Academic journal article Genetics

A Functional Module of Yeast Mediator That Governs the Dynamic Range of Heat-Shock Gene Expression

Academic journal article Genetics

A Functional Module of Yeast Mediator That Governs the Dynamic Range of Heat-Shock Gene Expression

Article excerpt

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-ΔHSE1, 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-ΔHSE1 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-ΔHSE1, 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-ΔHSE1. 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.

WHEN exposed to thermal or chemical stress, orV V ganisms 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) Hsfl (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 (GROSS et al. 1993; ERKINE et al. 1996), or whether HSF plays a more direct role in recruiting transcriptional coactivators under noninducing conditions, is unknown.

HSF is of additional interest, given that it can activate its target genes in the absence of several key general transcription factors (GTFs). These include Taf9 (TAFnI?, constituent of both SAGAand TFIID), Medl7/Srb4 and Med22/Srb6 (both subunits of Mediator), TFIIA, Kin28 (TFIIH kinase), and even the C-terminal domain (CTD) of the large subunit of RNA polymerase II (ApONE et al. 1998; LEE and Lis 1998; McNEiL et al. 1998; MOQTADERI et al. 1998; CHOU et al. 1999). Moreover, activated HSF has been shown to mediate gene-wide histone displacement and can do so in the absence of prominent chromatin remodeling (Swi/Snf), histone modification (Setl, Gcn5), and transcriptional elongation (Pafl) complexes (ZHAO et al. 2005). These observations suggest the possibility that HSF uses a novel route for transcriptional activation of its target genes, a notion supported by artificial recruitment experiments. …

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