A multiplicity of coactivators is required by Gcn4p at individual promoters in vivo

Abstract

Transcriptional activators interact with multisubunit coactivators that modify chromatin structure or recruit the general transcriptional machinery to their target genes. Budding yeast cells respond to amino acid starvation by inducing an activator of amino acid biosynthetic genes, Gcn4p. We conducted a comprehensive analysis of viable mutants affecting known coactivator subunits from the Saccharomyces Genome Deletion Project for defects in activation by Gcn4p in vivo. The results confirm previous findings that Gcn4p requires SAGA, SWI/SNF, and SRB mediator (SRB/MED) and identify key nonessential subunits of these complexes required for activation. Among the numerous histone acetyltransferases examined, only that present in SAGA, Gcn5p, was required by Gcn4p. We also uncovered a dependence on CCR4-NOT, RSC, and the Paf1 complex. In vitro binding experiments suggest that the Gcn4p activation domain interacts specifically with CCR4-NOT and RSC in addition to SAGA, SWI/SNF, and SRB/MED. Chromatin immunoprecipitation experiments show that Mbf1p, SAGA, SWI/SNF, SRB/MED, RSC, CCR4-NOT, and the Paf1 complex all are recruited by Gcn4p to one of its target genes (ARG1) in vivo. We observed considerable differences in coactivator requirements among several Gcn4p-dependent promoters; thus, only a subset of the array of coactivators that can be recruited by Gcn4p is required at a given target gene in vivo.

FIG. 1.

Analysis of SM resistance and physical and genetic assays used to confirm the identity of deletion strains. (A) Tenfold serial dilutions of the isogenic WT, gcn4Δ, and relevant deletion strains grown in SC medium were spotted to SC (control) medium or SC−ILV medium containing SM at 0.25, 0.5, 1.0, and 2.0 μg/ml. Growth on each plate after 3 days at 25°C was scored and adjusted for slow growth on SC medium, and these values were used to generate semiquantitative scores expressed as a percentage of the WT score on the same plate (SM resistance). (B) Schematic representation of PCR confirmation of deletions. For each gene of interest an upstream primer (A) or downstream primer (D) was used in conjunction with internal primers (B or C, respectively) to identify the WT allele or with primers specific for the kanMX4 sequences (primer KanB or KanC, respectively) to identify the deletion. All primer sequences were the same as those used by the Saccharomyces Genome Deletion Project for confirmation of the deletions (listed at http://www-sequence.stanford.edu/group/yeast_deletion_project/deletions3.html). (C) Example of the complementation assay used to confirm the identity of mutants displaying SM resistance less than 85% of that of the WT. A plasmid containing the WT gene corresponding to the relevant deletion (in this case GAL11; see Table 2 for a complete list of plasmids), or the empty vector, was introduced into the mutant strain, and transformants were tested by replica plating for sensitivity to SM.

FIG. 2.

Phenotypes of deletion mutants lacking subunits of chromatin-modifying complexes. The SM resistance measured as described for Fig. 1A and induced UAS_GCRE-CYC1-lacZ expression measured as described in Table 1 for each deletion strain are shown graphically as percentages of the WT values. The values for the UAS_GCRE-CYC1-lacZ reporter are averages for three independent transformants induced with SM. Standard deviations, all less than 20% of the mean values, have been omitted for clarity. (A) HAT-containing complexes; (B) chromatin-remodeling complexes.

FIG. 3.

Phenotypes of mediator and related complexes and miscellaneous mutants. (A) Venn diagram depicting the relationships among the subunits of CCR4-NOT, SRB/MED, the Paf1 complex, and THO/TREX; (B) graphic representation of the phenotypes of deletion mutants lacking subunits of these complexes, as shown in Fig. 2; (C) phenotypes of the mbf1Δ strain analyzed as for panel B.

FIG. 4.

Northern analysis of authentic Gcn4p target genes in a typical subset of deletion mutants. Total RNA was isolated for each strain under the inducing and noninducing conditions described in Table 1, and equal amounts of RNA were subjected to Northern analysis, probing for ACT1, ARG1, HIS4, ILV2, and SNZ1 mRNAs. Adjacent lanes contain RNA samples isolated from two independent cultures for each strain. The hybridization signals were quantified with a PhosphorImager (Molecular Dynamics, with ImageQuant 5.2 software), and the values obtained for SNZ1 (A), HIS4 (B), and ARG1 (C) were normalized to the corresponding ACT1 signals. The resulting ratios calculated for the mutant strains were normalized to the ratio measured in the WT, and the normalized ratios are plotted in histograms beneath the corresponding lanes of each blot.

FIG. 5.

Western analysis of Gcn4p levels in Gcn⁻ mutants. Single-copy plasmid p2382 (CEN) or high-copy-number plasmid pHQ1239 (2μm) harboring the GCN4-HA₃ allele was introduced into the WT and Gcn⁻ deletion strains, and the WT strain was also transformed with empty CEN or 2μm vector. (A and B) Extracts were prepared from two transformants of each strain induced with SM. Two amounts (20 and 50 μg) of total protein, labeled as relative protein (Rel. Prot.) amounts of 1 and 2.5, respectively, were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with Gcd6p antibodies and anti-HA antibodies. Lanes 1 and 2 contain samples from the WT strain bearing the empty vector. All Western blotting was carried out two or more times, and representative results are presented for the ccr4Δ (A) and not5Δ (B) mutants. All results are summarized in Table 5. (C) Most mutants with lowered levels of HA₃-Gcn4p, such as the not5Δ strain, maintain their SM^s phenotypes after transformation with the high-copy-number GCN4-HA₃ plasmid (2μm). The not5Δ and gcn4Δ mutants and the WT strain containing p2382 (CEN), pHQ1239 (2μm), or empty vector were replica plated to SC−ILV medium containing 1 μg of SM per ml and incubated at 25°C for 3 days.

FIG. 6.

Deletions of chromatin-modifying enzymes do not generally produce Gcn⁻ phenotypes (A to C). The indicated mutant and WT strains were tested for sensitivity to SM as shown in Fig. 1A.

FIG. 7.

GST-Gcn4p interacts specifically with the RSC and CCR4-NOT complexes but not with the Paf1 complex in cell extracts. Equal amounts of GST, GST-Gcn4p (GST-WT), and GST-Gcn4p containing 10 alanine substitutions in the activation domain (GST-10Ala) were incubated with WCEs from yeast strains grown in YPD medium. The GST proteins were precipitated with glutathione Sepharose, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and detected by Western analysis. (A) Equal amounts of WCEs from WT (BY4741), rsc1Δ (strain 4686), and rsc2Δ (strain 5266) strains were incubated with the GST proteins. Blots were probed with antibodies against the proteins listed on the left of the upper three panels. The bottom two panels depict Ponceau S staining of total proteins. Input lanes contained 1% of the WCEs used in pull-down assays. (B) Same as panel A except that WCEs were from derivatives of WT strain BY4741 expressing Myc₁₃-Rsc1p (top three panels) or Myc₁₃-Rsc2p (lower three panels), detected with Myc antibodies. (C) GST proteins were incubated with yeast WCE from WT strain BY4741, and the pull-down assays were probed with antibodies against the proteins listed beside the three panels, subunits of CCR4-NOT (Caf16p), SRB/MED (Srb7), and TFIID (TAF11/TAF40). (D) The GST proteins were incubated with WCEs from strains derived from BY4741 expressing Myc₁₃-tagged forms of Dhh1p (DHH1-myc), Ccr4p (CCR4-myc), or Paf1p (PAF1-myc). The tagged proteins were detected with Myc antibody, and SRB/MED interaction was detected with Srb7p antibody. Input lanes contained 10% of the WCEs used in the pull-down assays.

FIG. 8.

Evidence from ChIP analysis that Gcn4p recruits seven coactivators to the ARG1 promoter in vivo. (A) gcn4Δ strains expressing different Myc-tagged coactivator subunits, all derived from the gcn4Δ strain 249, were transformed with the high-copy-number GCN4 plasmid pHQ1239 or empty vector. The resulting transformants and gcn4Δ strain 249 containing vector or the GCN4-myc plasmid pSK-1 were induced with SM and treated with formaldehyde. Chromatin was sheared, heated to reverse the cross-links, and immunoprecipitated with anti-Myc antibodies. The amounts of coprecipitated DNA containing the ARG1 UAS or POL1 ORF (upper panels [IP]) and the corresponding amounts in the input chromatin samples (lower panels) were measured by quantitative PCR. Representative results are shown in lanes 3 to 16 for the high-copy-number GCN4 (even-numbered lanes) and vector (odd-numbered lanes) transformants of strains containing the indicated Myc₁₃-tagged subunits. Lanes 1 and 2 show representative results from the gcn4Δ strain transformed with vector or the GCN4-myc plasmid. (B and C) For each ChIP experiment, the PCR products were quantified by phosphorimaging analysis, and the ratio of UAS_ARG1 signals in the immunoprecipitated samples to those in the input samples was calculated and normalized for the corresponding ratio calculated for the POL1 signals. The resulting normalized ratio (IP_UASARG1/input_UASARG1)/(IP_POL1/input_POL1) obtained for the GCN4 strain was divided by the corresponding normalized ratio calculated for the gcn4Δ strain. The resulting values obtained in three or more independent experiments for each tagged strain were averaged, and the mean values and standard errors were plotted in the histograms as the ratios of the amounts of UAS_ARG1 specifically associated with each tagged subunit in GCN4 versus gcn4Δ cells.

FIG. 9.

Summary of effects of coactivator deletions on induction by Gcn4p of multiple target genes. The table includes only those mutants for which we assayed expression of HIS3-GUS or Gcn4p target gene transcripts by Northern analysis. The data shown here are from Table 1 (SM^r and induced UAS_GCRE-CYC1-lacZ expression), Table 3 (induced HIS3-GUS expression), and Table 4 (induced SNZ1, HIS4, ARG1, and ILV2 mRNAs). Data that are in italics in the tables are highlighted in red here; those underlined and in boldface with an asterisk, underlined and in boldface, or underlined only in the tables are shown in dark green, light green, or yellow, respectively. Briefly, data highlighted in dark and light green provide strong and suggestive evidence, respectively, that Gcn4p activation of the promoter is impaired by the mutation. Data highlighted in yellow indicate reduced promoter function in the mutant that cannot be attributed specifically to a defect in activation by Gcn4p. Data highlighted in red indicate greater-than-WT expression of the promoter in the mutant.