Components of MTOCs in Δ<i>scp3</i>.

<p>(A, B) Each protein tagged with GFP (Alp4 in A, Mto1 in B) was observed in EMM medium at 30°C. Sad1-mCherry was used as a marker of SPB. The bar indicates 5 μm.</p

Relationship between Ase1 and CIPC.

<p>(A) Ase1 tagged with GFP was expressed from the native promoter in wild-type cultured in EMM medium containing DMSO or 260 μM CIPC for 5 hours at 30°C. Sad1-mCherry was used as an SPB maker. The bars indicate 5 μm. (B) The Δ<i>ase1</i> strain expressing GFP-Atb2 from the native <i>nda3</i> promoter integrated at the <i>lys1</i> locus and Sad1-mCherry from the native locus was cultured in EMM medium containing DMSO or 260 μM CIPC for 5 hours at 30°C. The bars indicate 5 μm. (C) Each strain was streaked on YES medium with or without CIPC (260 μM) and incubated at 30°C for 5 days.</p

Relationship between Ase1 and Scp3.

<p>(A) Interphase MTs were observed in the Δ<i>ase1</i> strain overexpressing <i>scp3</i>⁺ or the Δ<i>scp3</i> strain overexpressing <i>ase1</i>⁺. Each gene was expressed from the <i>nmt</i> promoter of pREP81 in EMM medium at 30°C. (B) Nonparametric Mann-Whitney <i>U</i> test of (A). The red lines are the median. More than 150 microtubules were observed for each condition. (C) Mitotic cells overexpressing <i>ase1</i>⁺ were observed. <i>ase1</i>⁺ expression was induced for 18 hours. GFP-Atb2 expressed from the <i>nda3</i> promoter integrated at the <i>lys1</i> locus was used as a maker of microtubules and Sad1-mCherry for SPB. (D) Cells with normal spindle (upper) and with multiple SPBs (bottom) were counted for each strain. (E) <i>scp3</i>⁺ was overexpressed in the Δ<i>ase1</i> strain. Aberrant mitotic spindles are marked with arrowheads. The bars indicate 5 μm.</p

Microtubules in <i>cps3–81</i> treated with CIPC.

<p>The <i>cps3–81</i> mutant expressing GFP-Atb2 and Sad1-mCherry, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120109#pone.0120109.g001" target="_blank">Fig. 1A</a>, was treated with DMSO (control) or 260 μM CIPC for 5 hours in EMM medium at 30°C. The bar indicates 5 μm.</p

The effect of Scp3 overexpression.

<p>(A) Each strain expressing GFP-Atb2 and Sad1-mCherry, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120109#pone.0120109.g001" target="_blank">Fig. 1A</a>, was first grown in EMM containing thiamine at 30°C. Thiamine was washed out to induce expression of the <i>scp3</i>⁺ gene from the <i>nmt</i> promoter of pREP81. Each strain was cultured for 20 hours, and CIPC (260 μM) was added for 5 hours. The bar indicates 5 μm. (B) The angle between the long cell axis and each MT was measured for the analysis of microtubule orientation. More than 150 microtubules were observed under each condition. (C) The effect of CIPC to the <i>cps3–81</i> mutant was analyzed by Nonparametric Mann-Whitney <i>U</i> test. The <i>cps3–81</i> mutants transformed with an empty vector were grown in the presence or absence of CIPC for 5 hours were compared. The red lines are the median.</p

Microtubules in the wild-type strain treated with CIPC.

<p>(A) GFP-tagged α-tubulin (GFP-Atb2) as a microtubule marker and Sad1 tagged with mCherry (Sad1-mCherry) as an SPB marker were expressed from their native promoters. The cells were grown at 30°C and treated with 260 μM or 300 μM CIPC for 5 hours in EMM medium. DMSO was used as a solvent. Misoriented MTs are marked with arrowheads. The bar indicates 5 μm. (B) The wild-type strain in metaphase was observed in the presence of CIPC. The bar indicates 5 μm.</p

Abnormal microtubule orientation in Δ<i>scp3</i>.

<p>(A) The <i>∆scp3</i> strain expressing GFP-Atb2 and Sad1-mCherry from the native promoters was treated with DMSO or 260 μM CIPC for 5 hours in EMM medium at 30°C. The bar indicates 5 μm. (B) Microtubule orientation was analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120109#pone.0120109.g002" target="_blank">Fig. 2B</a>. (C) Nonparametric Mann-Whitney <i>U</i> test of (B). The red lines are the median.</p

Effect of Mediator disruption on euchromatic genes.

<p>Venn diagram showing the number of transcripts whose expression levels are increased (up) or decreased (down) >1.5-fold in mutants compared to the wild-type. P-values were calculated using Fisher's exact test. (A) Transcripts of <i>med18Δ-w</i> (left circles) vs. <i>med18Δ-p</i> (right circles) mutants (top) and <i>med20Δ-w</i> (left circles) vs. <i>med20Δ-p</i> (right circles) mutants (bottom). (B) Transcripts of <i>med18Δ-w</i> (left circles) vs. <i>dcr1Δ</i> (right circles) mutants (upper left), <i>med20Δ-w</i> (left circles) vs. <i>dcr1Δ</i> (right circles) mutants (upper right), <i>med18Δ-p</i> (left circles) vs. <i>dcr1Δ</i> (right circles) mutants (lower left), and <i>med20Δ-p</i> (left circles) vs. <i>dcr1Δ</i> (right circles) mutants (lower right).</p

Med18/Mediator is required for Rrp6/Exosome-dependent H3K9 methylation at the pericentromere.

<p>(A) 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. (B) ChIP analysis of H3K9me (left panel) and Swi6 (right panel) at <i>dh</i> repeats or <i>imr1L::ura4⁺</i> relative to <i>act1</i> or a tRNA genes (<i>trnaasn.05</i>), respectively. Error bars show the standard error of the mean (n = 3). (C) ChIP analysis of H3K9me (left panel) and Swi6 (right panel) at the <i>dh</i> repeats relative to <i>act1</i> or a gene-free region, respectively. Error bars show the standard error of the mean (n = 3). (D) ChIP analysis of Rrp6-13myc at <i>dh</i> repeats (left panel), <i>act1</i> (middle panel) and <i>fbp1</i> (right panel). Enrichment relative to the input whole cell extract (WCE) in the indicated strains are shown. Error bars represent the standard error of the mean (n = 3).</p

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.</p