Mediator regulates non-coding RNA transcription at fission yeast centromeres

Abstract

BackgroundIn fission yeast, centromeric heterochromatin is necessary for the fidelity of chromosome segregation. Propagation of heterochromatin in dividing cells requires RNA interference (RNAi) and transcription of centromeric repeats by RNA polymerase II during the S phase of the cell cycle.ResultsWe found that the Med8-Med18-Med20 submodule of the Mediator complex is required for the transcriptional regulation of native centromeric dh and dg repeats and for the silencing of reporter genes inserted in centromeric heterochromatin. Mutations in the Med8-Med18-Med20 submodule did not alter Mediator occupancy at centromeres; however, they led to an increased recruitment of RNA polymerase II to centromeres and reduced levels of centromeric H3K9 methylation accounting for the centromeric desilencing. Further, we observed that Med18 and Med20 were required for efficient processing of dh transcripts into siRNA. Consistent with defects in centromeric heterochromatin, cells lacking Med18 or Med20 displayed elevated rates of mitotic chromosome loss.ConclusionsOur data demonstrate a role for the Med8-Med18-Med20 Mediator submodule in the regulation of non-coding RNA transcription at Schizosaccharomyces pombe centromeres. In wild-type cells this submodule limits RNA polymerase II access to the heterochromatic DNA of the centromeres. Additionally, the submodule may act as an assembly platform for the RNAi machinery or regulate the activity of the RNAi pathway. Consequently, Med8-Med18-Med20 is required for silencing of centromeres and proper mitotic chromosome segregation.

Mediator Directs Co-transcriptional Heterochromatin Assembly by RNA Interference-Dependent and -Independent Pathways

“…Recently, two reports showed that MHD was important for heterochromatin formation at pericentromeres [34], [35]. However, there are several discrepancies between their data and ours.…”

“…Indeed, recent reports suggest a negative role of MHD subunits (Med18 and Med20) in heterochromatic transcription, based on the observation of an increase in the transcription of pericentromeric ncRNA in Mediator mutants [34], [35]. However, it is difficult to state conclusively whether the observed increase is due to the direct effects of the absence of Mediator because it is also possible that the deletion of Mediator subunits causes disruption of the heterochromatin, which secondarily induces an increase in transcription.…”

“…These results indicate that MHD is somehow involved in siRNA production after transcription of ncRNA. MHD is localized at the transcribed region in pericentromeric repeats (Figure S4; [34], [35] and is required for transcription in heterochromatin, suggesting that it directly functions in the coupling of transcription of heterochromatic ncRNA by RNAPII and processing of the siRNA by RNAi machinery. Retention of RNAi factors at the pericentromeric repeats in med18Δ cells and the requirement for MHD in heterochromatin formation via the artificial tethering of the RITS complex to RNA suggest that MHD functions after RITS associates with heterochromatic repeats and/or target RNA.…”

A systematic genetic screen identifies new factors influencing centromeric heterochromatin integrity in fission yeast

“…Among these mutants were deletions of two proteins that, although not annotated in PomBase as affecting chromatin silencing at centromeres, have previously been reported to have a modest impact on centromeric heterochromatin integrity: the mediator subunit Med20 [40], and CRL adaptor protein Ddb1 [27]. Aside from these two mutants whose centromeric silencing defects have already been characterised, we identified five mutants with no previously described effects on heterochromatin silencing.…”

The Mediator complex: a central integrator of transcription

“…In the yeast S. pombe , Mediator has been linked to pericentromeric heterochromatin formation 143 . These studies suggest that Mediator functions by coordinating pol II-dependent ncRNA transcription with the RNAi machinery to help form and maintain heterochromatin domains 143-145 . Similarly, Mediator has been implicated in telomeric heterochromatin maintenance in the budding yeast S. cerevisiae .…”