Mediator is required for heterochromatic silencing at the pericentromere.

<p>(A) Schematic of fission yeast centromere 1. Locations of <i>ura4</i> and <i>ade6</i> reporter genes inserted within the pericentromeric region are shown (<i>imr1L::ura4⁺</i> and <i>otr1R::ade6</i>). Black bars indicate the location of primers or probes used for ChIP, RT-PCR and northern analysis. (B) Silencing assay at the pericentromere. Shown are the results of serial dilutions of the indicated strains spotted onto non-selective media (N/S), medium with 5-fluoroorotic acid (5-FOA), and medium with a limited amount of adenine (Low Ade) to assay <i>ura4⁺</i> and <i>ade6⁺</i> expression. (C) Spots on Low Ade medium using Mediator mutants (<i>med18Δ</i>, <i>med20Δ</i>, <i>med8-K9</i> and <i>med31-H1</i>), which are defective in heterochromatic silencing at the pericentromere. (D) Silencing assay at the pericentromere. Shown are the results of serial dilutions of the indicated strains spotted onto N/S, 5-FOA and Low Ade media to assay <i>ura4⁺</i> and <i>ade6⁺</i> expression. <i>w</i> indicates white epiclones (<i>med18Δ-w</i> and <i>med20Δ-w</i>), and <i>p</i> indicates pink epiclones (<i>med18Δ-p</i> and <i>med20Δ-p</i>). (E) Northern Analysis of <i>dh</i>, <i>otr1R::ade6</i> and <i>imr1L::ura4</i> forward strand transcripts in wild-type (<i>wt</i>) and mutant cells using oligonucleotide probes. rRNA was used as a loading control. (F) Quantitative RT-PCR analysis of <i>cen dh</i> forward transcript levels relative to a control <i>act1⁺</i>, normalized to the wild type in the indicated strains. Error bars show the standard error of the mean (n = 3).