Role of Mediator in Regulating Pol II Elongation and Nucleosome Displacement in Saccharomyces cerevisiae

Abstract

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...

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

“…Diploids were then sporulated, and tetrads were dissected; spores containing the ewe mutation were identified based on growth phenotype and confirmed by sequencing analysis (DNA Facility, Iowa State University, Ames, IA). Sequences of these recessive mutations were determined and are as follows: med10-1001, N64D point mutation followed by 94-amino acid C-terminal truncation; med19-1001, 112-amino acid C-terminal truncation; and med21-1002, L76P point mutation (21,41 (34). Plasmids encoding Hsf1 truncation mutants were the gift of Peter Sorger (MIT, Cambridge, MA).…”

“…Chromatin Immunoprecipitation-ChIP was performed essentially as described previously (21). Briefly, 300 -350-ml mid-log cell cultures were used in heat shock time course experiments, and 50-ml aliquots were removed at each time point.…”

“…Shock-Previous genetic analysis has implicated a role for Mediator in regulating heat shock gene transcription in S. cerevisiae (20,21,37). To gain insight into mechanism, we asked if both core Mediator and its reversibly associated Kinase module were recruited to Hsf1-regulated genes in response to heat stress and, if so, how rapidly.…”

“…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.).…”

“…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).…”