Comparison of RNA profiling between wild type, med20â, rrp6â and med20â/rrp6â in Schizosaccharomyces pombe
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ABSTRACT:
ORGANISM(S): Schizosaccharomyces pombe
SUBMITTER: Jonas Carlsten
PROVIDER: E-GEOD-73792 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Mediator is a co-regulator of RNA polymerase II (Pol II), transducing signals from regulatory elements and transcription factors to the general transcription machinery at the promoter. We here demonstrate that Med20 influences ribosomal protein expression in fission yeast. In addition, loss of Med20 leads to an accumulation of aberrant readthrough tRNA transcripts. The aberrant transcripts are polyadenylated and targeted for degradation by the exosome. Similarly, other specialized RNA molecules, such as snRNA, snoRNA and rRNA are also accumulated in the absence of Med20. We suggest that fission yeast Mediator takes part in a regulatory pathway of Pol III transcripts.
2016-01-01 | GSE73792 | GEO
Project description:At Schizosaccharomyces pombe centromeres, non-coding RNAs are transcribed by RNA polymerase II and processed by the RNAi machinery to direct heterochromatin formation. Mediator is a regulator of RNA polymerase II transcription and we here demonstrated that it also controls the levels of centromeric transcripts. Loss of the Mediator subunit Med20 increases transcription of non-coding RNAs and disrupts the centromeric heterochromatin structure. We have used tilling arrays to study the gene expression changes in Med20 deletionVs WT and Med20 dcr1 double mutant Vs Dcr1 mutant in exponentially growing fission yeast cells to detect how the Med20 gene affect centromeric expression.
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Project description:At Schizosaccharomyces pombe centromeres, non-coding RNAs are transcribed by RNA polymerase II and processed by the RNAi machinery to direct heterochromatin formation. Mediator is a regulator of RNA polymerase II transcription and we here demonstrated that it also controls the levels of centromeric transcripts. Loss of the Mediator subunit Med20 increases transcription of non-coding RNAs and disrupts the centromeric heterochromatin structure. We have used tilling arrays to study the gene expression changes in Med20 deletionVs WT and Med20 dcr1 double mutant Vs Dcr1 mutant in exponentially growing fission yeast cells to detect how the Med20 gene affect centromeric expression. RNA samples were taken from exponentially growing fission yeast cells and reverse transcribed cDNA from the RNA are hybridized to affymetrix pombe tilling arrays: wild type,med20 mutant, Dcr1 mutants and med20,dcr1 double mutant.
2012-11-06 | E-GEOD-32310 | biostudies-arrayexpress
Project description:CENP-A chromatin the foundation for kinetochore assembly in all eukaryotes. CENP-A is incorporated outside of S phase and depends on its chaperone HJURPScm3, and Mis18 in vertebrates and fission yeast. The recruitment of Mis18 and HJURPScm3 to centromeres is cell cycle regulated. Vertebrate Mis18 associates with Mis18BP1KNL2 and RbAp46/48Mis16/Hat2. Mis18BP1KNL2 is critical for the recruitment of Mis18 and HJURPScm3 to vertebrate centromeres. However, no ortholog of Mis18BP1KNL2 has been identified in fission yeast, consequently it remains unknown how the key Cnp1CENP-A loading factor Mis18 is recruited. We identify two novel Mis18-interacting proteins (Eic1 and Eic2) in fission yeast. Our analyses show that Eic1 and Eic2 are components of a Mis18 complex. Eic1 is essential to maintain normal Cnp1CENP-A levels at centromeres and therefore is crucial for kinetochore integrity whereas Eic2 is dispensable. Eic1 also associates with components of the large constitutive CCAN/Sim4/Ctf19 complex. Our findings suggest that Eic1 is the functional counterpart of Mis18BP1KNL2 and acts as a link with constitutive kinetochore components to allow the temporally regulated recruitment of the Mis18 Cnp1CENP-A loader. 100bp paired end ChIP-Seq analysis of GFP tagged Eic1, Eic2, Scm3, and Mis18 with sonicated whole cell extract input DNA in asynchornous Schizosaccharomyces pombe cells.
2014-05-19 | E-GEOD-54685 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE32310: Transcriptome analysis of mediator Med20 mutant and dcr1 mutant in S.pombe GSE35524: Mediator promotes CENP-A incorporation at fission yeast centromeres [ChIP-seq] Refer to individual Series
2012-11-06 | E-GEOD-35527 | biostudies-arrayexpress
Project description:At Schizosaccharomyces pombe centromeres, heterochromatin formation is required for de novo incorporation of the histone H3 variant CENP-A/Cnp1, which in turn directs kinetochore assembly and ultimately chromosome segregation during mitosis. Noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (Pol II) directs heterochromatin formation via the RNAi machinery, but also through RNAiindependent RNA processing factors. Control of centromeric ncRNA transcription is therefore a key factor for proper centromere function. We here use transcriptional profiling, gene inactivation experiments, and chromatin immunoprecipitation analyses to demonstrate that the Mediator complex directs ncRNA transcription and regulates centromeric heterochromatin formation in fission yeast. Mediator co-localizes with Pol II at centromeres and loss of the Mediator subunit Med20 causes a dramatic increase in pericentromeric transcription and desilencing of the core centromere. As a consequence, heterochromatin formation is impaired both via the RNAi dependent and independent pathways, resulting in loss of CENP-A/Cnp1 from the core centromere, defect kinetochore function, and a severe chromosome segregation defect. Interestingly, the increased centromeric transcription observed in med20Δ appears to directly block CENP-A/Cnp1 incorporation and inhibition of Pol II transcription can suppress the observed phenotypes. Our data thus identify Mediator as a crucial regulator of ncRNA transcription at fission yeast centromeres and add another crucial layer of regulation to centromere function.
2012-11-06 | GSE35524 | GEO
Project description:At Schizosaccharomyces pombe centromeres, heterochromatin formation is required for de novo incorporation of the histone H3 variant CENP-A/Cnp1, which in turn directs kinetochore assembly and ultimately chromosome segregation during mitosis. Noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (Pol II) directs heterochromatin formation via the RNAi machinery, but also through RNAiindependent RNA processing factors. Control of centromeric ncRNA transcription is therefore a key factor for proper centromere function. We here use transcriptional profiling, gene inactivation experiments, and chromatin immunoprecipitation analyses to demonstrate that the Mediator complex directs ncRNA transcription and regulates centromeric heterochromatin formation in fission yeast. Mediator co-localizes with Pol II at centromeres and loss of the Mediator subunit Med20 causes a dramatic increase in pericentromeric transcription and desilencing of the core centromere. As a consequence, heterochromatin formation is impaired both via the RNAi dependent and independent pathways, resulting in loss of CENP-A/Cnp1 from the core centromere, defect kinetochore function, and a severe chromosome segregation defect. Interestingly, the increased centromeric transcription observed in med20Δ appears to directly block CENP-A/Cnp1 incorporation and inhibition of Pol II transcription can suppress the observed phenotypes. Our data thus identify Mediator as a crucial regulator of ncRNA transcription at fission yeast centromeres and add another crucial layer of regulation to centromere function. 3 samples examined: wild type chromatin incubated with beads as the non antibody control, wild type chromatin incubated with RNA Polymerase II CTD domain antibody and Protein G beads, and TAP-Med7 cells chromatin incubated with IgG beads.
2012-11-06 | E-GEOD-35524 | biostudies-arrayexpress
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.
2008-06-01 | E-GEOD-11397 | biostudies-arrayexpress
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
2008-06-01 | GSE11397 | GEO
Project description:Background: Heterochromatin at the pericentromeric repeats in fission yeast is assembled and spread by an RNAi-dependent mechanism, which is coupled to the transcription of non-coding RNA from the repeats by RNA polymerase II. In addition, Rrp6, a component of the nuclear exosome, also contributes to heterochromatin assembly and is coupled to non-coding RNA transcription. The multi-subunit complex Mediator, which directs initiation of RNA polymerase II-dependent transcription, has recently been suggested to function after initiation in processes such as elongation of transcription and splicing. However, the role of Mediator in the regulation of chromatin structure is not well understood. Results: We investigated the role of Mediator in pericentromeric heterochromatin formation and found that deletion of specific subunits of the head domain of Mediator compromised heterochromatin structure. The Mediator head domain was required for Rrp6-dependent heterochromatin nucleation at the pericentromere and for RNAi-dependent spreading of heterochromatin into the neighboring region. In the latter process, Mediator appeared to contribute to efficient processing of siRNA from transcribed non-coding RNA, which was required for efficient spreading of heterochromatin. Furthermore, the head domain directed efficient transcription in heterochromatin. Conclusions: These results reveal a pivotal role for Mediator in multiple steps of transcription-coupled formation of pericentromeric heterochromatin. This observation further extends the role of Mediator to co-transcriptional chromatin regulation. Gene expression profile at exponentially-growing phase.in the fission yeast deletion mutants of pmc6, med20 and dcr1.
2013-08-31 | E-GEOD-43543 | biostudies-arrayexpress