A triad of subunits from the Gal11/tail domain of Srb mediator is an in vivo target of transcriptional activator Gcn4p

Abstract

The Srb mediator is an important transcriptional coactivator for Gcn4p in the yeast Saccharomyces cerevisiae. We show that three subunits of the Gal11/tail domain of mediator, Gal11p, Pgd1p, and Med2p, and the head domain subunit Srb2p make overlapping contributions to the interaction of mediator with recombinant Gcn4p in vitro. Each of these proteins, along with the tail subunit Sin4p, also contributes to the recruitment of mediator by Gcn4p to target promoters in vivo. We found that Gal11p, Med2p, and Pgd1p reside in a stable subcomplex in sin4Delta cells that interacts with Gcn4p in vitro and that is recruited independently of the rest of mediator by Gcn4p in vivo. Thus, the Gal11p/Med2p/Pgd1p triad is both necessary for recruitment of intact mediator and appears to be sufficient for recruitment by Gcn4p as a free subcomplex. The med2Delta mutation impairs the recruitment of TATA binding protein (TBP) and RNA polymerase II to the promoter and the induction of transcription at ARG1, demonstrating the importance of the tail domain for activation by Gcn4p in vivo. Even though the Gal11p/Med2p/Pgd1p triad is the only portion of Srb mediator recruited efficiently to the promoter in the sin4Delta strain, this mutant shows high-level TBP recruitment and wild-type transcriptional induction at ARG1. Hence, the Gal11p/Med2p/Pgd1p triad may contribute to TBP recruitment independently of the rest of mediator.

FIG. 1.

Overlapping contributions of Srb mediator tail subunits and Srb2p in binding to recombinant GST-Gcn4p in vitro. Equal amounts of GST, WT GST-Gcn4p (WT), and GST-Gcn4-10Ala present in Escherichia coli extracts were incubated with 1 mg of the appropriate yeast WCEs and bound to glutathione-Sepharose resin. The bound fractions plus an aliquot of the WCE (input) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subunits of Srb mediator, SAGA, or SWI/SNF listed on the left were detected by Western analysis. The bottom section of panel B shows Ponceau S staining of the Western blots to reveal the amounts of GST proteins precipitated in the assays. (A) Binding assays were conducted using equal amounts of WCEs from yeast strains BY4741 (WT), 1742 (gal11Δ), 4393 (pgd1Δ), LS01 (med2Δ), 1976 (sin4Δ), 4734 (srb5Δ), 6611 (srb2Δ), 3119 (rox3Δ), and 5489 (med1Δ). (B) Binding assays were conducted using WCEs from strains BY4741 (WT), LS20 (pgd1Δ med2Δ), LS11 (pgd1Δ sin4Δ), LS10 (pgd1Δ gal11Δ), LS03 (gal11Δ med2Δ), LS12 (med2Δ sin4Δ), and LS09 (gal11Δ sin4Δ). (C) Binding assays were conducted using WCEs from strains BY4741 (WT), LS22 (srb2Δ srb5Δ), LS24 (srb2Δ pgd1Δ), LS04 (srb2Δ gal11Δ), and LS08 (srb2Δ sin4Δ).

FIG. 2.

Coimmunoprecipitation analysis of Srb mediator integrity in mutant strains. WCEs from the appropriate yeast strains were immunoprecipitated with monoclonal c-myc antibodies. The immune complexes were collected, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subjected to Western analysis to detect the proteins listed on the left of each panel or with monoclonal c-myc antibodies to detect the myc-tagged proteins. I, 4% of the input WCE; P, the total pellet fraction from the immunoprecipitate; S, 10% of the supernatant fraction. The bands evident in the immunoprecipitates from the three strains lacking SRB2 probed with Srb2p antibodies in lanes 5, 8, and 11 of panel B result from nonspecific reactions with the immunoglobulin G light chain that occur variably in these experiments. The following yeast strains were analyzed: BY4741 (SIN4) and FZY120 (SIN4-myc) (A); SIN4-myc strains FZY120 (WT), FZY177 (srb2Δ srb5Δ), FZY167 (srb2Δ gal11Δ), FZY179 (srb2Δ pgd1Δ), FZY165 (gal11Δ med2Δ), FZY169 (gal11Δ pgd1Δ), and FZY175 (pgd1Δ med2Δ) (B); BY4741 (SRB6 SIN4), MSY118 (SRB6-myc SIN4), MSY119 (SRB6-myc sin4Δ), and FZY254 (SRB6-myc srb2Δ) (C); BY4741 (GAL11; WT), FZY122 (GAL11-myc; WT), FZY124 (GAL11-myc sin4Δ), FZY156 (GAL11-myc pgd1Δ), FZY152 (GAL11-myc med2Δ), and FZY250 (GAL11-myc srb2Δ) (D); and BY4741 (MED2; WT), LS56 (MED2-myc; WT), LS57 (MED2-myc pgd1Δ), LS58 (MED2-myc sin4Δ), MSY41 (MED2-myc gal11Δ), and FZY401 (MED2-myc srb2Δ) (E).

FIG. 3.

The Gal11p/Med2p/Pgd1p subcomplex in sin4Δ extracts binds to GST-Gcn4p in vitro. (A) WCEs from the appropriate yeast strains were immunoprecipitated (IP) with myc or HA antibodies as indicated. The immune complexes were subjected to Western analysis to detect the proteins listed on the left using c-myc antibodies to detect myc-Gal11p and HA antibodies to detect HA-Med2p and HA-Pgd1p. I, 4% of the input WCE; P, the total pellet fraction from the immunoprecipitate; S, 10% of the supernatant fraction. The following yeast strains were employed: FZY122 (SIN4 GAL11-myc), MSY14 (SIN4 GAL11-myc PGD1-HA), MSY13 (SIN4 GAL11-myc MED2-HA), LS47 (sin4Δ GAL11-myc PGD1-HA), and LS45 (sin4Δ GAL11-myc MED2-HA). (B) GST pull-down assays were carried out as described for Fig. 1 using the following yeast strains: MSY14 (GAL11-myc PGD1-HA SIN4), MSY13 (GAL11-myc MED2-HA SIN4), LS45 (GAL11-myc MED2-HA sin4Δ), and LS47 (GAL11-myc PGD1-HA sin4Δ). White ovals mark the locations of Med4p in lanes 3 and 7; the bands beneath them in lanes 3, 7, 11, and 15 are cross-reacting species.

FIG. 4.

ChIP analysis of myc-Srb6p and myc-Gal11p recruitment by Gcn4p in mutants lacking different Srb mediator subunits. (A and D) gcn4Δ strains expressing myc-Srb6p (A) or myc-Gal11p (D) and containing the indicated mediator subunit deletions were transformed with high-copy-number GCN4 plasmid pHQ1239 (+ lanes) or empty vector YEplac195 (− lanes), grown for 3 h in SC lacking Ura, Arg, and Ilv and containing 0.5 μg of SM/ml, and subjected to ChIP analysis using antimyc antibodies. The immunoprecipitated DNA containing the ARG1 UAS or POL1 ORF (IP) and the corresponding input amounts (input) were measured by quantitative PCR. Three different amounts of input samples were analyzed for the GCN4 and gcn4Δ strains with WT mediator subunits, shown in the first six lanes, to demonstrate the linear response of the PCR signals to the amounts of input DNA. (B and E) The PCR products from the experiments described for panels A and D, respectively, were quantified by phosphorimaging analysis, and the ratios of signals in the IP to the input samples for the ARG1 UAS were normalized for the corresponding ratios for POL1 (normalized %IP). The resulting values from three PCR amplifications of chromatin immunoprecipitated from two independent cultures analyzed for each SRB6-myc (B) or GAL11-myc (E) strain were averaged, and the mean values and standard errors were plotted in the histograms. The percentages below the histograms give the proportions of the WT Gcn4p-dependent binding of the myc-tagged protein to the ARG1 UAS, calculated by subtracting the normalized ratios for the gcn4Δ strain from the normalized ratios for the GCN4 strain for that mutant and dividing by the corresponding value obtained for the WT pair of GCN4 and gcn4Δ strains. (C and F) The procedures described for panels B and E were employed to analyze binding of myc-Srb6p (C) or myc-Gal11p (F) to the SNZ1 UAS. The following yeast strains were employed in panels A to C: FZY232 (gcn4Δ SRB6-myc), FZY234 (gcn4Δ SRB6-myc sin4Δ), FZY274 (gcn4Δ SRB6-myc gal11Δ), FZY276 (gcn4Δ SRB6-myc pgd1Δ), FZY278 (gcn4Δ SRB6-myc med2Δ), and FZY270 (gcn4Δ SRB6-myc srb2Δ). The strains analyzed in panels D to F were FZY306 (gcn4Δ GAL11-myc), FZY308 (gcn4Δ GAL11-myc sin4Δ), FZY260 (gcn4Δ GAL11-myc pgd1Δ), FZY304 (gcn4Δ GAL11-myc med2Δ), and FZY264 (gcn4Δ GAL11-myc srb2Δ).

FIG. 5.

ChIP analysis of myc-Med2p, myc-Srb5p, and myc-Sin4p recruitment by Gcn4p in mutants lacking different Srb mediator subunits. ChIP analysis was conducted as described in Fig. 4 using gcn4Δ strains transformed with either empty vector YEplac195 (gcn4Δ), high-copy-number GCN4 plasmid pHQ1239 (h.c.GCN4), or single-copy GCN4 plasmid p2382 (s.c.GCN4). The following strains were employed: LS56 (gcn4Δ MED2-myc), LS58 (gcn4Δ MED2-myc sin4Δ), MSY41 (gcn4Δ MED2-myc gal11Δ), LS57 (gcn4Δ MED2-myc pgd1Δ), and FZY401 (gcn4Δ MED2-myc srb2Δ) (A and B); MSY120 (SRB5-myc) and MSY121 (SRB5-myc sin4Δ) (C); and FZY217 (gcn4Δ SIN4-myc), FZY219 (gcn4Δ SIN4-myc gal11Δ), FZY223 (gcn4Δ SIN4-myc pgd1Δ), FZY221 (gcn4Δ SIN4-myc med2Δ), and FZY225 (gcn4Δ SIN4-myc srb2Δ) (D and E).

FIG. 6.

Northern analysis of ARG1 and SNZ1 mRNA expression in mutants lacking single or double tail domain subunits. (A) Total RNA was isolated from strains grown under the inducing conditions described for Fig. 4 and subjected to Northern analysis, probing for ARG1 and SNZ1 mRNAs and for SCR1 RNA as a loading control. Adjacent lanes derive from duplicate cultures of the same strains. The ARG1 and SNZ1 signals were quantified with a phosphorimager and normalized for the cognate SCR1 signals. The normalized average for the gcn4Δ strain was subtracted from the normalized averages for all other strains, and the differences were plotted in the histogram as the percentages of the corresponding difference obtained for WT. The light and dark bars for each strain indicate the results obtained from duplicate cultures, as described above. (B) Percentages of WT levels of ARG1 and SNZ1 mRNAs in mediator mutants grown under noninducing conditions. The analysis for panel A was carried out on the same strains grown in the absence of SM. The Northern signals for each mutant were expressed as percentages of the corresponding value for WT and are listed below the mutant genotype. The following strains were analyzed: BY4741 (GCN4), 249 (gcn4Δ), 1742 (GCN4 gal11Δ), LS01 (GCN4 med2Δ), 4393 (GCN4 pgd1Δ), LS03 (GCN4 gal11Δ med2Δ), LS10 (GCN4 gal11Δ pgd1Δ), LS20 (GCN4 med2Δ pgd1Δ), 1976 (GCN4 sin4Δ), LS09 (GCN4 sin4Δ gal11Δ), LS12 (GCN4 sin4Δ med2Δ), and LS11 (GCN4 sin4Δ pgd1Δ).

FIG. 7.

ChIP analysis of Gcn4p-dependent binding of TBP and Rpb1p to the ARG1 promoter and coding sequences in med2Δ and sin4Δ mutants. The following gcn4Δ strains were transformed with high-copy-number plasmid pHG1239 (GCN4) or empty vector (gcn4): FZY232 (gcn4Δ SRB6-myc), FZY234 (gcn4Δ SRB6-myc sin4Δ), and FZY278 (gcn4Δ SRB6-myc med2Δ) ChIP analysis was done as described for Fig. 4 except that antibodies against TBP or Rpb1p were used for the immunoprecipitations and primers to amplify the ARG1 TATA element (A and B) or 3′ end of the ARG1 ORF (C) were included.