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Transcriptome analysis of mediator Med20 mutant and dcr1 mutant in S.pombe

ABSTRACT: 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.

ORGANISM(S): Schizosaccharomyces pombe

PROVIDER: GSE32310 | GEO | 2012/11/06

SECONDARY ACCESSION(S): PRJNA155507

REPOSITORIES: GEO

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Transcriptome analysis of mediator Med20 mutant and dcr1 mutant in S.pombe

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

Mediator promotes CENP-A incorporation at fission yeast centromeres [ChIP-seq]

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

Mediator directs co-transcriptional heterochromatin assembly by RNAi-dependent and -independent pathways.

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

Mediator promotes CENP-A incorporation at fission yeast centromeres [ChIP-seq]

2012-11-06 | GSE35524 | GEO

Effects of RNAi protein overexpression in mutant cells on H3K9 dimethylation in fission yeast cells

Project description:We test the effects of overexpressing RNAi proteins Dcr1, Rdp1, or Ago1 in wildtype, dcr1∆, ago1∆, or rdp1∆ cells. We find that overexpression does not generally affect H3K9me2 at euchromatic loci, but there are varying effects in H3K9me2 on centromeric heterochromatin.

2013-12-26 | GSE52444 | GEO

Mediator promotes CENP-A incorporation at fission yeast centromeres

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

Comparison of RNA profiling between wild type, med20∆, rrp6∆ and med20∆/rrp6∆ in Schizosaccharomyces pombe

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

DamID for Swi6, Rdp1 and Dcr1 in Schizosaccharomyces pombe

Project description:In the fission yeast Schizosaccharomyces pombe, the RNA interference (RNAi) pathway is required to generate small interfering RNAs (siRNAs) that mediate heterochromatic silencing of centromeric repeats. Here we demonstrate that RNAi also functions to repress genomic elements other than constitutive heterochromatin. Using DamID (DNA adenine methyltransferase identification) we show that Dcr1 and Rdp1 physically associate with some euchromatic genes, non-coding RNA (ncRNA) genes, and retrotransposon long terminal repeats (LTRs), and that this association is independent of the Clr4 histone methyltransferase. Physical association of RNAi with chromatin is sufficient to trigger a silencing response but not to assemble heterochromatin. The mode of silencing at the newly identified RNAi targets is consistent with a co-transcriptional gene silencing model as proposed earlier and functions with trace amounts of siRNAs. We anticipate that similar mechanisms could also be operational in other eukaryotes.

2010-09-25 | GSE24360 | GEO

Comparison of RNA profiling between wild type, med20â, rrp6â and med20â/rrp6â in Schizosaccharomyces pombe

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. Â 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. RNA-sequencing of wt, med20â, rrp6â, med20â/rrp6â strains using wt as control. Each strain was sequenced using three biological replicas. The results were analyzed for differential expression.

2016-07-03 | E-GEOD-73792 | biostudies-arrayexpress

Effects of RNAi protein overexpression in mutant cells on H3K9 dimethylation in fission yeast cells

Project description:We test the effects of overexpressing RNAi proteins Dcr1, Rdp1, or Ago1 in wildtype, dcr1M-bM-^HM-^F, ago1M-bM-^HM-^F, or rdp1M-bM-^HM-^F cells. We find that overexpression does not generally affect H3K9me2 at euchromatic loci, but there are varying effects in H3K9me2 on centromeric heterochromatin. Examination of genome-wide enrichment in H3K9 dimethylation in fission yeast haploid cells

2013-12-26 | E-GEOD-52444 | biostudies-arrayexpress

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