Mediator is required for transcriptional activation in heterochromatin.

<p>(A) Schematic of the fission yeast mating-type locus. Location of the <i>ura4</i> reporter inserted within the <i>cenH</i> is shown (<i>kint2::ura4⁺</i>). Black bars indicate the location of primers or probes used for ChIP, RT-PCR and northern analysis. (B) ChIP analysis of H3K9me and Swi6 at <i>cenH dh</i> repeats or <i>kint2::ura4⁺</i>, each relative to <i>act1</i> or <i>trnaasn.05</i>. Error bars show the standard error of the mean (n = 3). (C) Silencing assay at the mating-type locus. The results of serial dilutions of the indicated strains spotted onto N/S, 5-FOA media and medium without uracil (-URA) for the silencing of <i>ura4</i> are shown. Note that PMGS (PM medium containing L-glutamic acid as a nitrogen source instead of ammonium chloride) plates were used as N/S plates. (D) Quantitative RT-PCR analysis of <i>cenH dh</i> repeats or <i>kint2::ura4⁺</i> forward transcript levels relative to a control <i>SPRRNA.48</i>, whose levels were normalized to that of the wild-type in the indicated strains. Error bars show the standard error of the mean (n = 3). (E) ChIP analysis of RNAPII at <i>cenH dh</i> repeats or <i>kint2::ura4⁺</i> relative to <i>SPRRNA.48</i>. Error bars show the standard error of the mean (n = 3). P values were determined using a two-sided Student's t-test. Note that in Figures B, D and E, <i>h⁻</i> strains (<i>h⁻, h⁻clr4Δ</i>), which do not have <i>cenH</i> sequence, were included to show the primers used in the experiments only detect <i>cenH</i> and do not detect the pericentromeric repeats.