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 heatshock 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 heatshock gene expression is unknown. In an effort to identify these factors, we isolated spontaneous extragenic suppressors of hsp82-DHSE1, 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-DHSE1 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-DHSE1, 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-DHSE1. 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. W HEN exposed to thermal or chemical stress, organisms respond by vigorously transcribing genes encoding heat-shock proteins (HSPs). HSPs function as molecular chaperones and protect the cellalong with ubiquitin, proteases, metallothioneins, and antioxidant enzymes-from damage caused by the expression of misfolded proteins. In the yeast Saccharomyces cerevisiae, the expression of heat-responsive genes is stimulated by the sequence-specific transcriptional activator heat-shock factor (HSF) Hsf1 (ScHSF) (Sorger and Pelham 1988;Nieto-Sotelo et al. 1990;Sorger 1990). In response to metabolic, oxidative, or osmotic stress, the transcription of a number of HSP genes is additionally enhanced by the gene-specific activators Msn2/Msn4 and Skn7 (Boy-Marcotte et al. 1998;Treger et al. 1998;Gasch et al. 2000;Raitt et al. 2000;Amoros and Estruch 2001;Kandror et al. 2004). Nonetheless, the only activator known to promote basal heat-shock gene transcription is HSF (McDaniel et al. 1989;Park and Craig 1989;Erkine et al. 1996). Whether this basal expression is an indirect consequence of HSF's role in establishing and maintaining a nucleosomeremodeled (''nucleosome-free'') structure over the transcription start site Erkine et al. 1996), or whether HSF plays a more direct role in recruiting transcriptional coactivators under n...

Mediator Recruitment to Heat Shock Genes Requires Dual Hsf1 Activation Domains and Mediator Tail Subunits Med15 and Med16

“…Spontaneous ewe suppressor mutations, derived from strain HS1004 (SLY101 background) (20), were transferred to BY4741 by allelic replacement, creating strains termed SYK1000, S7JJ2a, and N2JJ1a, respectively. To generate these strains, we used a modification of the cloning-free PCR-based allele replacement strategy described by Rothstein and colleagues (40).…”

“…The quantity of cDNA present in each sample Ref. 20 was determined by subtracting the background signal (without reverse transcriptase), followed by normalization to the SCR1 transcript. Amplicon coordinates were as follows: SSA4, ϩ815 to ϩ946; HSP104, ϩ1223 to ϩ1394; ZPR1, ϩ720 to ϩ809; and HSP82, ϩ2134 to ϩ2228.…”

“…Dissociation of the Middle module from the core complex (as seen in med19⌬ mutants) results in defects in both basal and activated transcription in vitro (16). In addition, genetic analysis indicates that the Middle module has roles in both transcriptional activation and repression in vivo (17)(18)(19)(20) and participates in postrecruitment steps in transcription, including promoter escape and Pol II elongation (21). The Tail module is composed * This work was supported by National Science Foundation Grants MCB-0747227 and MCB-1025025 (to D. S. G.).…”

“…scHsf1 is of additional interest because it bypasses the requirement for a number of critical general transcription factors, including Kin28 (TFIIH kinase), Taf9 (a subunit of both TFIID and SAGA), TFIIA, and even the CTD of Pol II (reviewed in Ref. 20). In contrast, at least one coactivator is critically required for HSP gene activation, Mediator (20,21,37).…”

“…20). In contrast, at least one coactivator is critically required for HSP gene activation, Mediator (20,21,37). Mediator is robustly recruited to the HSP gene promoter regions in both yeast (38) and Drosophila, and in the case of Drosophila, this recruitment precedes that of Pol II (39).…”