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Functional studies of the Yeast Mediator Tail Module Subunits

ABSTRACT: The yeast Mediator complex can be divided into three modules, designated Head, Middle and Tail. Tail comprises the Med2, Med3, Med5, Med15 and Med16 protein subunits, which are all encoded by genes that are individually non-essential for viability. In cells lacking Med16, Tail is displaced from Head and Middle. However, inactivation of MED5/MED15 and MED15/MED16 are synthetically lethal, indicating that Tail performs essential functions as a separate complex even when it is not bound to Middle and Head. We have used the N-Degron method to create temperature sensitive (ts) mutants in the Mediator tail subunits Med5, Med15 and Med16 to study the immediate effects on global gene expression when each subunit is individually inactivated, and when MED5/15 or MED15/16 are inactivated together. All Degron constructs were expressed from their normal chromosomal location under the control of their respective endogenous promoters. We isolated RNA from each strain as early as 45 minutes after changing from the permissive to the restrictive growth conditions to minimize possible secondary effects on gene expression that are not directly caused by the Degron construct(s).

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Luke Selth

PROVIDER: E-GEOD-47712 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Functional studies of the Yeast Mediator Tail Module Subunits

Project description:The yeast Mediator complex can be divided into three modules, designated Head, Middle and Tail. Tail comprises the Med2, Med3, Med5, Med15 and Med16 protein subunits, which are all encoded by genes that are individually non-essential for viability. In cells lacking Med16, Tail is displaced from Head and Middle. However, inactivation of MED5/MED15 and MED15/MED16 are synthetically lethal, indicating that Tail performs essential functions as a separate complex even when it is not bound to Middle and Head. We have used the N-Degron method to create temperature sensitive (ts) mutants in the Mediator tail subunits Med5, Med15 and Med16 to study the immediate effects on global gene expression when each subunit is individually inactivated, and when MED5/15 or MED15/16 are inactivated together.

2013-06-07 | GSE47712 | GEO

Connection of core and and tail Mediator modules restrains transcription from TFIID-dependent promoters [nsRNA-seq]

Project description:The Mediator coactivator complex is divided into four modules: head, middle, tail, and kinase. Deletion of the architectural subunit Med16 separates core Mediator (cMed), comprising the head, middle, and scaffold (Med14), from the tail. However, the direct global effects of tail/cMed disconnection are unclear. We find that rapid depletion of Med16 downregulates genes that require the SAGA complex for full expression, consistent with their reported tail dependence, but also moderately overactivates TFIID-dependent genes in a manner partly dependent on the separated tail, which remains associated with upstream activating sequences. Suppression of TBP dynamics via removal of the Mot1 ATPase partially restores normal transcriptional activity to Med16-depleted cells, suggesting that cMed/tail separation results in an imbalance in the levels PIC formation at SAGA-requiring and TFIID-dependent genes. We suggest that the preferential regulation of SAGA-requiring genes by tailed Mediator helps maintain a proper balance of transcription between these genes and those more dependent on TFIID.

2021-07-30 | GSE169747 | GEO

Connection of core and and tail Mediator modules restrains transcription from TFIID-dependent promoters [ChEC-seq]

2021-07-30 | GSE169746 | GEO

Connection of core and tail Mediator modules restrains transcription from TFIID-dependent promoters [ChIP-Seq]

Project description:The Mediator coactivator complex is divided into four modules: head, middle, tail, and kinase. Deletion of the architectural subunit Med16 separates core Mediator (cMed), comprising the head, middle, and scaffold (Med14), from the tail. However, the direct global effects of tail/cMed disconnection are unclear. We find that rapid depletion of Med16 downregulates genes that require the SAGA complex for full expression, consistent with their reported tail dependence, but also moderately overactivates TFIID-dependent genes in a manner partly dependent on the separated tail, which remains associated with upstream activating sequences. Suppression of TBP dynamics via removal of the Mot1 ATPase partially restores normal transcriptional activity to Med16-depleted cells, suggesting that cMed/tail separation results in an imbalance in the levels of PIC formation at SAGA-requiring and TFIID-dependent genes. We suggest that the preferential regulation of SAGA-requiring genes by tailed Mediator helps maintain a proper balance of transcription between these genes and those more dependent on TFIID.

2021-07-30 | GSE171981 | GEO

Connection of core and and tail Mediator modules restrains transcription from TFIID-dependent promoters [ChEC-seq Med16-AID]

2021-07-30 | GSE179765 | GEO

Tail and Kinase Modules Differently Regulate Core Mediator Recruitment and Function In Vivo

Project description:Mediator is a highly conserved transcriptional coactivator organized into four modules, namely Tail, Middle, Head and Kinase (CKM). Previous work suggests regulatory roles for Tail and CKM, but an integrated model for these activities is lacking. Here, we analyzed the genome-wide distribution of Mediator subunits in wild-type and mutant yeast cells in which RNA polymerase II promoter escape is blocked allowing detection of transient Mediator forms. We found that while all modules are recruited to upstream activated regions (UAS), assembly of Mediator within the pre-initiation complex is accompanied by the release of CKM. Interestingly, our data show that CKM regulates Mediator-UAS interaction rather than Mediator-promoter association. In addition, while Tail is required for Mediator recruitment to UAS, Tail-less Mediator nevertheless interacts with core promoters. Collectively, our data suggest that the essential function of Mediator is mediated by Head and Middle at core promoters, while Tail and CKM play regulatory roles.

2016-10-25 | GSE81107 | GEO

A Dynamic Model for the Function of Mediator Modules In Vivo

Project description:Mediator is a highly conserved transcriptional coactivator organized into four modules, namely Tail, Middle, Head and Kinase (CKM). Previous work suggests regulatory roles for Tail and CKM, but an integrated model for these activities is lacking. Here, we analyzed the genome-wide distribution of Mediator subunits in wild-type and mutant yeast cells in which RNA polymerase II promoter escape is blocked allowing detection of transient Mediator forms. We found that while all modules are recruited to upstream activated regions (UAS), assembly of Mediator within the pre-initiation complex is accompanied by the release of CKM. Surprisingly, our data show that CKM regulates Mediator-UAS interaction rather than Mediator-promoter association. In addition, while Tail is required for Mediator recruitment to UAS, Tail-less Mediator nevertheless interacts with core promoters. Collectively, our data suggest that the essential function of Mediator is mediated by Head and Middle at core promoters, while Tail and CKM play regulatory roles.

2016-10-20 | GSE81127 | GEO

The polymorphic PolyQ tail protein of the Mediator Complex, Med15, regulates variable response to stress

Project description:The Mediator is composed of multiple of proteins in the head, body, tail and CDK subunits conserved from yeast to humans. However, not all the components are required for transcription. Components of the tail subunit are not essential but to varying degrees are required for changes in transcription to stress. While some stresses are familiar such as heat, desiccation, and starvation, others are exotic yet elicit a physical stress response. MCHM is a hydrotrope that induces growth arrest in yeast. By exploiting genetic variation, specifically in Med15, between yeast strains, we found that Med15 with the polyQ expansion conferred MCHM sensitivity. This sensitivity was not from a loss of function as the reciprocal hemizygous hybrids were all sensitive, suggesting that there is an incompatibility between Mediator complexes from genetic divergent yeast strains. Transcriptomics from yeast expressing the incompatible Med15 changed expression in diverse pathways. Expansion of polyQ tracts in Med15 resulted in multiple isoforms which were mostly from likely post-translational modifications. Stability of Med15 was dependent on Ydj1, a chaperone and the incompatible Med15 allele was expressed at lower levels and less stable than the compatible Med15 allele. Med15 is tethered to the rest of the Mediator complex via Med2 and 3. Deletion of either Med2 or Med3 changes the Med15 isoform patterns in the in a similar manner whereas deletion of Med5, a distal component of the tail did not change the pattern. The med2 and 3 mutants are similarly sensitive to MCHM while med5 mutants are not. Differences between the Med15 alleles extend to responses to different stresses. Whereas the incompatible allele of Med15 improved growth to chemicals that produce free radicals, and the compatible allele of Med15 improved growth to reducing agents, caffeine, and hydroxyurea. This may reflect the positive and negative role that Med15 has in transcription.

2020-04-06 | GSE129898 | GEO

Functional metabolomics as a tool to analyze Mediator function and structure in plants

Project description:Mediator is a multiprotein transcriptional co-regulator complex composed of four modules; Head, Middle, Tail, and Kinase. It conveys signals from promoter-bound transcriptional regulators to RNA polymerase II and thus plays an essential role in eukaryotic gene regulation. We describe subunit localization and activities of Mediator in Arabidopsis through metabolome and transcriptome analyses from a set of Mediator mutants. Functional metabolomic analysis based on the metabolite profiles of Mediator mutants using multivariate statistical analysis and heat-map visualization shows that different subunit mutants display distinct metabolite profiles, which cluster according to the reported localization of the corresponding subunits in yeast. Based on these results, we suggest localization of previously unassigned plant Mediator subunits to specific modules. We also describe novel roles for individual subunits in development, and demonstrate changes in gene expression patterns and specific metabolite levels in med18 and med25, which can explain their phenotypes. We find that med18 displays levels of phytoalexins normally found in wild type plants only after exposure to pathogens. Our results indicate that different Mediator subunits are involved in specific signaling pathways that control developmental processes and tolerance to pathogen infections.

2018-01-30 | MTBLS450 | MetaboLights

ChIP-seq of Mediator subunits in Mediator mutants and wild type yeast

Project description:Mediator subunits Med17 (Srb4) and Med15 (Gal11) were subjected to ChIP in wild type yeast, med18 (srb5), med20 (srb2), and med2 med3 med15 (triple mutant) yeast, all in kin28-anchor away yeast after 1 hr rapamycin treatment to evict Kin28 from the nucleus. This results in inactivation of Kin28, which reduces Mediator turnover at promoters and allows stronger Mediator ChIP signal at promoters. <br></br>Both Med17 and Med15 were tagged with myc and precipitated using the monoclonal antibody against c-myc. Please note that a specific control for rapamycin treatment was not performed in this experiment, as that has been well documented in the literature for this experiment, using what is called the anchor away method. The untagged sample is a control for non-specific immunoprecipitation.

2017-12-01 | E-MTAB-5824 | biostudies-arrayexpress

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