Mediator dynamics during heat shock in budding yeast

Abstract

The Mediator complex is central to transcription by RNA polymerase II (Pol II) in eukaryotes. In budding yeast (Saccharomyces cerevisiae), Mediator is recruited by activators and associates with core promoter regions, where it facilitates preinitiation complex (PIC) assembly, only transiently before Pol II escape. Interruption of the transcription cycle by inactivation or depletion of Kin28 inhibits Pol II escape and stabilizes this association. However, Mediator occupancy and dynamics have not been examined on a genome-wide scale in yeast grown in nonstandard conditions. Here we investigate Mediator occupancy following heat shock or CdCl₂ exposure, with and without depletion of Kin28. We find that Pol II occupancy shows similar dependence on Mediator under normal and heat shock conditions. However, although Mediator association increases at many genes upon Kin28 depletion under standard growth conditions, little or no increase is observed at most genes upon heat shock, indicating a more stable association of Mediator after heat shock. Unexpectedly, Mediator remains associated upstream of the core promoter at genes repressed by heat shock or CdCl₂ exposure whether or not Kin28 is depleted, suggesting that Mediator is recruited by activators but is unable to engage PIC components at these repressed targets. This persistent association is strongest at promoters that bind the HMGB family member Hmo1, and is reduced but not eliminated in hmo1Δ yeast. Finally, we show a reduced dependence on PIC components for Mediator occupancy at promoters after heat shock, further supporting altered dynamics or stronger engagement with activators under these conditions.

Figure 1.

Effect of heat shock on Mediator association. (A) Heat maps and line graphs depicting normalized occupancy of the Mediator tail module subunit, Med15, in kin28AA yeast treated with rapamycin and the parent strain YFR1321, also treated with rapamycin, before and after 15 min of heat shock, at 42 Hsf1 targets and 213 Msn2-4 targets and 137 RP genes (see Methods; Supplemental Table S2). (B) Box and whisker plots showing the ratios of Med15 occupancy with and without Kin28 depletion for the approximately 300 genes showing the highest Med15 occupancy in Kin28-depleted cells without or with heat shock; ratios are also shown for RP genes, Hsf1 targets, and Msn2-4 targets in heat-shocked cells. (C) Browser scans showing Med15 occupancy upstream of BAP2 and UBI4 in kin28AA yeast and the parent strain YFR1321, both treated with rapamycin, with and without heat shock. UBI4 is a target of Hsf1, whereas BAP2 is not a target of Hsf1 or Msn2-4. Scale, in reads per million mapped reads, is indicated for each scan. (D) Heat maps and line graphs depicting occupancy of the Mediator tail module subunit, Med15, in BY4741 yeast, kin28AA yeast treated with rapamycin, and the parent strain YFR1321, also treated with rapamycin, in the absence of heat shock, at “UAS genes” (see text and Supplemental Table S2) and RP genes.

Figure 2.

Mediator association persists at RP genes after heat shock. (A) Browser scans showing normalized occupancy of Med15 upstream of RPL42A and RPS4B in kin28AA yeast and the parent strain YFR1321, both treated with rapamycin, with and without heat shock (top four scans) or Rap1 in strain BY4741 (bottom scan). Scale, in reads per million mapped reads, is indicated for each scan. (B) Heat maps and line graphs depicting normalized occupancy of the Mediator head module subunit, Med18, in kin28AA yeast treated with rapamycin, with and without heat shock, at Hsf1 targets and RP genes. (C) Heat maps and line graphs depicting normalized occupancy of Mediator subunits Med15 (tail) and Med18 (head), and Rap1, at Hsf1 target genes and RP genes in BY4741. The signal observed at transcribed ORF regions (seen at Hsf1 targets under heat shock conditions, and at RP genes under non-heat-shocked conditions) is a ChIP artifact frequently observed at highly transcribed ORFs (Eyboulet et al. 2013; Park et al. 2013; Teytelman et al. 2013; Jeronimo and Robert 2014; Knoll et al. 2020).

Figure 3.

Mediator association persists at non-RP genes repressed by heat shock. (A) Heat maps and line graphs showing normalized occupancy of Pol II and Med15 at non-RP UAS genes (see text) having Pol II occupancy decreased by at least twofold upon heat shock (“UAS down”) (Supplemental Table S2) or having Pol II occupancy unchanged or increased upon heat shock (“UAS not down”) (Supplemental Table S2) in rapamycin-treated YFR1321, the parent strain to kin28AA yeast, with and without heat shock. (B) Heat maps and line graphs showing normalized occupancy of Med15 (tail) and Med18 (head) at “UAS down” and “UAS not down” genes in kin28AA yeast treated with rapamycin, with and without heat shock. (C) Browser scans showing Med15 and Pol II occupancy at ILV1 and TEF1 genes in kin28AA yeast (“−Kin28”) or the parent strain YFR1321 (“+Kin28”), both treated with rapamycin, with or without heat shock. Scale, in reads per million mapped reads, is indicated for each scan. Note that Pol II occupancy is reduced at both genes upon heat shock; the vertical dashed lines emphasize the shift of the Med15 peak toward the promoters only when Kin28 is depleted in the absence of heat shock.

Figure 4.

Persistent Mediator association preferentially occurs at Hmo1-binding RP genes repressed by heat shock. (A) Heat maps and line graphs showing normalized occupancy of Med15 and Med18 in kin28AA yeast treated with rapamycin, with and without heat shock, at RP genes divided into Hmo1-binding, non-Hmo1-binding, and Abf1-binding genes (Supplemental Table S2). (B) Browser scans showing occupancy of Med15 and Med18 in kin28AA yeast treated with rapamycin, with and without heat shock, and Rap1 and Hmo1 in non-heat-shocked yeast, at RPS24A and RPL21A (Hmo1-binding) and RPL40B and RPS9B (non-Hmo1-binding). Scale, in reads per million mapped reads, is indicated for each scan. (C) Heat maps and line graphs showing normalized occupancy of Med15 and Med18 at UAS genes, Hsf1 targets, and Rap1-binding RP genes that do or do not bind Hmo1, in wild-type (BY4741) and hmo1Δ yeast after 15 min of heat shock.

Figure 5.

Effect of depleting PIC components on Mediator association with gene promoters. (A) Heat maps and line graphs showing normalized occupancy of Med15 (tail) and Med18 (head) at TATA-containing, Taf1-depleted promoters from the 1000 genes with highest Pol II occupancy (228 genes); TATA-less, Taf1-enriched genes excluding RP genes from the 1000 genes with highest Pol II occupancy (330 genes); and RP genes after depletion of Kin28 alone or together with Taf1, TBP, or Rpb3, as indicated. (B) Heat maps and line graphs showing normalized occupancy of Med15 (tail) and Med18 (head) at the approximately 300 genes with highest Pol II occupancy after heat shock, Hsf1 targets, and RP genes after depletion of Kin28 alone or together with Taf1, TBP, or Rpb3, as indicated, without or with 15 min of heat shock, as indicated.

Figure 6.

Effect of CdCl₂ exposure on Pol II and Mediator occupancy. (A) Heat maps and line graphs showing normalized occupancy of Pol II at 50 strongly induced genes (Supplemental Table S2; Momose and Iwahashi 2001) and RP genes in the anchor-away parent strain, YFR1321. (B) Heat maps and line graphs showing normalized occupancy by Med15 (tail module) and Med18 (head module) at 50 strongly induced genes, the 500 genes having the highest ratio of induced to uninduced Pol II occupancy, and RP genes. (C) Browser scans showing Med15 occupancy in reads per million mapped reads after Kin28 depletion in unstressed cells, cells exposed to CdCl₂, and after 15 min of heat shock, as indicated, at the RPS4B and RPL42A loci. Scale, in reads per million mapped reads, is indicated for each scan.