Saccharomyces cerevisiae
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA360382 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR5164660.fastq.gz | Fastqsanger.gz | |||
| SRR5164670.fastq.gz | Fastqsanger.gz | |||
| SRR5164661.fastq.gz | Fastqsanger.gz | |||
| SRR5164671.fastq.gz | Fastqsanger.gz | |||
| SRR5164662.fastq.gz | Fastqsanger.gz |
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Project description: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.
2017-07-13 | GSE93190 | GEO
Project description:Mediator is a transcriptional co-activator recruited to enhancers by DNA-binding activators, and it also interacts with RNA polymerase (Pol) II as part of the preinitiation complex (PIC). We demonstrate that a single Mediator complex associates with the enhancer and core promoter in vivo, indicating that it can physically bridge these transcriptional elements. However, the Mediator kinase module associates strongly with the enhancer, but not with the core promoter, and it dissociates from the enhancer upon depletion of the TFIIH kinase. Severing the kinase module from Mediator by removing the connecting subunit Med13 does not affect Mediator association at the core promoter, but increases occupancy at enhancers. Thus, Mediator undergoes a compositional change in which the kinase module, recruited via Mediator to the enhancer, dissociates from Mediator to permit association with Pol II and the PIC. As such, Mediator acts as a dynamic bridge between the enhancer and core promoter.
2016-10-31 | GSE82082 | GEO
Project description:The Cdk8 kinase module (CKM) is a dissociable part of the coactivator complex Mediator that regulates RNA polymerase II (Pol II transcription. The CKM has negative and positive functions in gene transcription that remain poorly understood at the mechanistic level. Here, we prepare recombinant CKM from the yeast Saccharomyces cerevisiae and show that it binds core Mediator (cMed) to sterically inhibit cMed binding to the Pol II preinitiation complex (PIC) in vitro. We further show that the Cdk8 kinase activity of CKM counteracts CKM-cMed interaction,thereby releasing CKM and enabling Mediator to bind the PIC. Finally, we report that the kinase activity of Cdk8 is required for gene activation during heat shock in vivo, but not under steady state growth conditions. These results converge with previous literature on a model for CKM function. In this model, CKM negatively regulates Mediator function at upstream activating sequences by preventing Mediator binding to the PIC at the promoter. During gene activation, Cdk8 kinase activity may release Mediator and allow its binding to the PIC, thereby stimulating transcription initiation and accounting for the positive function of CKM.
2021-06-16 | GSE161140 | GEO
Project description:The fission yeast Mcs6-Mcs2-Pmh1 complex (homologous to metazoan Cdk7-cyclin HMat1) performs dual functions: in cell cycle control, as a CDK-activating kinase; and in transcription by RNA polymerase (Pol) II, as part of TFIIH. It is unknown whether that linkage serves to coordinate gene expression with cell division. Mutants in mcs6 and pmh1 arrest with incomplete cell separation and decreased phosphorylation of the Pol II large subunit. Gene expression profiling by microarray hybridization revealed that a defined subset (~5%) of genes was repressed by Mcs6 complex impairment, whereas the majority was refractory. The repression signature included a cell-cycle cluster implicated in cytokinesis and cell separation, and overlapped with those of the cell-separation mutants sep1, sep10 and sep15 (relative to wild-type cells). The gene sep10 encodes the homolog of a protein found in metazoan Mediator-like complexes, and sep15 encodes an established component of the Mediator. In mcs6 or pmh1 mutants, sep10+, which also encodes a component of the transcriptional machinery, becomes essential for viability. Finally, mcs6+ also interacts genetically with sep1+, which encodes a forkhead transcription factor required for periodic transcription during mitosis and cell division. Thus, the Mcs6 complex (a direct activator of the cell-cycle engine) also helps govern the cell-cycle transcriptional program.
2004-11-01 | E-MEXP-191 | biostudies-arrayexpress
Project description:The fission yeast Mcs6-Mcs2-Pmh1 complex (homologous to metazoan Cdk7-cyclin HMat1) performs dual functions: in cell cycle control, as a CDK-activating kinase; and in transcription by RNA polymerase (Pol) II, as part of TFIIH. It is unknown whether that linkage serves to coordinate gene expression with cell division. Mutants in mcs6 and pmh1 arrest with incomplete cell separation and decreased phosphorylation of the Pol II large subunit. Gene expression profiling by microarray hybridization revealed that a defined subset (~5%) of genes was repressed by Mcs6 complex impairment, whereas the majority was refractory. The repression signature included a cell-cycle cluster implicated in cytokinesis and cell separation, and overlapped with those of the cell-separation mutants sep1, sep10 and sep15 (relative to wild-type cells). The gene sep10 encodes the homolog of a protein found in metazoan Mediator-like complexes, and sep15 encodes an established component of the Mediator. In mcs6 or pmh1 mutants, sep10+, which also encodes a component of the transcriptional machinery, becomes essential for viability. Finally, mcs6+ also interacts genetically with sep1+, which encodes a forkhead transcription factor required for periodic transcription during mitosis and cell division. Thus, the Mcs6 complex (a direct activator of the cell-cycle engine) also helps govern the cell-cycle transcriptional program.
2004-11-01 | E-MEXP-192 | biostudies-arrayexpress
Project description:The fission yeast Mcs6-Mcs2-Pmh1 complex (homologous to metazoan Cdk7-cyclin HMat1) performs dual functions: in cell cycle control, as a CDK-activating kinase; and in transcription by RNA polymerase (Pol) II, as part of TFIIH. It is unknown whether that linkage serves to coordinate gene expression with cell division. Mutants in mcs6 and pmh1 arrest with incomplete cell separation and decreased phosphorylation of the Pol II large subunit. Gene expression profiling by microarray hybridization revealed that a defined subset (~5%) of genes was repressed by Mcs6 complex impairment, whereas the majority was refractory. The repression signature included a cell-cycle cluster implicated in cytokinesis and cell separation, and overlapped with those of the cell-separation mutants sep1, sep10 and sep15 (relative to wild-type cells). The gene sep10 encodes the homolog of a protein found in metazoan Mediator-like complexes, and sep15 encodes an established component of the Mediator. In mcs6 or pmh1 mutants, sep10+, which also encodes a component of the transcriptional machinery, becomes essential for viability. Finally, mcs6+ also interacts genetically with sep1+, which encodes a forkhead transcription factor required for periodic transcription during mitosis and cell division. Thus, the Mcs6 complex (a direct activator of the cell-cycle engine) also helps govern the cell-cycle transcriptional program.
2004-11-01 | E-MEXP-193 | biostudies-arrayexpress
Project description:The conserved core domain of the TATA binding protein (TBP) interacts with multiple partners forming the complexes required for transcription by RNA Polymerases I, II and III. We use genetically modified mouse embryonic fibroblasts to show that many TBP core domain mutants complement loss of endogenous TBP, but this often results in a slow growth phenotype. Two TBP mutations, R188E and K243E, disrupt the TBP-BTAF1 interaction and B-TFIID complex formation. Transcriptome and ChIP-seq analyses show that loss of B-TFIID does not affect global genomic distribution of TBP, but positively or negatively affects TBP and/or RNA Polymerase II (Pol II) recruitment to a selected set of promoters. We identify a set of promoters where wild-type TBP assembles a partial inactive preinitiation complex lacking Pol II and TAF1. Our results suggest that an exchange of the B-TFIID complex in wild-type cells for TFIID in R188E and K243E mutant cells at these primed promoters recruits Pol II to activate their expression. We also observe that both Wt and mutant TBP can occupy promoters without concurrent Pol II recruitment and active transcription. Our data reveal a novel regulatory mechanism involving the formation of a partial preinitiation complex that primes the promoter for productive preinitiation complex formation in mammalian cells.
2012-03-01 | GSE27908 | GEO
Project description:Transcription by RNA polymerase (Pol) II requires assembly of a preinitiation complex (PIC) composed of general transcription factors (GTFs) bound at the core promoter. In vitro, the TATA-binding protein (TBP), TFIIB, TFIIF, and Pol II are essential for PIC formation and transcriptional initiation, whereas TFIIA, TFIIE, and TFIIH are not required under certain conditions. In addition, the PIC is stable in the absence of nucleotide triphosphates, and sequential addition of GTFs results in a series of partial PICs. Here, we analyze the roles of all GTFs in yeast cells by measuring PIC formation and Pol II transcription upon depletion of individual GTFs. All GTFs are essential for TBP binding and Pol II transcription in vivo, suggesting that partial PICs composed of GTF subsets do not exist at appreciable levels. In contrast, TBP-associated factors (TAFs) contribute to Pol II transcriptional activity at most (and perhaps all) genes, but TAF-independent transcription occurs at a substantial level, particularly at promoters lacking canonical TATA elements. Lastly, unlike the case in vitro, PICs are not observed in cells deprived of uracil, and presumably UTP, suggesting a mechanism that removes transcriptionally inactive PICs from promoters.
2019-02-27 | GSE122734 | GEO
Project description:Nuclear pore complexes (NPCs) influence gene expression besides their established function in nuclear transport. The TREX-2 complex localizes to the NPC basket and affects gene-NPC interactions, transcription and mRNA export. How TREX-2 regulates the gene expression machinery is unknown. Here, we show that TREX-2 interacts with the Mediator complex, an essential regulator of RNA Polymerase (Pol) II. Structural and biochemical studies identify a conserved region on TREX-2, which directly binds the Mediator Med31/Med7N submodule. TREX-2 regulates assembly of Mediator with its Cdk8 kinase and is required for recruitment and site-specific phosphorylation of Pol II. Transcriptome and phenotypic profiling confirm that TREX-2 and Med31 are functionally interdependent. TREX-2 additionally uses its Mediator-interacting surface to regulate mRNA export suggesting a mechanism for coupling transcription initiation and early steps of mRNA processing at the Mediator level. In sum, we provide insight into how NPC-associated adaptor complexes can access the core transcription machinery. RNAseq was performed from WT, sac3∆, cdk8∆ and Sac3 R288D mutant cells. For each strain triplicates were analyzed. WT strain was sac3∆ transformed with pRS315 SAC3 WT
2015-08-27 | E-GEOD-67431 | biostudies-arrayexpress
Project description:In Saccharomyces cerevisiae short non-coding RNA (ncRNA) generated by RNA Polymerase II (Pol II) are terminated by the NRD complex consisting of Nrd1, Nab3 and Sen1. We now show that Pcf11, a component of the cleavage and polyadenylation complex (CPAC), is generally required for NRD-dependent transcription termination through the action of its CTD interacting domain (CID). Pcf11 localizes downstream of Nrd1 on NRD terminators, and its recruitment depends on Nrd1. Furthermore mutation of the Pcf11 CID results in Nrd1 retention on chromatin, delayed degradation of ncRNA and restricts Pol II CTD Ser2 phosphorylation and Sen1-Pol II interaction. Finally, the pcf11-13 and sen1-1 mutant phenotypes are very similar as both accumulate RNA:DNA hybrids and display Pol II pausing downstream of NRD terminators. We predict a mechanism whereby Nrd1 and Pcf11 exchange on chromatin facilitates Pol II pausing and CTD Ser2-P phosphorylation. This in turn promotes Sen1 activity that is required for NRD-dependent transcription termination in vivo. ChIP-seq with antibody against pol II in wild type and Pcf11 mutants: Pcf11-2, Pcf11-9 and Pcf11-13 grown at 25C and 37C along with input samples
2015-04-02 | E-GEOD-67483 | biostudies-arrayexpress