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 med18 Δ, med20 Δ 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 dcr1 Δ strain is shown for comparison. The strains for this figure were: WT (FY498), med18Δ (MT42), med20Δ (MT26), med8ts (MT31) med1 Δ (MT13), med27 Δ (MT11), med31 Δ (MT14), med12 Δ (MT6), and dcr1 Δ (TP480).