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 pre-initiation complex (PIC) assembly, only transiently prior to 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 exhibits similar dependence on Mediator under normal and heat shock conditions. However, while 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. 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 hmo1∆ 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.

Function and dynamics of the Mediator complex: novel insights and new frontiers

“…The large majority (129 out of 137) of RP genes depend on the general regulatory factor Rap1 for their activation, and Mediator occupancy peaks coincided with Rap1 binding sites for RP genes repressed by heat shock. This was true even under conditions of Kin28 depletion, consistent with the absence of a PIC at the repressed RP genes [ 71 , 72 ]. Further investigation revealed persistent Mediator association at UAS regions not only at RP genes, but also at many other genes repressed by heat shock.…”

“…Altered Mediator dynamics following stress induction have also been observed in ChIP-seq experiments [ 71 ]. First, conspicuous ChIP-seq peaks were observed at UASs of genes activated by Hsf1 and Msn2/4 (activators of heat shock and stress-induced genes, respectively) after heat shock, consistent with earlier work reporting strong Mediator ChIP signal at genes induced by heat shock or galactose [ 21 ].…”

“…Mediator occupancy was previously reported at UAS regions of two repressed genes, ROX1 and ZRT2 , in yeast grown under anaerobic conditions, and at genes repressed by treatment with sulfometuron methyl, which mimics starvation conditions [ 22 , 24 ]. Persistent association at RP genes repressed by cadmium chloride administration was also observed [ 71 ]. The large majority (129 out of 137) of RP genes depend on the general regulatory factor Rap1 for their activation, and Mediator occupancy peaks coincided with Rap1 binding sites for RP genes repressed by heat shock.…”

“…All browser scans were produced using the integrative genome viewer [ 85 ]. Data is from [ 31 , 71 ].…”

Reliance of Host-Encoded Regulators of Retromobility on Ty1 Promoter Activity or Architecture

“…Gene ontology enrichment analysis was performed using the Gene Ontology Term Finder at the Saccharomyces genome database (SGD) (Cherry et al, 2012) (Paul et al, 2015) and PRJNA657372 (Sarkar et al, 2022).…”