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. 2007 Jun;27(11):3900-10.
doi: 10.1128/MCB.00089-07. Epub 2007 Apr 2.

Selective requirement for SAGA in Hog1-mediated gene expression depending on the severity of the external osmostress conditions

Meritxell Zapater  1 , Marc Sohrmann, Matthias Peter, Francesc Posas, Eulàlia de Nadal
Affiliations

Selective requirement for SAGA in Hog1-mediated gene expression depending on the severity of the external osmostress conditions

Meritxell Zapater et al. Mol Cell Biol. 2007 Jun.

Abstract

Regulation of gene expression by the Hog1 stress-activated protein kinase is essential for proper cell adaptation to osmostress. Hog1 coordinates an extensive transcriptional program through the modulation of transcription. To identify systematically novel components of the transcriptional machinery required for osmostress-mediated gene expression, we performed an exhaustive genome-wide genetic screening, searching for mutations that render cells osmosensitive at high osmolarity and that are associated with reduced expression of osmoresponsive genes. The SAGA and Mediator complexes were identified as putative novel regulators of osmostress-mediated transcription. Interestingly, whereas Mediator is essential for osmostress gene expression, the requirement for SAGA is different depending on the strength of the extracellular osmotic conditions. At mild osmolarity, SAGA mutants show only very slight defects on RNA polymerase II (Pol II) recruitment and gene expression, whereas at severe osmotic conditions, SAGA mutants show completely impaired RNA Pol II recruitment and transcription of osmoresponsive genes. Thus, our results define an essential role for Mediator in osmostress gene expression and a selective role for SAGA under severe osmostress. Our results indicate that the requirement for a transcriptional complex to regulate a promoter might be determined by the strength of the stimuli perceived by the cell through the regulation of interactions between transcriptional complexes.

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Figures

FIG. 1.
FIG. 1.
SAGA complex is essential for osmostress gene expression at high osmolarity. (A) Mutations on specific SAGA components render cells osmosensitive. Wild type (wt) and indicated mutant strains were spotted on YPD plates without (Control) or with 1.2 M NaCl (NaCl) or 2.2 M sorbitol (Sorbitol). Growth was scored after 3 days. (B) Deletion of genes encoding components of SAGA complex causes delay in transcription under mild osmostress conditions. The wild-type strain and indicated mutant strains were grown to mid-log phase in rich medium and then subjected to osmotic shock (0.4 M NaCl) for indicated times. Total RNA was assayed by Northern blotting for STL1, CTT1, and RPL28 (as a loading control). (C and D) Deletion of genes encoding components of SAGA complex abolishes transcription under severe osmostress conditions. Wild-type and mutant strains were grown to mid-log phase in rich medium and then subjected to osmotic shock (1.2 M NaCl or 1.8 M sorbitol) for indicated times. Total RNA was assayed as for panel B.
FIG. 2.
FIG. 2.
SAGA is required under severe stress conditions irrespective of the activator responsible for osmostress gene induction. (A) Wild-type (wt) and hot1Δ strains were grown to mid-log phase in rich medium and then subjected to osmotic shock (1.2 M NaCl) for indicated times. Total RNA was assayed by Northern blotting for STL1 and RDN18-1 (as a loading control). (B) Wild-type and msn2/4Δ strains were grown as for panel A. Total RNA was assayed by Northern blotting for CTT1 and RDN18-1 (as a loading control).
FIG. 3.
FIG. 3.
Osmostress gene expression is not affected by mutations of TFIID complex. Wild-type (wt), taf1ts, or taf2ts strain was grown at 25°C until an OD660 of 0.5 was reached, shifted to 37°C for 90 min, and then subjected to osmotic stress (1.2 M NaCl) for indicated times. Total RNA was assayed by Northern blotting for STL1, CTT1, and RDN18-1 (as a loading control). Expression of TFIID-dependent gene TRX1 was monitored by Northern blotting with the wild-type or taf1ts strain without NaCl treatment (lower panel).
FIG. 4.
FIG. 4.
Mediator is critical for gene expression in response to osmostress. (A) Deletion of genes encoding for components of Mediator complex render cells osmosensitive. The wild-type (wt) and indicated mutant strain were spotted on YPD plates with or without 1.2 M NaCl (NaCl) or 2.2 M sorbitol (Sorbitol). Growth was scored after 3 days. (B) Deletion of genes encoding Mediator subunits causes impaired transcription already under mild osmostress conditions. Wild-type and indicated mutant strains were grown to mid-log phase in rich medium and then subjected to osmotic shock (0.4 M NaCl) for indicated times. Total RNA was assayed by Northern blotting for STL1, CTT1, HSP12, and RPL28 (as a loading control). (C and D) Inactivation of Srb4 Mediator subunit causes impaired transcription under mild and severe osmostress conditions. Wild-type and srb4ts strains were grown at 25°C until an OD660 of 0.5 was reached and shifted to 37°C for 90 min. Cultures were then subjected to osmotic stress (0.4 or 1.2 M NaCl) for indicated times. Total RNA was assayed as for panel B.
FIG. 5.
FIG. 5.
Hog1 is required for recruitment of SAGA, Mediator, and RNA Pol II to osmostress promoters under mild and severe osmotic conditions. (A) Recruitment of SAGA, Mediator, and RNA Pol II depends on Hog1 MAPK under mild osmostress conditions. Wild-type (wt) or hog1Δ mutant strain expressing Spt20-Myc (left) or Srb4-HA (center) or non-epitope tagged (right) was grown to mid-log phase and treated with 0.4 M NaCl for 5 min. Proteins were immunoprecipitated with anti-Myc, HA, or RNA Pol II antibody. Binding to STL1 promoter was determined by ChIP. n-fold induction of treated (open bars) or untreated (filled bars) cultures normalized to a telomere- or GAL1-specific band (internal controls) is shown. (B) Recruitment of SAGA, Mediator, and RNA Pol II depends on Hog1 MAPK under severe osmostress conditions. Strains as in panel A were grown and treated with 1.2 M NaCl for 75 min. Samples for ChIP analyses were processed as for panel A.
FIG. 6.
FIG. 6.
Recruitment of SAGA, Mediator, and RNA Pol II at osmoresponsive promoters occurs after Hog1 binding to STL1 promoter. Wild-type strains carrying Hog1-Myc, Spt20-Myc, or Srb4-HA were grown to mid-log phase and treated with 1.2 M NaCl for indicated times. Proteins were immunoprecipitated with anti-Myc, HA, or RNA Pol II antibody (W8G16). Binding to STL1 promoter was determined by ChIP. Relative intensities of PCR fragments normalized to the telomere-specific band (internal control) are shown. Data represent means and standard deviations for three independent experiments.
FIG. 7.
FIG. 7.
Interaction of Hog1 with STL1 promoter initiates sequential recruitment of proteins at osmostress promoters. (A) Recruitment of Hog1 to osmostress promoters is independent of SAGA and Mediator complexes. Wild-type, hot1Δ, spt20Δ, or pgd1Δ strain expressing Hog1-HA was grown and subjected to 0.4 M NaCl for 5 min (left panel) or 1.2 M NaCl for 75 min (right panel). Binding to STL1 promoter was determined by ChIP. n-fold induction of treated (open bars) or untreated (filled bars) cultures normalized to a telomere-specific band (internal control) is shown. (B) SAGA and Mediator complexes are recruited independently to osmostress promoters at mild osmostress conditions. Wild-type, pgd1Δ, or sin4Δ strain expressing Spt20-HA (left) or wild-type or spt20Δ strain expressing Srb4-HA (right) was grown and subjected to osmostress (0.4 M NaCl for 5 min). Binding to STL1 promoter was determined as for panel A. (C) Recruitment of Mediator to osmostress promoters is reduced in SAGA mutants under severe osmostress conditions. Strains as in panel B were grown and treated with 1.2 M NaCl for 75 min. Binding to STL1 promoter was determined as for panel A.
FIG. 8.
FIG. 8.
Recruitment of SAGA and Mediator complexes also occurs at coding regions of stress-responsive gene upon induction. (A) SAGA complex is recruited to the coding region of STL1 upon osmotic stress. Wild-type strains expressing Ada2-Myc or Spt20-Myc were grown and subjected to mild (0.4 M NaCl for 5 min, left) or severe (1.2 M NaCl for 75 min, right) osmotic stress. Binding to the STL1 ORF was determined by ChIP. n-fold induction of treated (open bars) or untreated (filled bars) cultures normalized to a telomere- or GAL1-specific band (internal controls) is shown. (B) Mediator complex is recruited at the coding region of osmostress genes. Wild-type and spt20Δ strains expressing Srb4-HA were grown and subjected to mild (0.4 M NaCl for 5 min, left) or severe (1.2 M NaCl for 75 min, right) osmotic stress. Binding to the STL1 ORF was determined as for panel A. (C) Binding of Tbp1 is restricted to promoter regions of osmostress genes. The wild-type strain expressing Tbp1-Myc was grown and subjected to mild (0.4 M NaCl for 5 min, left) or severe (1.2 M NaCl for 75 min, right) osmotic stress. Binding to the STL1 promoter and ORF was determined as for panel A.
FIG. 9.
FIG. 9.
Hog1 coprecipitates with SAGA and Mediator complexes. (A) TAP-tagged Ada2, Ada1, Spt20, Spt3 or Spt8 strain expressing GST or GST-Hog1 was subjected to brief osmotic shock (10 min, 0.4 M NaCl). GST proteins were pulled down by glutathione-Sepharose 4B, and the presence of TAP proteins was probed by immunoblotting using anti-TAP (PAP; Sigma) (upper panel). Prec., precipitated proteins. Total extract represents <20% of total input protein (middle panel). Amounts of precipitated GST proteins were detected using anti-GST (lower panel). (B) Interaction of SAGA with Hog1 is conserved in Mediator-deficient strain. Wild-type (wt) or pgd1Δ strain bearing Spt20-HA was transformed with plasmid expressing GST or GST-Hog1 and grown in selective medium. Cultures were either untreated or treated with 0.4 M NaCl for 10 min. GST proteins were pulled down as in panel A. (C) Interaction of Mediator with Hog1 is impaired in SAGA-deficient strain under severe osmostress conditions. Wild-type or spt20Δ strain bearing Srb4-Myc was transformed with plasmid expressing GST or GST-Hog1 and grown in selective medium. Cultures were either untreated or treated with 0.4 M NaCl for 10 min or 1.2 M NaCl for 100 min. GST proteins were pulled down as in panel A.
FIG. 10.
FIG. 10.
(A) Integrity of Mediator complex is maintained in spt20Δ mutant strain under severe stress conditions. Extracts from Srb4-Myc- and Med2-TAP-tagged strains were subjected to gel filtration through Superose 6 HR 10/30 column. Fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and proteins detected with antibodies against Myc and TAP epitopes. wt, wild type. (B) Coimmunoprecipitation of Mediator with Hog1 occurs in non-chromatin-associated complexes. The wild-type strain bearing Srb4-Myc was transformed with plasmid expressing GST or GST-Hog1 and grown in selective medium. Cultures were treated with either 0.4 M NaCl for 10 min or 1.2 M NaCl for 100 min. Protein extracts were untreated (−) or treated (+) with DNase for 30 min at 37°C before GST proteins were pulled down by glutathione-Sepharose beads. Srb4-Myc-containing (top panel) or GST-containing (bottom panel) proteins were detected by Western blotting using antibodies against Myc and GST. Prec., precipitated proteins. Total extract represents <20% of total input protein (middle panel). (C) SAGA and Mediator complexes coprecipitate in Hog1-independent manner. Wild-type or hog1Δ strain bearing Spt20-Myc was transformed with plasmid expressing GST or GST-Srb4 and grown in selective medium. Cultures were treated with 1.2 M NaCl for 100 min. GST proteins were pulled down by glutathione-Sepharose beads. Spt20-Myc-containing (top panel) or GST-containing (bottom panel) proteins were detected by Western blotting using antibodies against Myc and GST. Total extract represents <20% of total input protein (middle panel).
FIG. 11.
FIG. 11.
Whereas Mediator is essential to recruit Pol II at the STL1 promoter upon stress, SAGA is required selectively under severe osmostress conditions. (A) Recruitment of Pol II is impaired by deletion of Mediator PGD1 gene even under mild osmostress conditions. Wild-type or pgd1Δ strain was grown to mid-log phase and subjected to mild osmotic stress treatment (0.4 M NaCl for 5 min). Pol II was immuprecipitated with W8G16 antibody. Binding to STL1 promoter was determined by ChIP. n-fold induction of treated (open bars) or untreated (filled bars) cultures normalized to a telomere-specific band (internal control) is shown. (B) Recruitment of RNA Pol II to promoters is slightly delayed in a SAGA mutant under mild osmostress conditions. Wild-type (filled circles) or spt20Δ (open circles) strain was treated with 0.4 M NaCl for indicated times. ChIP analyses were performed as for panel A. Data from one representative experiment out of three are shown. (C) Recruitment of RNA Pol II to promoters is dramatically impaired in a SAGA mutant under severe osmostress conditions. Strains as in panel B were treated with 1.2 M NaCl for indicated times. ChIP analyses were performed as for panel A. Data from one representative experiment out of three are shown. (D) Recruitment of Tbp1 at the STL1 promoter is abolished under severe osmostress in SAGA-deficient cells. Wild-type or spt20Δ strain expressing Tbp1-Myc was grown and subjected to severe osmotic stress (1.2 M NaCl) for indicated times. Binding to STL1 promoter was determined by ChIP. n-fold-induction of treated or untreated cultures normalized to a GAL1-specific band (internal control) is shown.
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