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. 2012 Nov 21;5(1):19.
doi: 10.1186/1756-8935-5-19.

Mediator regulates non-coding RNA transcription at fission yeast centromeres

Michael Thorsen  1 , Heidi Hansen, Michela Venturi, Steen Holmberg, Genevieve Thon
Affiliations

Mediator regulates non-coding RNA transcription at fission yeast centromeres

Michael Thorsen et al. Epigenetics Chromatin. .

Abstract

Background: In 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.

Results: We 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.

Conclusions: Our 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.

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Figures

Figure 1
Figure 1
Centromeric silencing is alleviated by mutations in the Med8-Med18-Med20 submodule. (A) Schematic representation of S. pombe centromere 1. The insertion site of the ura4+ reporter used below (imr1R(NcoI)::ura4+), the probe for siRNA detection in Figure 3, and amplicons for the various PCRs performed in this study are shown. One position of the putative dg promoter (pro) is indicated relative to the outer repeats (dg and dh) of centromere 1. The crossed line represents an array of dg and dh repeats next to the innermost repeats (imr) and the central core (cnt). (B-D) Ten-fold serial dilutions of cell suspensions were spotted onto the indicated media. Plates were incubated at 33°C for (B) and (D) and at 37°C for the med8ts mutant in (C). Expression of ura4+ permits growth in the absence of uracil and causes sensitivity to 5-FOA. Reduced growth on 5-FOA for the med18Δ, med20Δ and med8ts mutants indicates derepression of heterochromatic silencing in these three strains. In contrast, deletion of other non-essential Mediator subunits in (D) does not alter growth on 5-FOA. (E) Quantification of ura4+ transcript by RT-qPCR confirms derepression of imr1R(NcoI)::ura4+ in the med18Δ and med20Δ mutants. The actin transcript (act1+) was used for normalization. A dcr1Δ strain is shown for comparison. The strains for this figure were: WT (FY498), med18Δ (MT42), med20Δ (MT26), med8ts (MT31) med1Δ (MT13), med27Δ (MT11), med31Δ (MT14), med12Δ (MT6), and dcr1Δ (TP480).
Figure 2
Figure 2
Mutations in the Med8-Med18-Med20 submodule cause an accumulation of centromeric transcripts. (A) The steady-state level of centromeric non-coding RNA was estimated by RT-PCR in the indicated mutants. The actin transcript was used as reference. (B) RT-qPCR shows that the dh transcript accumulates in med18Δ and med20Δ strains. (C) Strand specific RT-PCR shows that med18Δ and med20Δ strains have wild-type ratios of forward to reverse transcripts. (D) Northern blot analysis shows that the length of major centromeric transcripts is unchanged in the mutants. The strains for this figure were: WT (FY498), med18Δ (MT42), med20Δ (MT26), med8ts (MT31), and dcr1Δ (TP480).
Figure 3
Figure 3
siRNA levels in med18+and med20+deletion strains. (A) Representative Northern blot of siRNA in wild type and indicated mutants. Total RNA was run on a 17.5% polyacrylamide/7M urea gel, blotted and hybridized as described in Materials and Methods. Ethidium-bromide staining of the same RNA preparations was used as loading control. (B) Quantification of the blots (n = 4) *P <0.05; **P =5.2e to −12. The strains for this figure were: WT (FY498), med18Δ (MT42), med20Δ (MT26), and dcr1Δ (TP480).
Figure 4
Figure 4
Mediator occupancy (Med7-TAP) at the centromeric dg promoter and dg repeat in med18+and med20+deletion strains. ChIP analyses show that the relative Mediator occupancy at (A) the centromeric promoter as well as at (B) the dg repeat is unchanged in med18Δ and med20Δ mutant strains. The strains for this figure were: WT (FY498), med18Δ (MT42), and med20Δ (MT26).
Figure 5
Figure 5
RNA Pol II occupancy at the centromeric promoter and dg repeat in med18+and med20+deletion strains. ChIP analyses show that compared to wild-type, the RNA Pol II occupancy at (A) the dg centromeric promoter as well as at (B) the dg repeat is increased in med18+ and med20+ deletion strains. For comparison, the RNA Pol II occupancy in a clr4Δ deletion strain is also shown. *P <0.004; **P <1e to −6. The strains for this figure were: WT (FY498), med18Δ (MT42), and med20Δ (MT26), and clr4Δ (PG3423).
Figure 6
Figure 6
Mutations in the Med8-Med18-Med20 submodule compromise H3K9 methylation at the centromeric dg promoter. ChIP analyses show that the level of H3K9 dimethylation at the centromeric dg promoter is reduced in med18Δ and med20Δ mutants relative to wild-type. A clr4Δ strain was processed in parallel for comparison. *P <0.003. The strains for this figure were: WT (FY498), med18Δ (MT42), med20Δ (MT26), and clr4Δ (PG3423).
Figure 7
Figure 7
Deletion of med18+or med20+impairs centromere function. (A) A non-essential mini-chromosome, Ch16m23::ura4+-Tel[72], is frequently lost in strains deleted for med18+, med20+ or clr4+. Cells containing the mini-chromosome form white colonies on medium with low concentration of adenine while cells lacking the mini-chromosome form red colonies. Loss of the mini-chromosome in the first cell division after plating results in a half-sectored colony. (B) Deletion of med18+ or med20+ renders the cells sensitive to the microtubule destabilizing agent thiobendazole (12 μg/ml). The strains for this figure were: WT (FY520), med18Δ (TP527), med20Δ (TP527), and clr4Δ (PG3420).
Figure 8
Figure 8
Model illustrating the effect of the Med8-Med18-Med20 submodule on heterochromatin. Med8-Med18-Med20 may block recruitment of RNA Pol II to the centromeric chromatin by interacting with Rpb4/Rpb7. Additionally, the submodule may stimulate the activity of RNAi and thus influence the methylation level of H3K9 in centromeric chromatin. Further, Med8-Med18-Med20 in concert with Rpb1/Rpb2 may decide the fate of non-coding transcripts by directing them towards the RNAi machinery or to other downstream processes. See text for details.
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