Mediator recruitment to heat shock genes requires dual Hsf1 activation domains and mediator tail subunits Med15 and Med16

Abstract

The evolutionarily conserved Mediator complex is central to the regulation of gene transcription in eukaryotes because it serves as a physical and functional interface between upstream regulators and the Pol II transcriptional machinery. Nonetheless, its role appears to be context-dependent, and the detailed mechanism by which it governs the expression of most genes remains unknown. Here we investigate Mediator involvement in HSP (heat shock protein) gene regulation in the yeast Saccharomyces cerevisiae. We find that in response to thermal upshift, subunits representative of each of the four Mediator modules (Head, Middle, Tail, and Kinase) are rapidly, robustly, and selectively recruited to the promoter regions of HSP genes. Their residence is transient, returning to near-background levels within 90 min. Hsf1 (heat shock factor 1) plays a central role in recruiting Mediator, as indicated by the fact that truncation of either its N- or C-terminal activation domain significantly reduces Mediator occupancy, whereas removal of both activation domains abolishes it. Likewise, ablation of either of two Mediator Tail subunits, Med15 or Med16, reduces Mediator recruitment to HSP promoters, whereas deletion of both abolishes it. Accompanying the loss of Mediator, recruitment of RNA polymerase II is substantially diminished. Interestingly, Mediator antagonizes Hsf1 occupancy of non-induced promoters yet facilitates enhanced Hsf1 association with activated ones. Collectively, our observations indicate that Hsf1, via its dual activation domains, recruits holo-Mediator to HSP promoters in response to acute heat stress through cooperative physical and/or functional interactions with the Tail module.

FIGURE 1.

Robust yet transient recruitment of holo-Mediator to HSP gene promoter regions. A–D, in vivo cross-linking analysis of representative Head, Middle, Tail, and Kinase subunits at the indicated regions of the SSA4, HSP104, and ZPR1 genes either prior to or for the indicated times following heat shock (30 °C to 39 °C upshift). For recovery (R), cells were heat-shocked at 39 °C for 15 min and then downshifted to 23 °C for 25 min. The indicated subunits were detected using TAP antibody; mock IP signal (beads alone) was subtracted from each ChIP signal. The abundance of each subunit at the promoter, ORF, and 3′-UTR of each gene was quantified relative to its abundance at the promoter at T = 0 min, which was assigned a value of 1. Depicted are means ± S.D. (error bars); n ≥ 3 independent samples.

FIGURE 2.

Mutations in Tail subunits Med14, Med15, and Med16 diminish Hsf1 occupancy of heat shock-induced promoters, whereas mutations in the Mediator Head, Middle, and Kinase modules have no effect. A, Hsf1 ChIP analysis of HSP promoters under non-inducing and 15 min heat shock-inducing conditions in isogenic MED⁺ and med14/rgr1-Δ2 strains DY150 and DY2694. The assay was conducted as in Fig. 1 except that an Hsf1-specific polyclonal antibody was used; preimmune signal was subtracted from each ChIP signal. The abundance of Hsf1 in response to heat shock was quantified relative to its abundance at each promoter under non-heat shock conditions (T = 0 min). Depicted are means ± S.D. (error bars); n ≥ 3. B, dynamic range of Hsf1 occupancy at HSP promoters in MED⁺ and rgr1-Δ2 strains during the first 15 min of heat shock (data from A). C, Hsf1 occupancy of HSP promoters in isogenic MED⁺ and med15Δ cells (BY4741 background) either maintained at 30 °C (0 min) or subjected to a 39 °C heat shock for the indicated times. Shown are means ± S.D., n = 3. D, as in C, except isogenic MED⁺ and med16Δ cells were examined. E and F, as in C, except Hsf1 occupancy of SSA4 and HSP82 promoters was assayed in MED⁺ and isogenic med19-1001, med10-1001, med21-1002, and cdk8Δ strains. Asterisks signify a significant difference in Hsf1 occupancy between WT and mutant (p < 0.05; two-tailed t test; equal variance).

FIGURE 3.

med16Δ diminishes recruitment of the Tail subunit Med3 to induced HSP promoters while obviating recruitment of subunits representative of the other three modules. A, occupancy of TAP-tagged Head, Middle, Tail and Kinase subunits within the SSA4 promoter in isogenic MED⁺ and med16Δ cells in a heat shock time course. TAP ChIP analysis was conducted as described in the legend to Fig. 1. Occupancy of each subunit in the MED⁺ strain at T = 0 min was assigned a value of 1; all other values (MED⁺ and med16Δ) are normalized to it. Only ChIP values above the beads alone background are shown. Depicted are means ± S.D. (error bars); n = 3. B–D, as in A, except Mediator subunit occupancy at the HSP104, HSP82, and ZPR1 promoters is depicted.

FIGURE 4.

med15Δ drastically reduces Tail subunit recruitment to induced HSP promoters, whereas the med15Δ med16Δ double mutation eliminates it. A, Med3-TAP occupancy of the SSA4, HSP104, and ZPR1 promoters in MED⁺, med15Δ, and med15Δ med16Δ cells in a heat shock time course conducted and analyzed as in Fig. 3. Depicted are means ± S.D. (error bars); n = 3. B, Western blot analysis of Med3-TAP in the same WT, med16Δ, med15Δ, and med15Δ med16Δ strains analyzed above. Pgk1 was used as loading control. Med3-TAP levels were quantified using a Storm 860 PhosphorImager, and the normalized values, relative to Pgk1, are displayed in the adjacent graph.

FIGURE 5.

med15Δ and med16Δ single mutations drastically reduce Head subunit Med17 recruitment to induced HSP promoters, whereas the med15Δ med16Δ double mutation eliminates it. Med17 occupancy of HSP promoters in MED⁺, med16Δ, med15Δ, and med15Δ med16Δ cells in a heat shock time course conducted and analyzed as in Fig. 3. Med17 was detected using a polyclonal anti-Med17/Srb4 antibody; mock IP signal (beads alone) was subtracted from each ChIP signal. Depicted are means ± S.D. (error bars); n = 3. Asterisks signify Med17 occupancy in the double mutant that is significantly less than that observed in either single mutant (p < 0.05; two-tailed t test; equal variance).

FIGURE 6.

Tail subunit mutations reduce Pol II recruitment to, and mRNA expression of, HSP genes during the early stages of heat shock. A (top panels), Pol II occupancy at the SSA4 promoter and ORF either prior to or for the indicated times following an instantaneous 39 °C heat shock (recovery (R) conducted as in Fig. 1). Pol II abundance was determined by ChIP using a CTD polyclonal antibody; preimmune background was subtracted from each immune signal. Net values for each time point/strain combination were normalized to the MED⁺ strain at T = 0 min; promoter and ORF were quantified separately. Depicted are means ± S.D. (error bars); n = 3. Bottom panels, RT-qPCR analysis of SSA4 mRNA levels in MED⁺, med16Δ, med15Δ, and med15Δ med16Δ strains. SSA4 mRNA/SCR1 RNA quotients, normalized to the MED⁺ 20 min quotient (which was set to 1.0), are shown. Note that SCR1 is a structural RNA (Pol III transcript). Depicted are means ± S.D. (n = 2); each MED⁺ versus mutant pairwise comparison was conducted separately. B–D, Pol II occupancy at HSP104, HSP82, and ZPR1 promoter and ORF regions and associated mRNA expression levels were determined as in A.

FIGURE 7.

Both N- and C-terminal activators of Hsf1 are required for efficient Mediator recruitment to HSP promoters, whereas either one or the other suffices for transcription. A, domain maps of Hsf1⁺ and its C- and N-terminally truncated derivatives (DNA-binding domain (DBD), trimerization domain (3-mer), and repression domain (CE2)). B, growth assay of HSF1⁺, HSF1^ΔCTA, HSF1^ΔNTA, and HSF1^{ΔNTA ΔCTA} strains. 5-Fold serial dilutions were spotted onto rich medium, and cells were grown at the indicated temperatures for 2–3 days. C and D, Hsf1 and Med4-TAP ChIP analysis of the SSA4 promoter either prior to or following instantaneous heat shock in the indicated isogenic strains, conducted as in Figs. 1 and 2. Means ± S.D. (error bars) are depicted; n = 3. E, RT-qPCR analysis of SSA4 expression under non-inducing and 15-min heat shock-inducing conditions in the indicated isogenic strains. SSA4 mRNA/SCR1 RNA quotients, normalized to the WT 0 min quotient (which was set to 1.0), are shown; means ± S.D. are depicted (n = 2). F–N, factor occupancy and mRNA expression levels of HSP104, HSP82, and ZPR1 analyzed as in C–E.

FIGURE 7.

Both N- and C-terminal activators of Hsf1 are required for efficient Mediator recruitment to HSP promoters, whereas either one or the other suffices for transcription. A, domain maps of Hsf1⁺ and its C- and N-terminally truncated derivatives (DNA-binding domain (DBD), trimerization domain (3-mer), and repression domain (CE2)). B, growth assay of HSF1⁺, HSF1^ΔCTA, HSF1^ΔNTA, and HSF1^{ΔNTA ΔCTA} strains. 5-Fold serial dilutions were spotted onto rich medium, and cells were grown at the indicated temperatures for 2–3 days. C and D, Hsf1 and Med4-TAP ChIP analysis of the SSA4 promoter either prior to or following instantaneous heat shock in the indicated isogenic strains, conducted as in Figs. 1 and 2. Means ± S.D. (error bars) are depicted; n = 3. E, RT-qPCR analysis of SSA4 expression under non-inducing and 15-min heat shock-inducing conditions in the indicated isogenic strains. SSA4 mRNA/SCR1 RNA quotients, normalized to the WT 0 min quotient (which was set to 1.0), are shown; means ± S.D. are depicted (n = 2). F–N, factor occupancy and mRNA expression levels of HSP104, HSP82, and ZPR1 analyzed as in C–E.

FIGURE 8.

Pol II occupancy of HSP genes in HSF1⁺, HSF1^ΔCTA, HSF1^ΔNTA, and HSF1^ΔNTAΔCTA strains. A, Pol II occupancy of the SSA4 promoter and ORF either prior to or following a 15-min heat shock. Pol II ChIP analysis was conducted and quantified as described in the legend to Fig. 6. Depicted are means ± S.D. (error bars); n = 3. Note that the extracts used for this analysis were the same as those used for the Hsf1 and Med4-TAP ChIP assays presented in Fig. 7. B–D, Pol II occupancy of the HSP104, HSP82, and ZPR1 promoter and ORF regions as in A.

FIGURE 9.

Factor occupancy of a typical yeast HSP gene under non-inducing, acutely inducing, and chronically inducing conditions. The location and abundance of the indicated factors, as inferred from kinetic ChIP assays (this study and Refs. 21, 38, 57, 70, 75), are depicted. The increase in fractional occupancy is symbolized by an increase in the number of symbols (Hsf1, nucleosomes) and/or boldness of font and color. Note that the enhanced promoter occupancy of Hsf1, Mediator, SAGA, Pol II, and general transcription factors (GTFs) in response to heat shock is very rapid and may occur in a nearly simultaneous fashion. Although Mediator occupancy of HSP genes in non-induced and chronically heat-shocked cells is typically <10% of that seen in acutely induced ones (e.g. see Figs. 1 and 5), other factors may be present, albeit at reduced levels, under these conditions (e.g. see Fig. 6) and are so indicated. In addition, whereas Mediator is exclusively recruited to the promoter, SAGA is recruited to both promoter and coding regions (57). Disc-shaped objects, nucleosomes; Ac, hyperacetylated forms of H3 and H4; Ser2-P and Ser5-P, phosphorylated CTD forms of Pol II (phospho-CTD states have not been determined for chronic heat shock conditions).