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Polymerase pausing induced by sequence-specific RNA binding protein drives heterochromatin assembly (ChIP-Seq)

ABSTRACT: In S. pombe, transcripts derived from the pericentromeric dg and dh repeats promote heterochromatin formation via RNAi as well as an RNAi-independent mechanism involving the RNAPII-associated RNA-binding protein Seb1 and RNA processing activities. We show that Seb1 promotes long-lived RNAPII pauses at pericentromeric repeat regions and their presence correlates with the heterochromatin-triggering activities of the corresponding dg and dh DNA fragments. Globally increasing RNAPII stalling by other means induces the formation of novel large ectopic heterochromatin domains. Such ectopic heterochromatin occurs even in cells lacking RNAi. These results uncover Seb1-mediated polymerase stalling as a signal necessary for heterochromatin nucleation.

ORGANISM(S): Schizosaccharomyces pombe

PROVIDER: GSE114535 | GEO | 2018/07/03

REPOSITORIES: GEO

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Polymerase pausing induced by sequence-specific RNA binding protein drives heterochromatin assembly (RNA-Seq)

Project description:In S. pombe, transcripts derived from the pericentromeric dg and dh repeats promote heterochromatin formation via RNAi as well as an RNAi-independent mechanism involving the RNAPII-associated RNA-binding protein Seb1 and RNA processing activities. We show that Seb1 promotes long-lived RNAPII pauses at pericentromeric repeat regions and their presence correlates with the heterochromatin-triggering activities of the corresponding dg and dh DNA fragments. Globally increasing RNAPII stalling by other means induces the formation of novel large ectopic heterochromatin domains. Such ectopic heterochromatin occurs even in cells lacking RNAi. These results uncover Seb1-mediated polymerase stalling as a signal necessary for heterochromatin nucleation.

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Polymerase pausing induced by sequence-specific RNA binding protein drives heterochromatin assembly (PAR-CLIP)

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Polymerase pausing induced by sequence-specific RNA binding protein drives heterochromatin assembly (NET-Seq)

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Direct sequencing of polyadenylation sites in fission yeast S. pombe

Project description:We sequenced the 3' ends of polyadenylated transcripts among total RNA in fission yeast wildtype and rdp1∆ cells and mapped the 3'-most nucleotide prior to polyadenylation. We find that mRNA-coding open reading frames have a low number of polyadenylation sites with most of the reads, whereas pericentromeric dg and dh repeats have a high number of polyadenylation sites throughout the transcribed region, mapping to both strands. direct sequencing of polyadenylated 3' ends of RNA (Helicos)

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Direct sequencing of polyadenylation sites in fission yeast S. pombe

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The Nrd1-like protein Seb1 coordinates co-transcriptional 3 end processing and polyadenylation site selection

Project description:Three studies comprising of a strand specific RNA-seq analysis of the fission yeast transcriptome upon depletion of the termiantion factor Seb1 (2 repeats). RNAPII ChIP-seq in fission yeast upon depletion of the termiantion factor Seb1 (1 IP and 1 input). Seb1 ChIP-seq in fission yeast (HTP, 2 IP and 2 input).

2016-07-31 | E-MTAB-4827 | 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

RNAi promotes heterochromatic silencing through replication-coupled release of RNA pol II (RNA seq)

Project description:Heterochromatin comprises tightly compacted repetitive regions of eukaryotic chromosomes. The inheritance of heterochromatin through mitosis requires RNA interference (RNAi), which guides histone modification during the DNA replication phase of the cell cycle. Here, we show that the alternating arrangement of origins of replication and non-coding RNA in pericentromeric heterochromatin results in competition between transcription and replication. Co-transcriptional RNAi releases RNA polymerase II (PolII), allowing completion of DNA replication by the leading strand DNA polymerase, and associated histone modifying enzymes which spread heterochromatin with the replication fork. In the absence of RNAi, stalled forks are repaired by homologous recombination without histone modification. small RNA cloning from wild type and dcr1-deleted strains

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Mkt1 is required for RNAi-mediated silencing and establishment of heterochromatin in fission yeast

Project description:Constitutive domains of repressive heterochromatin are maintained within the fission yeast genome through self-reinforcing mechanisms involving histone methylation and small RNAs. Non-coding RNAs generated from heterochromatic regions are processed into small RNAs by the RNA interference pathway, and are subject to silencing through both transcriptional and post-transcriptional mechanisms. While the pathways involved in maintenance of the repressive heterochromatin state are reasonably well understood, less is known about the requirements for its establishment. Here we describe a novel role for the post-transcriptional regulatory factor Mkt1 in establishment of heterochromatin at pericentromeres in fission yeast. Loss of Mkt1 does not affect maintenance of existing heterochromatin, but does affect its recovery following depletion, as well as de novo establishment of heterochromatin on a mini-chromosome. Pathway dissection revealed that Mkt1 is required for RNAi-mediated post-transcriptional silencing, downstream of small RNA production. Mkt1 physically associates with pericentromeric transcripts, and is additionally required for maintenance of silencing and heterochromatin at centromeres when transcriptional silencing is impaired. Our findings provide new insight into the mechanism of RNAi-mediated post-transcriptional silencing in fission yeast, and unveil an important role for post-transcriptional silencing in establishment of heterochromatin that is dispensable when full transcriptional silencing is imposed.

2019-11-28 | PXD015484 | Pride

RNAi promotes heterochromatic silencing through replication-coupled release of RNA pol II (ChIP-seq)

Project description:Heterochromatin comprises tightly compacted repetitive regions of eukaryotic chromosomes. The inheritance of heterochromatin through mitosis requires RNA interference (RNAi), which guides histone modification 1 during the DNA replication phase of the cell cycle2. Here, we show that the alternating arrangement of origins of replication and non-coding RNA in pericentromeric heterochromatin results in competition between transcription and replication. Co-transcriptional RNAi releases RNA polymerase II (PolII), allowing completion of DNA replication by the leading strand DNA polymerase, and associated histone modifying enzymes3 which spread heterochromatin with the replication fork. In the absence of RNAi, stalled forks are repaired by homologous recombination without histone modification. RNA PolII ChIP from wild type and dcr1-deleted samples in exponentially growing and HU-arrested cultures.

2011-07-29 | E-GEOD-31016 | biostudies-arrayexpress

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