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Evidence that Mediator is essential for Pol II transcription but not a required component of the preinitiation complex in vivo

ABSTRACT: Mediator, a transcriptional co-activator complex, is recruited to enhancers by DNA-binding activators via its tail module, and it interacts with RNA polymerase (Pol) II as part of the preinitiation complex (PIC) via its head module. Although Mediator has been described as a general transcription factor, it is unclear if it is essential for Pol II transcription and/or is a required component of the PIC in vivo. We comprehensively analyze this issue by measuring Pol II transcription upon rapid depletion of individual or multiple Mediator subunits from the nucleus. Depletion of individual subunits, even those essential for cell growth, causes a general but only modest decrease in Pol II transcription. Transcriptional effects are stronger on SAGA-dependent genes as opposed to TFIID-dependent genes. Thus, Mediator modules that associate either with the enhancer or with the core promoter confer partial transcriptional activity. Furthermore, Pol II transcription can occur when Mediator is not detected at core promoters. In contrast, simultaneous depletion of all Mediator modules causes a drastic decrease in Pol II transcription that is roughly comparable to the effect observed upon anchor-away-mediated depletion of Pol II or TBP. These results provide strong evidence that Mediator is essential for Pol II transcription in vivo, but that it is not a required component of the PIC.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE93190 | GEO | 2017/07/13

SECONDARY ACCESSION(S): PRJNA360382

REPOSITORIES: GEO

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Project description:Transcriptional repression of ribosomal components and tRNAs is coordinately regulated in response to a wide variety of environmental stresses. Part of this response involves the convergence of different nutritional and stress signaling pathways on Maf1, a protein that is essential for repressing transcription by RNA polymerase (pol) III in Saccharomyces cerevisiae. Here we identify the functions buffering yeast cells that are unable to down-regulate transcription by RNA pol III. MAF1 genetic interactions identified in screens of non-essential gene-deletions and conditionally-expressed essential genes reveal a highly interconnected network of 64 genes involved in ribosome biogenesis, RNA pol II transcription, tRNA modification, ubiquitin-dependent proteolysis and other processes. A survey of non-essential MAF1 synthetic sick/lethal (SSL) genes identified six gene-deletions that are defective in transcriptional repression of ribosomal protein (RP) genes following rapamycin treatment. This subset of MAF1 SSL genes included MED20 which encodes a head module subunit of the RNA pol II Mediator complex. Genetic interactions between MAF1 and subunits in each structural module of Mediator were investigated to examine the functional relationship between these transcriptional regulators. Gene expression profiling identified a prominent and highly selective role for Med20 in the repression of RP gene transcription following treatments with rapamycin, chlorpromazine and tunicamycin and in post-diauxic cells. In addition, attenuated repression of RP genes by rapamycin was observed in a strain deleted for the Mediator tail module subunit Med16. The data suggest that Mediator and Maf1 function in parallel pathways to negatively regulate RP mRNA and tRNA synthesis. Keywords: genetic modification, stress response We generated 12 microarray profiles from fluor-reversed replicates of wild-type and med20? strains with or without treatment with rapamycin and under 5 other conditions that repress ribosomal protein gene transcription. The effect of rapamycin on strains deleted for 3 other Mediator subunits was also assessed relative to wild-type. See Willis et al., (2008) in revision.

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Evidence that Mediator is essential for Pol II transcription but not a required component of the preinitiation complex in vivo

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