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

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

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 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

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

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

Effect of Exp4 on Sox9 binding to its target gene.

<p>(A) The effect of Exp4 depletion on the recruitment of Sox9 to its endogenous target gene. U2OS cells were treated with control siRNA (open bar) or siRNA for Exp4 (solid bar). After a 48 h incubation, fixed chromatin was prepared from both groups of treated cells and subjected to immunoprecipitation with anti-Sox9 antibody. The <i>Col2a1</i> enhancer regions recovered in the two precipitated fractions were quantified by PCR. As a control, <i>GAPDH</i> genes recovered in the two precipitated fractions were quantified. The relative amount of enhancer DNA is shown: the amount of <i>Col2a1</i> enhancer DNA obtained from the control siRNA cells is set at 100. (B) The DNA sequence of the probe used for DNA pull-down contains a biotinylated 18 base-pair Sox9 binding sequence of the <i>Col2a1</i> enhancer region. (C) DNA pull-down assay with the indicated amounts (ng) of His-Sox9 and FLAG-Exp4. His-Sox9 and FLAG-Exp4 were incubated with a 5′ biotinylated double-stranded DNA probe. The complex was pulled down with streptavidin-agarose and the streptavidin-agarose bead bound fraction and flow-through fractions (FT) were analyzed by Western blotting.</p

Model of the functions of Mediator in pericentromeric heterochromatin assembly in fission yeast.

<p>Mediator localizes to the pericentromeres to recruit RNAPII for efficient transcription. Furthermore, Mediator regulates processing of transcribed ncRNA by RNAi machineries, which directs heterochromatin formation. In addition, Mediator is also required for Rrp6-dependent heterochromatin formation. For details, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003677#s3" target="_blank">Discussion</a>.</p