase; gene expression; stress response; transfer RNA (tRNA).

FIGURE 1.

Ctt1-GFP is a good reporter of the oxidative stress response. A, schematic representation of chromosomal ctt1 tagged with GFP. B, ctt1-GFP strain is not sensitive to oxidative stress. Serial dilutions from cultures of strains 972 (WT), AV18 (Δsty1), EP198 (Δctt1), and PG102 (ctt1-GFP) were spotted onto rich plates without (YES) or with H₂O₂. C, relative levels of ctt1 mRNA by Northern blot. Total RNA from strains 972 (WT) and EP102 (ctt1-GFP), either untreated (0) or treated with 1 mm H₂O₂ for the indicated times, was analyzed by Northern blot with probes for ctt1. rRNA and act1 are shown as loading controls. D, rich media cultures of strains 972 (WT) and PG102 (ctt1-GFP), either untreated (0) or treated with 1 mm H₂O₂ for the indicated times, were analyzed to determine Ctt1-GFP protein levels by Western blot using polyclonal antibody against GFP. Sty1 is shown as a loading control. E, rich media cultures of strains 972 (WT), PG102 (ctt1-GFP), and ES10 (ctt1-GFP Δsty1), either untreated (0) or treated with 1 mm H₂O₂ for the indicated times, were analyzed to determine Ctt1-GFP fluorescence levels by flow cytometry. Results are expressed as FITC/FCS ratio, normalized to the levels of untreated wild-type ctt1-GFP cells, with an assigned value of 1. S.D. were calculated from biological duplicates.

FIGURE 2.

Flow cytometry-based screening of a fission yeast deletion collection using fluorescence of Ctt1-GFP as a reporter. A, schematic representation of the construction and flow cytometry analysis of the Ctt1-GFP-expressing library of deletion strains. B, rich media cultures of strain PG102 (ctt1-GFP), either untreated or treated with 1 mm H₂O₂ for 60 min, were distributed in the 96 wells of a flow cytometry plate, and fluorescence along the complete plate was determined. Results are expressed as FITC/FCS ratio, normalized to the values at well number 1, with an assigned value of 1. A trend line is drawn in red. C, graphs show Ctt1-GFP FITC/FCS ratio corresponding to all deletion collection strains in a logarithmic scale, either untreated (left) or treated with 1 mm H₂O₂ for 60 min (right). Components of some complexes are differentially colored. The names of some deletion strains with the lowest or highest levels of Ctt1-GFP fluorescence are indicated.

FIGURE 3.

Paf1 and Set1 complexes regulate the levels of ctt1-GFP mRNA. A, deletion strains of Paf1 complex subunits are sensitive to oxidative stress. Serial dilutions from cultures of strains 972 (WT), MS98 (Δatf1), JE21 (Δprf1), JE23 (Δpaf1), and JE22 (Δleo1) were spotted onto rich plates without (YES) or with 2 mm of H₂O₂. B, arrest in the growth of the same strains as in A was calculated by subtracting the time required to reach an A₆₀₀ of 0.5 in the presence or absence of 2 mm of H₂O₂. C, deletion strains of Set1 complex subunits are sensitive to oxidative stress. Serial dilutions from cultures of strains 972 (WT), MS98 (Δatf1), JE28 (Δset1), JE31 (Δspf1), JE29 (Δswd1), JE30 (Δswd3), JE27 (Δshg1), and JE32 (Δash2) were spotted onto rich plates without (YES) or with 2 mm of H₂O₂. D, arrest in the growth of the same strains as in C was calculated as in B. E, rich media cultures of strains PG111 (WT ctt1-GFP), JE42 (Δset1 ctt1-GFP), JE45 (Δspf1 ctt1-GFP), JE43 (Δswd1 ctt1-GFP), JE44 (Δswd3 ctt1-GFP), JE41 (Δshg1 ctt1-GFP), and JE46 (Δash2 ctt1-GFP) either untreated (Unt.) or treated with 1 mm H₂O₂ for 60 min were analyzed to determine Ctt1-GFP fluorescence levels by flow cytometry. Results are expressed as FITC/FCS ratio, normalized to the levels of untreated or treated wild-type cells, with an assigned value of 1. F, relative levels of ctt1 mRNA by Northern blot. Total RNA from strains as in C was analyzed by Northern blot with probes for ctt1. rRNA is shown as loading control. B, D, and E, S.D. were calculated from biological duplicates.

FIGURE 4.

Trm112 and some of its partners display defects in oxidative stress tolerance. A, rich media cultures of strains PG111 (WT ctt1-GFP), JE48 (Δtrm112 ctt1-GFP), JE37 (Δmtq2 ctt1-GFP), JE39 (Δtrm9 ctt1-GFP), JE47 (Δtrm11 ctt1-GFP), and PG163 (Δbud23 ctt1-GFP) either untreated (Unt.) or treated with 1 mm H₂O₂ for 60 min were analyzed to determine Ctt1-GFP fluorescence level by flow cytometry. Results are expressed as a FITC/FCS ratio, normalized to the levels of untreated or treated wild-type cells, with an assigned value of 1. B, scheme illustrating tRNA modifications that require Trm112-methylase dimers. C, serial dilutions from cultures of strains 972 (WT), MS98 (Δatf1), JE34 (Δtrm112), JE23 (Δmtq2), JE25 (Δtrm9), JE33 (Δtrm11), and PG141 (Δbud23) were spotted onto rich plates without (YES) or with 2 mm and 5 mm of H₂O₂. D, arrest in the growth of strains 972 (WT), MS98 (Δatf1), JE34 (Δtrm112), JE23 (Δmtq2), and JE25 (Δtrm9) was calculated as described in Fig. 3B. A and D, S.D. were calculated from biological duplicates.

FIGURE 5.

Trm112-Trm9 is necessary for efficient Atf1 translation upon oxidative stress. A, deletion of trm9 or trm112 affects transcription of ctt1. Cultures of strains 972 (WT), JE25 (Δtrm9), and JE34 (Δtrm112) were treated with 1 mm H₂O₂ for the indicated times. Total RNA was analyzed by Northern blot with probes for ctt1 and act1. rRNA and act1 were used as loading controls. B, scheme illustrating the activation of the stress gene expression program by Sty1 and Atf1. MAP, mitogen-activated protein. C, Trm9 and Trm112 methyltransferases are responsible for formation of the terminal methyl group of mcm⁵U₃₄ (5-methoxycarbonylmethyluridine). D, absence of Sin3/Elp3, Trm9, or Trm112 barely affects transcription of the atf1 gene. Cultures of strains 972 (WT), IV16 (Δelp3/sin3), JE25 (Δtrm9), and JE34 (Δtrm112) were treated with 1 mm H₂O₂ for the indicated times. Total RNA was analyzed by Northern blot with probes for atf1 and act1. E, absence of Sin3/Elp3, Trm9, or Trm112 affects Atf1 protein levels. Strains as in D were analyzed by Western blot using polyclonal anti-Atf1 antibodies. Anti-tubulin was used as the loading control. F, quantification of the relative mRNA (left) and protein levels (right) of the blots in D and E, respectively. Unt., untreated. S.D. were calculated from biological duplicates.

FIGURE 6.

Overexpression of tRNA_UUU suppresses the growth defects of Δtrm9 but not of Δtrm112 upon oxidative stress. A, B, and C, serial dilutions from cultures of strains 972 (WT), JF77 (Δelp3/sin3), JE49 (Δtrm9), and JE52 (Δtrm112) transformed with episomal plasmids p465 (ptRNA_UUU^Lys), p466 (ptRNA_CUU^Lys), or the empty vector pREP.42x, were spotted onto rich plates without (YES) or with 2 mm H₂O₂. D, E, and F, relative levels of tRNA overexpression by Northern blot. Total RNA from the same strains as in A, B, and C was analyzed by Northern blot with probes of dsDNA of the indicated tRNAs labeled at their antisense strand. act1 is shown as a loading control.

FIGURE 7.

Expression of a synthetic AAA-to-AAG atf1 gene renders wild-type Atf1 protein levels in cell lacking Trm9 or Trm112. A, schematic representation of HA-Atf1 and HA-Atf1_AAA-to-AAG proteins, expressed from the constitutive sty1 promoter. The relative positions of the 11 AAA and AAG lysine codons are indicated. B and C, vectors carrying the wild-type (HA-atf1) or mutated atf1 genes (HA-atf1_AAA-to-AAG) under the control of a constitutive promoter were integrated in the chromosomes of wild type and Δtrm9 and Δtrm112 strains. Rich media cultures of strains JF91 (sty1′::HA-atf1), JF94 (sty1′::HA-atf1_AAA-to-AAG), PG136 (Δtrm9 sty1′::HA-atf1_AAA-to-AAG), PG138 (Δtrm112 sty1′::HA-atf1_AAA-to-AAG), PG139 (Δtrm9 sty1′::HA-atf1), and PG140 (Δtrm112 sty1′::HA-atf1) either untreated (0) or treated with 1 mm H₂O₂ for the indicated times were analyzed to determine HA-atf1 mRNA levels by Northern blot using an anti-HA probe (B) or HA-Atf1 protein levels by Western blot using monoclonal antibody against HA (C). The graphs on the right panels indicate the relative levels of HA-atf1/act1 mRNAs (B) or HA-Atf1/tubulin protein levels (C).