A functional module of yeast mediator that governs the dynamic range of heat-shock gene expression

Abstract

We report the results of a genetic screen designed to identify transcriptional coregulators of yeast heat-shock factor (HSF). This sequence-specific activator is required to stimulate both basal and induced transcription; however, the identity of factors that collaborate with HSF in governing noninduced heat-shock gene expression is unknown. In an effort to identify these factors, we isolated spontaneous extragenic suppressors of hsp82-deltaHSE1, an allele of HSP82 that bears a 32-bp deletion of its high-affinity HSF-binding site, yet retains its two low-affinity HSF sites. Nearly 200 suppressors of the null phenotype of hsp82-deltaHSE1 were isolated and characterized, and they sorted into six expression without heat-shock element (EWE) complementation groups. Strikingly, all six groups contain alleles of genes that encode subunits of Mediator. Three of the six subunits, Med7, Med10/Nut2, and Med21/Srb7, map to Mediator's middle domain; two subunits, Med14/Rgr1 and Med16/Sin4, to its tail domain; and one subunit, Med19/Rox3, to its head domain. Mutations in genes encoding these factors enhance not only the basal transcription of hsp82-deltaHSE1, but also that of wild-type heat-shock genes. In contrast to their effect on basal transcription, the more severe ewe mutations strongly reduce activated transcription, drastically diminishing the dynamic range of heat-shock gene expression. Notably, targeted deletion of other Mediator subunits, including the negative regulators Cdk8/Srb10, Med5/Nut1, and Med15/Gal11 fail to derepress hsp82-deltaHSE1. Taken together, our data suggest that the Ewe subunits constitute a distinct functional module within Mediator that modulates both basal and induced heat-shock gene transcription.

Figure 1.

HSF localizes to the yeast nucleus under both noninducing and heat-shock-activating conditions. Strain HSF1-GFP was cultivated and photographed under non-heat-shock (30°) or heat-shock (37° for 5 min) conditions as indicated.

Figure 2.

Structural phenotypes of hsp82 alleles and suppressor screen strategy. (A) Chromatin structure of the wild type and mutant hsp82 promoter. At the wild-type allele, HSF occupies HSE1 prior to heat shock, and all three HSEs following it. Its promoter nucleosomes are highly remodeled (DNase I hypersensitive) under non-heat-shock conditions and displaced, along with ORF nucleosomes, upon heat shock (depicted) (Zhao et al. 2005). At the hsp82-ΔHSE1 allele, HSF occupancy is reduced 20-fold relative to HSP82⁺ and its promoter nucleosomes (shaded green) are stable and precisely positioned. They are not detectably altered by heat shock. Nucleosome +1, located downstream of the initiation site, is also detectable at HSP82⁺ under noninducing conditions. (B) Selection/screen strategy employed in this study. Depicted are the three hsp82 alleles (all chromosomal integrants) present in strain HS1004 and the primary phenotypes of the parental strain and suppressor mutants.

Figure 2.

Structural phenotypes of hsp82 alleles and suppressor screen strategy. (A) Chromatin structure of the wild type and mutant hsp82 promoter. At the wild-type allele, HSF occupies HSE1 prior to heat shock, and all three HSEs following it. Its promoter nucleosomes are highly remodeled (DNase I hypersensitive) under non-heat-shock conditions and displaced, along with ORF nucleosomes, upon heat shock (depicted) (Zhao et al. 2005). At the hsp82-ΔHSE1 allele, HSF occupancy is reduced 20-fold relative to HSP82⁺ and its promoter nucleosomes (shaded green) are stable and precisely positioned. They are not detectably altered by heat shock. Nucleosome +1, located downstream of the initiation site, is also detectable at HSP82⁺ under noninducing conditions. (B) Selection/screen strategy employed in this study. Depicted are the three hsp82 alleles (all chromosomal integrants) present in strain HS1004 and the primary phenotypes of the parental strain and suppressor mutants.

Figure 3.

ewe suppressors strongly activate noninduced transcription of the hsp82/lacZ reporter. (A) A representative suppressor of each complementation group was grown in rich medium at 30° to early log phase and then split into two aliquots, noninduced (NHS) and heat shock induced (HS). The latter culture was subjected to a 30° → 39° thermal upshift for 45 min and allowed to recover at 30° for 20 min; then both cultures were harvested, whole-cell extracts were isolated, and β-gal levels were determined as described in materials and methods. Depicted are means ±SEM (n = 3). Strains used were HS1004 (WT), HS1005 (ewe1/sin4), J1 (ewe2/rgr1), S5 (ewe3/med7), S2 (ewe4/srb7), J15 (ewe5/nut2), and J34 (ewe6/rox3). (B) Fold inducibility of hsp82-ΔHSE1 in the wild-type strain and spontaneous ewe mutants (derived from the data of A). Dashed line indicates a level of 1.0 (no induction).

Figure 4.

Disruption of GAL11, SRB2, SRB10, or NUT1 has little or no affect on hsp82-ΔHSE1 expression. Wild-type (WT) and mutant strains were cultivated, split into NHS and HS aliquots, and assayed for β-gal activity as in Figure 3. (A) Noninduced hsp82-ΔHSE1/lacZ expression in WT (HS1001), gal11Δ (DAD3), srb2Δ (DAD2), and srb10Δ (RRG2) strains. (B) Heat-shock-induced expression of hsp82-ΔHSE1/lacZ (open bars) compared with nonshocked expression (solid bars) in the same strains. (C) hsp82-ΔHSE1/lacZ expression in WT and nut1Δ strains (HS1001 and JHD1, respectively) under NHS and HS conditions as indicated. (A–C) means ±SEM (n = 3).

Figure 5.

HSP gene expression of ewe suppressors under control and heat-shock-inducing conditions. Northern analysis of wild-type (WT; HS1004), rgr1 (J1), rox3 (J34), srb7 (S2), nut2 (J15), and med7 (S5) strains cultivated in rich medium to early log phase in 30°. Cultures were either maintained at 30° (basal) or shifted to 39° for 15 min (induced) prior to isolation of total cellular RNA. Steady-state HSP mRNA levels were detected using gene-specific probes; signals were normalized to those of ACT1 (y-axis values are quotients: HSP mRNA/ACT1 mRNA). ACT1 expression, in turn, was independently quantified in each suppressor mutant relative to the abundance of the pol III transcript, SCR1, and found to be unaffected (S. B. Kremer and D. S. Gross, data not shown). For all panels, means of two or three independent experiments ±SD are illustrated.

Figure 5.

HSP gene expression of ewe suppressors under control and heat-shock-inducing conditions. Northern analysis of wild-type (WT; HS1004), rgr1 (J1), rox3 (J34), srb7 (S2), nut2 (J15), and med7 (S5) strains cultivated in rich medium to early log phase in 30°. Cultures were either maintained at 30° (basal) or shifted to 39° for 15 min (induced) prior to isolation of total cellular RNA. Steady-state HSP mRNA levels were detected using gene-specific probes; signals were normalized to those of ACT1 (y-axis values are quotients: HSP mRNA/ACT1 mRNA). ACT1 expression, in turn, was independently quantified in each suppressor mutant relative to the abundance of the pol III transcript, SCR1, and found to be unaffected (S. B. Kremer and D. S. Gross, data not shown). For all panels, means of two or three independent experiments ±SD are illustrated.

Figure 6.

The C-terminal truncation mutant rgr1-Δ2 strongly enhances the basal transcription of both hsp82-ΔHSE1 and wild-type HSP genes, while drastically reducing their induced transcription. (A) hsp82-ΔHSE1/lacZ (β-gal) expression levels in RGR1⁺ (HS1004) and rgr1-Δ2 (SBK501) strains under noninducing and heat-shock-inducing conditions were determined as in Figure 3. Shown are means ±SEM (n = 4). (B) Dynamic range of expression of the strains analyzed in A. Dashed line indicates a level of 1.0 (no induction). (C and D). Northern analyses of RGR1⁺ (DY150) and rgr1-Δ2 (DY2694) strains maintained at 30° (C) or subjected to a 15-min 39° heat shock (D) using HSP-specific probes as in Figure 5. Illustrated are means ±SD (n = 2 or 3). (E) Dynamic range of heat-shock gene expression of RGR1⁺ and rgr1-Δ2 strains (data from C and D).

Figure 6.

The C-terminal truncation mutant rgr1-Δ2 strongly enhances the basal transcription of both hsp82-ΔHSE1 and wild-type HSP genes, while drastically reducing their induced transcription. (A) hsp82-ΔHSE1/lacZ (β-gal) expression levels in RGR1⁺ (HS1004) and rgr1-Δ2 (SBK501) strains under noninducing and heat-shock-inducing conditions were determined as in Figure 3. Shown are means ±SEM (n = 4). (B) Dynamic range of expression of the strains analyzed in A. Dashed line indicates a level of 1.0 (no induction). (C and D). Northern analyses of RGR1⁺ (DY150) and rgr1-Δ2 (DY2694) strains maintained at 30° (C) or subjected to a 15-min 39° heat shock (D) using HSP-specific probes as in Figure 5. Illustrated are means ±SD (n = 2 or 3). (E) Dynamic range of heat-shock gene expression of RGR1⁺ and rgr1-Δ2 strains (data from C and D).

Figure 7.

Basal transcription of PHO5 is elevated in the context of certain ewe mutations. Northern analysis of wild-type (WT; HS1004), rgr1 (J1), rox3 (J34), srb7 (S2), nut2 (J15), and med7 (S5) strains cultivated in rich YPDA medium to early log phase in 30°. PHO5 transcript levels were normalized to those of ACT1. Depicted are means of two independent experiments ±SD.

Figure 8.

Location of Ewe subunits within yeast pol II Mediator as depicted in an integrated interaction map. Subunit locations were deduced from the outcome of pairwise two-hybrid assays (Guglielmi et al. 2004 and references therein). Modified from Gugliemi et al. (2004) and presented here with permission.