Interactions between Cohesin and CHRIII in S. cerevisiae

<div><p>The centromere is indicated with a black circle; the smoothed data are indicated with a green line. 50-kb intervals are indicated by vertical grey lines.</p> <p>(A) Data generated from a <i>cdc16</i>-arrest ChIP for Mcd1-18Myc in W303a. The midpoint of each feature is used to represent the log₂ of the median red:green ratio (left y-axis) with a black line, high firing replication origins are indicated with black triangles, and previously mapped CARC2, CARC1, CARC3, CARC4, CARC5, and CARC6 (Laloraya et al., 2000) correspond to peaks 9, 10, 29, 30, 31, and 32, respectively. Peaks are located and numbered by PeakFinder (with the exception of telomeres) using the parameters described in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020259#s4" target="_blank">Materials and Methods</a>.</p> <p>(B) Smoothed data from <i>cdc16</i>-arrest ChIP for Mcd1/Scc1-6HA in A364a.</p> <p>(C) Smoothed data from <i>cdc16</i>-arrest ChIP for Smc3-6Myc in A364a.</p></div

Visual Representation of the Interactions between Mcd1-18Myc and the S. cerevisiae Genome in W303a

<p>For each of the 16 chromosomes the centromere is indicated with a black circle, the smoothed data (based on the log₂ of the ratio) is indicated with a green line (left y-axis), and the percent GC is indicated by a red line (right y-axis). Vertical grey bars mark 50-kb intervals. Peaks are located and numbered by PeakFinder (with the exception of telomeres) using the parameters described in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020259#s4" target="_blank">Materials and Methods</a>. For Chromosome XII, peaks 41 and 42 correspond to the previously described peaks CARL1 and CARL2 (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020259#pbio-0020259-Laloraya1" target="_blank">Laloraya et al. 2000</a>).</p

Meiotic Cohesin

<div><p>DSB data are shown in red, Rec8 data in black, and Mcd1 data in grey.</p> <p>(A) Ratios for meiotic cohesin are compared to mitotic cohesin in SK1 for kilobasepairs 440–461 on Chromosome XII. For the mitotic culture, cells were arrested with nocodazole. For meiotic cells, timepoints were collected every 2 h from hour 4 to hour 12 after transfer to SPM. The median ratio value was used to represent the data. Meiosis is slower in an SK1 strain with an HA-epitope-tagged Rec8 than in a wild-type strain (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020259#pbio-0020259-g009" target="_blank">Figure 9</a>). The gene structure for this locus is shown below the graph, with genes encoded by the Watson strand labeled on top and genes encoded by the Crick strand labeled on the bottom.</p> <p>(B) Ratios for meiotic cohesin are compared to mitotic cohesin and DSBs for kilobasepairs 295–345 on Chromosome XII.</p></div

Features of Peaks

<div><p>(A) Using all cohesin-binding peaks within 40 kb of a telomere ordered based on distance from the telomere, we calculated a five-point moving average for distance in kilobases from the telomere (x-axis) and plotted this as a function of the five-point moving average of the log₂ value for the associated peaks (y-axis).</p> <p>(B) Chromosome length (x-axis) is plotted as a function of the number of cohesin peaks (y-axis). A line was fitted using the least squares method and <i>R²</i> = 0.96.</p> <p>(C) The distance between peaks was put into 1-kb bins; the average distance between peaks is 10.9 kb and the median is 9.3 kb.</p></div

Cohesin Sites Mapped Using ChIP Followed by Semiquantitative PCR with Primers at 1-kb Intervals in a YAC Containing Human DNA

<div><p>(A) Cohesin binding for the entire YAC is shown.</p> <p>(B) Cohesin binding in the region spanning 135–180 kb is shown for the wild-type YAC (black diamonds) and for the YAC containing a replacement of the sequences at 156–162 kb with the gene encoding geneticin resistance (grey squares).</p></div

Meiotic Timecourse for an SK1 Strain Containing Rec8-3HA

<div><p>Cells were collected at the indicated timepoints throughout meiosis using the same experimental regime used to collect the binding sites of Rec8-3HA presented in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020259#pbio-0020259-g008" target="_blank">Figure 8</a>. The epitope tag appears to slow meiosis by 3–4 h as compared to an untagged strain. Three assays were developed to monitor culture synchrony during meiosis.</p> <p>(A) FACS profile of the <i>REC8-3HA</i> strain. Aliquots of cells were fixed with 70% EtOH, followed by FACS analysis.</p> <p>(B) Nuclear division of the <i>REC8-3HA</i> strain. Aliquots of cells were fixed with 1% formaldehyde for 1 h at room temperature. Nuclear DNA was stained by DAPI and visualized under a fluorescence microscope. At least 200 cells were scored at each timepoint.</p> <p>(C) Rec8-3HA protein level. Protein extracts were prepared and subjected to SDS-PAGE and Western blot. The Rec8-3HA protein level was detected by an anti-HA antibody (12CA5). The same blot was stripped and reprobed with anti-β-tubulin antibody to detect the level of β-tubulin, which served as a loading control.</p></div

Transcription Affects the Cohesin Peak at the Promoter of <i>GAL2</i>

<p>SGD coordinates 260–320 kb (x-axis) and a gene map are depicted for Chromosome XII. The strain 1827-22D (isogenic to the strain in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020259#pbio-0020259-g001" target="_blank">Figure 1</a> except <i>CDC16</i>) was grown with either 2% glucose (A) or 2% galactose (B) as the carbon source. Cultures were arrested with nocodazole, and ChIP chip was performed. The smoothed data (as the log₂ of the ratio) is depicted in green, the peaks found by PeakFinder are indicated with black dots, and the region corresponding to the <i>GAL2</i> promoter is indicated with a grey bar. Transcription of <i>GAL2</i> is up-regulated 42-fold in (B).</p