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. 2008 May;7(5):826-35.
doi: 10.1128/EC.00465-07. Epub 2008 Mar 28.

Transcription factors Pcr1 and Atf1 have distinct roles in stress- and Sty1-dependent gene regulation

Miriam Sansó  1 , Madelaine Gogol, José Ayté, Chris Seidel, Elena Hidalgo
Affiliations

Transcription factors Pcr1 and Atf1 have distinct roles in stress- and Sty1-dependent gene regulation

Miriam Sansó et al. Eukaryot Cell. 2008 May.

Abstract

The mitogen-activated protein kinase Sty1 is essential for the regulation of transcriptional responses that promote cell survival in response to different types of environmental stimuli in Schizosaccharomyces pombe. Upon stress activation, Sty1 reversibly accumulates in the nucleus, where it stimulates gene expression via the Atf1 transcription factor. The Atf1 protein forms a heterodimer with Pcr1, but the specific role of this association is controversial. We have carried out a comparative analysis of strains lacking these proteins individually. We demonstrate that Atf1 and Pcr1 have similar but not identical roles in S. pombe, since cells lacking Pcr1 do not share all the phenotypes reported for Deltaatf1 cells. Northern blot and microarray analyses demonstrate that the responses to specific stresses of cells lacking either Pcr1 or Atf1 do not fully overlap, and even though most Atf1-dependent genes induced by osmotic stress are also Pcr1 dependent, a subset of genes require only the presence of Atf1 for their induction. Whereas binding of Atf1 to most stress-dependent genes requires the presence of Pcr1, we demonstrate here that Atf1 can bind to the Pcr1-independent promoters in a Deltapcr1 strain in vivo. Furthermore, these analyses show that both proteins have a global repressive effect on stress-dependent and stress-independent genes.

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Figures

FIG. 1.
FIG. 1.
Atf1 and Pcr1 interact in vivo and display nuclear localization. (A) Schematic representation of Atf1 and Pcr1 proteins. The bZIP domains and the potential MAPK sites (S or T) are indicated. (B) HA-Atf1 and Pcr1 interact in vivo before and after stress. Strain 972 (no tag) or MS9 (Δatf1 with integrated, nmt-driven pHA-atf1), were treated (+) or not (−) for 30 min with 1 mM H2O2, and formaldehyde extracts were obtained. Ten milligrams of total protein extracts were immunoprecipitated with anti-HA antibodies (IP α-HA), and the resulting immunoprecipitates were analyzed by SDS-PAGE and blotted with anti-HA or anti-Pcr1 antibodies. As a loading control, 150 μg of whole-cell extracts were loaded (WCE). (C) Nuclear localization of GFP-Atf1 and GFP-Pcr1 proteins in Δatf1 and Δpcr1 strains. We used strains MS13 (Δatf1 with integrated, nmt-driven pGFP-atf1), MS14 (Δpcr1 with integrated, nmt-driven pGFP-atf1), MS16 (Δpcr1 with integrated, nmt-driven pGFP-pcr1), and MS15 (Δatf1 with integrated, nmt-driven, pGFP-pcr1). Cells were stained with 4′,6′-diamidino-2-phenylindole (DAPI) to label DNA (center panels). The cellular distributions of the fusion proteins under nonstressed conditions were determined by fluorescence microscopy (GFP) (lower panels). The same cells under differential interference contrast (Nomarski) optics are shown in the upper panels.
FIG. 2.
FIG. 2.
Pcr1 is a phosphoprotein. (A) Pcr1 is phosphorylated under unstressed conditions. Native extracts from MS7 cells (Δpcr1) transformed with p138.41x, expressing HA-Pcr1, were prepared and incubated with (+) or without (−) lambda phosphatase in the presence or absence of phosphatase inhibitors, as indicated. Pcr1 was detected after SDS-PAGE followed by Western blotting using polyclonal anti-Pcr1 antibodies. (B) Pcr1 is dephosphorylated upon oxidative stress. Strains MS7 with p138.41x (Δpcr1 + pHA-pcr1) or 972 (WT) were treated with 1 mM H2O2 stress for the times indicated. Native (to detect HA-Pcr1) or boiled (to detect endogenous Pcr1 in strain 972) protein extracts, obtained as described in Materials and Methods, were analyzed by Western blot analysis with anti-Pcr1 antibodies. Phosphorylated (Pcr1-P) and unphosphorylated (Prc1) protein forms, either HA tagged or untagged, are indicated with arrows. (C) Dephosphorylation of Pcr1 does not occur upon KCl stress. Strain MS7 with p138.41x (Δpcr1 + pHA-pcr1) was treated with 0.4 M KCl for 15 or 30 min or left untreated. The phosphorylation status of HA-Pcr1 was analyzed as panel B. (D) H2O2-dependent dephosphorylation of Pcr1 is Sty1 dependent. Strain NT224 transformed with p138.41x (Δsty1 + pHA-pcr1) was treated with 1 mM H2O2 stress for the times indicated. The phosphorylation status of HA-Pcr1 was analyzed as for panel B.
FIG. 3.
FIG. 3.
Atf1 but not Pcr1 is essential for survival in front of all major types of stresses. (A) Survival of wild-type, Δsty1, Δatf1, and Δpcr1 strains in response to heat shock and oxidative stress. Strains 972 (WT), AV18 (Δsty1), AV15 (Δatf1), and MS5 (Δpcr1) were grown in rich medium to a final OD600 of 0.5. Cells were then incubated at 45°C for 30 min or with 2 mM H2O2 for 1 h before plating on rich-medium agar plates. Survival was measured as a percentage of the colony number at time zero. The experiments were repeated at least three times with very similar results. (B) Analysis of the osmotic stress resistance of wild-type, Δsty1, Δatf1, Δpcr1, and Δatf1 Δpcr1 strains. Strains 972 (WT), AV18 (Δsty1), AV15 (Δatf1), MS5 (Δpcr1), and MS48 (Δatf1 Δpcr1) were grown in liquid minimal medium to a final OD600 of 0.5, and the number of cells indicated at the top of the panels was spotted onto minimal medium plates containing KCl, NaCl, or sorbitol at the indicated concentrations and incubated at 30°C for 3 to 4 days.
FIG. 4.
FIG. 4.
Pcr1 is required for transcription of most but not all stress response genes. (A) Transcription of many stress response genes requires the presence of both Atf1 and Pcr1 transcription factors. Cultures of strains 972 (WT), AV18 (Δsty1), AV15 (Δatf1), and MS5 (Δpcr1) were left untreated (−) or were treated for 30 min with 0.4 M KCl (K) or 1 mM H2O2 (H). Total RNA was extracted and analyzed by Northern blotting with probes for gpx1, ctt1, zym1, or cta3. (B) Transcription of hsp9, gpd1, or srx1 does not require Pcr1 for all stress situations. Strains and conditions are as described for panel A. cdc2 was used as a loading control.
FIG. 5.
FIG. 5.
Pcr1 does not regulate the levels of Atf1. (A) Atf1 protein levels are lower in Δsty1 and Δpcr1 mutants than in wild-type cells. TCA extracts from strains 972 (WT), AV18 (Δsty1), and MS5 (Δpcr1) were obtained from cultures grown in minimal medium before (0) or after a 10- or 30-min exposure to 0.4 M KCl. The relative amounts of Atf1 protein were determined by Western blotting with polyclonal anti-Atf1 antibodies (Atf1). Expression of tubulin was detected as a loading control (Tub2). (B) Atf1 protein levels are very similar in wild-type, Δsty1, and Δpcr1 cells when expressed from the Sty1-independent nmt promoter. Strains JM1066 (Δatf1), MS48 (Δpcr1 Δatf1), and NT224 (Δsty1) were transformed with plasmid p151.41x (containing the nmt-driven HA-atf1 gene, pHA-atf1), and TCA extracts were obtained before (−) or after 30 min with 0.4 M KCl (K) or 1 mM H2O2 (H). Cell lysates were analyzed by Western blotting with anti-Atf1 and anti-Tub2 antibodies as for panel A. RNA from the same strains was also isolated, and the expression of gpx1 was determined by Northern blotting (gpx1 mRNA). atf1 transcript levels are shown as a loading control (atf1 mRNA).
FIG. 6.
FIG. 6.
Atf1 is bound to promoters before and after stress. (A) Chromatin immunoprecipitation analysis of Atf1-HA and Pcr1-HA bound to stress promoters. Strains JM1821 (atf1-HA6H), MS65 (pcr1-HA) (both strains tagged at the chromosomal loci), or wild-type 972 with no plasmid (no tag) were grown, formaldehyde extracts were obtained, and chromatin bound to Atf1-HA or Pcr1-HA was isolated using anti-HA monoclonal antibodies (IP α-HA). Recovered DNA was assayed by PCR amplification with primers encompassing the hsp9 and cta3 promoters or the cdc18 ORF as a negative control. Two different concentrations of whole-cell extracts (WCE) were also analyzed with the primer pairs to demonstrate that the quantity of amplified DNA is dependent on the amount of input (WCE) (1/100 or 1/500). (B) Atf1-Pcr1 heterodimer binds to standard stress-dependent promoters in vivo. Strains JM1821 (atf1-HA6H) and MS65 (pcr1-HA) or wild-type strain 972 (no tag) was grown in the absence of stress (−) or presence of 0.4 M KCl (KCl) or 1 mM H2O2 (H) for 15 min. We obtained formaldehyde extracts, and performed chromatin immunoprecipitation of Atf1-HA6H and Pcr1-HA with anti-HA monoclonal antibodies (IP α-HA) to assay Atf1 and Pcr1 binding to specific DNA sites. PCR was performed with primers encompassing the classical CESR promoter cta3 and the cdc18 ORF as a negative control. (C) Atf1 binding is Pcr1 dependent in the cta3 promoter. Strains JM1066 (Δatf1) and MS48 (Δatf1 Δpcr1) transformed with nmt-driven p151.41x (pHA-atf1) or wild-type 972 with no plasmid (no tag) were grown as described for panel B. We obtained formaldehyde extracts, and chromatin immunoprecipitation of HA-Atf1 (IP α-HA) was performed as for panel B. (D) Pcr1 binding to Pcr1-independent stress promoters in vivo. Strains MS10 (Δpcr1 pHA-pcr1) transformed with the integrated, nmt-driven p139.81x′ (pHA-pcr1) or wild-type 972 with no plasmid (no tag) were grown in the presence (KCl) or not (−) of 0.4 M KCl for 15 min. Formaldehyde extracts and chromatin immunoprecipitation of HA-Pcr1 were performed as for panel B. Specific primers were used for PCR amplification of the gpd1, hsp9, and srx1 promoters, as well as the cdc18 ORF as a negative control. (E) Atf1 binding to Pcr1-independent stress promoters in vivo. The same experiment as for panel D was performed with strains JM1066 (Δatf1) and MS48 (Δatf1 Δpcr1) transformed with nmt-driven p151.41x (pHA-atf1) or 972 (WT), with no plasmid (no tag).
FIG. 7.
FIG. 7.
Microarray analysis of the responses of wild-type, Δatf1, and Δpcr1 cells to KCl stress. A Venn diagram of genes up-regulated more than twofold after 30 min of 0.4 M KCl stress is shown. mRNA from strains 972 (WT), AV15 (Δatf1), and MS5 (Δpcr1), grown in minimal medium, was analyzed by microarray hybridization. For the wild type, 170 genes were induced by KCl (A, D, F, and G); for Δatf1 cells, 58 genes were induced (B, D, E, and G); and for Δpcr1 cells, 84 genes were induced (C, E, F, and G). n-fold inductions upon stress are relative to mRNA levels for the untreated condition of each strain. The list of all A-to-G subsets of genes is provided in Table S1 in the supplemental material.
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