Mutations Disrupting Histone Methylation Have Different Effects on Replication Timing in <i>S. pombe</i> Centromere

<div><p>The fission yeast pericentromere comprises repetitive sequence elements packaged into heterchromatin marked by histone H3K9 methylation and Swi6 binding. Transient disruption of Swi6 during S phase allows a period of RNA synthesis which programs the RNAi machinery to maintain histone methylation. However, Swi6 is also required for early replication timing. We show that not only Swi6 but also the chromodomain protein Chp1 are delocalized during S phase. Different from loss of <i>swi6</i>, mutations that disrupt histone methylation in the centromere, <i>chp1</i>Δ and <i>clr4</i>Δ, undergo early DNA replication. However, timing is modestly delayed in RNAi mutants <i>dcr1</i>Δ or <i>rdp1</i>Δ, while <i>hrr1</i>Δ mutants resemble <i>swi6</i>Δ in their replication delay. Finally, we show that recruitment of RNA polymerase II in the centromere occurs independently of replication. These different effects indicate that replication timing is not simply linked to histone methylation.</p></div

Centromere replication is delayed in RNAi mutants.

<p>A, schematic showing the experimental procedure. Cells were arrested in G1 by nitrogen depletion and released into the cell cycle by refeeding. BrdU and HU were added at 1.5 hours after release. B-D, BrdU ChIP in different mutants at the early origin <i>ars2004</i> (B), and the centromere repeats <i>dg</i> (C), and <i>dh</i> (D). BrdU enrichment was calculated by the ratio of IP versus Input by semi-quantitative PCR and at least two independent experiments were performed. Each mutant compared to its 0 timepoint. Asterisks mark samples with BrdU signal significantly higher than the WT at 6 h timepoint with p<0.05 (Student's T test). Primers: #1041/1042(dg), #1033/1034 (dh), and #1257/1258 (ars2004).</p

The centromere replicates early in <i>clr4</i>Δ or <i>chp1</i>Δ mutants.

<p>A, the structure of the three <i>S. pombe</i> centromeres. Repetitive sequences <i>dg</i> and <i>dh</i> in the outer repeats (<i>otr</i>) are present in slightly different organization in each centromere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061464#pone.0061464-Tran1" target="_blank">[66]</a>. The length of <i>dg</i> or <i>dh</i> is around 4-6 kb. B, scheme of the experimental protocol. <i>cdc25-22</i> mutants were shifted to 36°C for 4 hours. One hour prior to release to 25°C, 10 mM of HU was added so that only early origins fire. Upon release, 100 µg/ml BrdU was added to label new DNA synthesis. C, incorporation of BrdU in the <i>dg</i> region was detected by BrdU enrichment, which was calculated by the ratio of IP versus Input by semi-quantitative PCR using primers #1536/1537 (dg). Three independent experiments were performed. Asterisks mark samples with BrdU signal significantly higher than the WT at 90 min with p<0.05 (Student's T test). The quality of synchronization is determined by flow cytomertry. There is no significant different among WT, <i>chp1</i>Δ and <i>clr4</i>Δ (Figure S1A).</p

Swi6 and Chp1 each delocalize from the centromere between M and S phase.

<p>Asynchronously growing cells containing CFP-Cnp1 Sad1-DsRed (FY4229), GFP-Swi6 Sad1-DsRed (FY3665), or Chp1-GFP Sad1-DsRed (FY5911) were imaged every three minutes and the images were projected as described in materials and methods. A, quantitation of the data. The first frame in which spindle pole body duplication was observed was assigned as “0”. The first frame in which re-association of the GFP marked protein with the SPB occurred, and was maintained for at least three frames, was recorded. The re-association timing of CFP-Cnp1 (green), GFP-Swi6 (purple), or Chp1-GFP (black) with Sad1-DsRed is summarized in this distribution. Y axis, number of cells; X axis, time (minutes) after duplication of Sad1-DsRed, which is used to mark the beginning of M phase. Representative frames of GFP-Swi6, or Chp1-GFP are provided here B-C, respectively; selected original movies are provided in Movies S1, S2, S3. Arrows show cells with delocalization of the GFP signal from the SPB.</p

Replication timing and polymerases recruitment in wild type and <i>swi6</i>Δ.

<p>Cells were synchronized by <i>cdc25-22</i> block and release. Two independent experiments were performed and the representative result is presented. The signals were determined by quantitative real-time PCR. A, position of PCR probes used in this experiment. B–C, recruitment of DNA polymerase alpha (blue line), RNA polymerase II (green line) and incorporation of BrdU (red line) in euchromatin and the centromere, respectively. Left Y axis, DNA polymerase alpha or RNA polymerase II enrichment; right Y axis, BrdU enrichment. Red arrow, early replication time-point; blue arrow, late replication time-point. Primers: #1257/1258 (ars2004), #1265/1266 (non-ars), #875/876 (AT2080), #1628/1629 (dg), #1183/1184 (dh), and #879/880 (cnt2).</p

Quantitative performance results of yeast segmentation algorithms.

<p>¹ Relative to all cells.</p><p>% pixel mismatch compared to gold standard cell contours, relative to all cells.² Defined as the percentage of cell contours with less than 10</p><p>³ Relative to all contours.</p><p><a href="mailto:[email protected]" target="_blank">[email protected]</a> GHz, 4 GB RAM).⁴ Average CPU time per image, measured using a regular desktop PC (Windows 7, Matlab R2011a, Intel(R) Core(TM) i7-2600 </p

Example results of distance transform-based procedure.

<p>Original images (left), pixel classification before (middle) and after (right) distance transform-based procedure. Blue pixels indicate background, red is cell membrane and green is cell interior region.</p

Segmentation results on well-known phenotypes.

<p>Cell contours are colored randomly.</p

Analysis of pixel intensity variation of the same specimen imaged at different z-focus position.

<p>Top plot: original image at one z-position with red contours indicating k-means cluster boundaries. Middle plot: the same image with color-coded cluster membership for pixels. Bottom plot: Mean intensity of each pixel cluster through z-position.</p

Examples of images before (left) and after (right) adaptive focus gradient correction and contrast enhancement.

<p>In the left panel the original images are overlaid with nucleus centroids (black and white spots) and the estimated <i>z</i> = 0 line (green line, where specimen plane intersects focal plane). For the nucleus centroids, black means its average intensity is lower than background, and white means it is brighter than background.</p