The chromatin structure of heat shock protein (HSP)-encoding genes undergoes dramatic alterations upon transcriptional induction, including, in extreme cases, domain-wide nucleosome disassembly. Here, we use a combination of gene knockout, in situ mutagenesis, chromatin immunoprecipitation, and expression assays to investigate the role of histone modification complexes in regulating heat shock gene structure and expression in Saccharomyces cerevisiae. Two histone acetyltransferases, Gcn5 and Esa1, were found to stimulate HSP gene transcription. A detailed chromatin immunoprecipitation analysis of the Gcn5-containing SAGA complex (signified by Spt3) revealed its presence within the promoter of every heat shock factor 1-regulated gene examined. The occupancy of SAGA increased substantially upon heat shock, peaking at several HSP promoters within 30 -45 s of temperature upshift. SAGA was also efficiently recruited to the coding regions of certain HSP genes (where its presence mirrored that of pol II), although not at others. Robust and rapid recruitment of repressive, Rpd3-containing histone deacetylase complexes was also seen and at all HSP genes examined. A detailed analysis of HSP82 revealed that both Rpd3(L) and Rpd3(S) complexes (signified by Sap30 and Rco1, respectively) were recruited to the gene promoter, yet only Rpd3(S) was recruited to its open reading frame. A consensus URS1 cis-element facilitated the recruitment of each Rpd3 complex to the HSP82 promoter, and this correlated with targeted deacetylation of promoter nucleosomes. Collectively, our observations reveal that SAGA and Rpd3 complexes are rapidly and synchronously recruited to heat shock factor 1-activated genes and suggest that their opposing activities modulate heat shock gene chromatin structure and fine-tune transcriptional output.The heat shock response is a regulated transcriptional response to elevated temperature and other environmental insults. It is essential for the viability of all organisms. In mammals, physiological stresses such as fever, inflammation, infection, ethanol toxicity, and tissue ischemia are countered by the heat shock response, which is regulated by heat shock factor 1 (Hsf1), 2 an evolutionarily conserved, trimeric transcriptional activator (reviewed in Ref. 1). Concomitant with its conferring protection from environmental stress, Hsf1 plays important roles in suppressing neurodegeneration (2) and in enhancing carcinogenesis (3). In many organisms, including Homo sapiens, Drosophila melanogaster, and Saccharomyces cerevisiae, Hsf1 is localized within the nucleus under both control and stressful conditions (4 -6). In the budding yeast S. cerevisiae, Hsf1 binds its high affinity heat shock-response elements (HSEs) under noninducing conditions (7), from which it locally opens chromatin structure and promotes constitutive transcription (8 -11). In addition, the protein inducibly binds low affinity HSEs, contributing to the substantial increase in target gene transcription that occurs in response to stress (4,(12)(13)(...

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 is a modular multisubunit complex that functions as a critical coregulator of RNA polymerase II (Pol II) transcription. While it is well accepted that Mediator plays important roles in the assembly and function of the preinitiation complex (PIC), less is known of its potential roles in regulating downstream steps of the transcription cycle. Here we use a combination of genetic and molecular approaches to investigate Mediator regulation of Pol II elongation in the model eukaryote, Saccharomyces cerevisiae. We find that ewe (expression without heat shock element) mutations in conserved Mediator subunits Med7, Med14, Med19, and Med21-all located within or adjacent to the middle module-severely diminish heat-shock-induced expression of the Hsf1-regulated HSP82 gene. Interestingly, these mutations do not impede Pol II recruitment to the gene's promoter but instead impair its transit through the coding region. This implies that a normal function of Mediator is to regulate a postinitiation step at HSP82. In addition, displacement of histones from promoter and coding regions, a hallmark of activated heat-shock genes, is significantly impaired in the med14 and med21 mutants. Suggestive of a more general role, ewe mutations confer hypersensitivity to the antielongation drug 6-azauracil (6-AU) and one of them-med21-impairs Pol II processivity on a GAL1-regulated reporter gene. Taken together, our results suggest that yeast Mediator, acting principally through its middle module, can regulate Pol II elongation at both heat-shock and non-heat-shock genes. IN eukaryotes, transcription of the DNA template into premRNA by RNA polymerase II (Pol II) occurs in a welldefined, stepwise fashion. First, chromatin, the nucleoprotein complex in which the DNA is packaged, must unfold into a 10 nm, beads-on-a-string filament, and for many genes a nucleosome-free region needs to be created over the core promoter (Venters and Pugh 2009). Both are achieved via activator-mediated recruitment of chromatin modification and remodeling enzymes (reviewed in Li et al. 2007). Once a permissive chromatin template has been created, Pol II and the other general transcription factors then bind the core promoter, where they are assembled into a preinitiation complex (PIC; formally analogous to the closed RNA polymerase complex in prokaryotes). Next, Pol II forms an open complex concomitant with ATP-dependent melting of the DNA strands and initiates transcription following phosphorylation of its C-terminal repeat domain (CTD) at Ser5 residues by the TFIIH kinase, Cdk7. Following synthesis of 25-30 nucleotides, Pol II pauses, allowing the nascent RNA to be capped. Finally, Pol II transitions to productive elongation, which requires Ser2 phosphorylation of the CTD. In metazoans, this is catalyzed by P-TEFb and in yeast by Bur1 and Ctk1 (reviewed in Saunders et al. 2006).A key regulator of many of the above steps is Mediator, an evolutionarily conserved, modular multiprotein complex. Mediator acts as a signal transducer through its interac...