The Chromosomal Association of the Smc5/6 Complex Depends on Cohesion and Predicts the Level of Sister Chromatid Entanglement

<div><p>The cohesin complex, which is essential for sister chromatid cohesion and chromosome segregation, also inhibits resolution of sister chromatid intertwinings (SCIs) by the topoisomerase Top2. The cohesin-related Smc5/6 complex (Smc5/6) instead accumulates on chromosomes after Top2 inactivation, known to lead to a buildup of unresolved SCIs. This suggests that cohesin can influence the chromosomal association of Smc5/6 via its role in SCI protection. Using high-resolution ChIP-sequencing, we show that the localization of budding yeast Smc5/6 to duplicated chromosomes indeed depends on sister chromatid cohesion in wild-type and <i>top2-4</i> cells. Smc5/6 is found to be enriched at cohesin binding sites in the centromere-proximal regions in both cell types, but also along chromosome arms when replication has occurred under Top2-inhibiting conditions. Reactivation of Top2 after replication causes Smc5/6 to dissociate from chromosome arms, supporting the assumption that Smc5/6 associates with a Top2 substrate. It is also demonstrated that the amount of Smc5/6 on chromosomes positively correlates with the level of missegregation in <i>top2-4</i>, and that Smc5/6 promotes segregation of short chromosomes in the mutant. Altogether, this shows that the chromosomal localization of Smc5/6 predicts the presence of the chromatid segregation-inhibiting entities which accumulate in <i>top2-4</i> mutated cells. These are most likely SCIs, and our results thus indicate that, at least when Top2 is inhibited, Smc5/6 facilitates their resolution.</p></div

Model describing the connection between chromosome segregation and the chromosomal association of Smc5/6 and cohesin.

<p>Summary of how the chromosomal association of Smc5/6 in metaphase (<i>left</i>) correlates with chromosome segregation in anaphase (<i>right</i>) in wild-type and <i>top2-4</i> cells, in which <i>top2-4</i> is inactivated either prior or after DNA replication. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004680#s3" target="_blank">discussion</a> for detailed description.</p

The chromosomal association of Smc6 does not depend on DSB formation or recombination.

<p>(<b>A</b> and <b>B</b>) Chromosome arm association of Smc6-FLAG in G2/M-arrested <i>mre11Δ</i>, <i>top2-4 mre11Δ</i>, <i>rad52Δ</i>, <i>top2-4 rad52Δ</i> cells as indicated, determined by ChIP-seq (<b>A</b>) and ChIP-qPCR (<b>B</b>). Samples were collected in G2/M after a synchronous S-phase at 35°C, nonpermissive for the <i>top2-4</i> allele. In (A), a region spanning 100–200 kb from the left telomere of chromosome 7 is shown. Panel details for (A) and (B) are described in the legend of <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004680#pgen-1004680-g002" target="_blank">Figure 2</a>. (<b>C</b>) Western blot of Rad53 and actin in wild-type and <i>top2-4</i> cells arrested in S-phase by HU or in G2/M by nocodazole, as indicated.</p

Chromosome segregation in wild-type, <i>top2-4</i>, <i>scc1-73</i> and <i>top2-4 scc1-73</i> cells.

<p>(<b>A</b>) Representation of experimental setup. Wild-type, <i>top2-4</i>, <i>scc1-73</i>, or <i>top2-4 scc1-73</i> cells harboring multiple copies of tetracycline operators at a specific chromosomal region, and expressing GFP-tagged tubulin and tdTomato-marked tetracycline repressors, were imaged during progression through one synchronous cell cycle under conditions which inactivated both mutant alleles. Elongation of the tubulin spindle was used as time point zero (0). Chromatid separation was defined as the moment when the tdTomato signal was split into two, segregation when each of the chromatid-marking dots was found in mother and bud cell, respectively. On the X-axis, bars at time point 0 represent the sum of separation events in the images collected at 0 and 0.5 minutes, time point 1 minute the sum of separation events in the frames of 1 and 1.5 minutes etc. Cells in which chromatids do not separate during the 70 minutes of imaging fall in into the category of events which are marked with an asterisk on the X-axis. If segregation occurs within 5 minutes of separation, the cells fall into the “green-bar-category” on the Z-axis (≤5 minutes). If segregation occurs more than 5 minutes after separation, the cells fall into the “red-bar-category” on the Z-axis (>5 minutes). Cells that do not segregate their chromatids during the entire 70 minutes of imaging are placed into the “black-bar-category” on the Z-axis (triangle). (<b>B-D</b>) Separation and segregation of chromosomes 4 (<b>B</b>), 1 (<b>C</b>), 5 (<b>D</b>) in wild-type and <i>top2-4</i> cells. (<b>E</b> and <b>F</b>) Separation and segregation of chromosomes 1 (<b>E</b>) and 5 (<b>F</b>) in <i>scc1-73</i> and <i>top2-4 scc1-73</i> cells. The tetracycline operators are integrated 35 kb from the centromeres on chromosomes 1, 5 and 4. This places these markers at 44, 117 and 1045 kb away from the telomeres. On chromosomes 5 and 4 the telomere proximal sites are placed 350 and 995 kb away from centromere, respectively. This places them at 72 and 85 kb away from respective telomeres.</p

Smc5/6 is enriched on replicated chromosomes in between convergently transcribed genes close to centromeres, and the level of enrichment increases if the centromeres are distant from a chromosome end.

<p>(<b>A</b>–<b>C</b>) Chromosomal localization of Smc6-FLAG during G1 (<b>A</b>) and S-phase (<b>C</b>), or bromodeoxyuridine (BrdU) incorporation (<b>B</b>), as determined by ChIP-seq. In (B and C), cells were arrested in G1 and subsequently released into media containing BrdU in the presence of hydroxyurea (HU), or at 18°C as indicated. Samples were collected in HU and 1 and 2 hours after the release. ChIP-seq analysis was performed using anti-FLAG (<b>A</b> and <b>C</b>), or anti-BrdU antibodies (<b>B</b>). (<b>D</b>) Chromosomal localization of Smc6-FLAG, Nse4-FLAG and Scc1-FLAG in G2/M-arrested cells. Samples were collected in a nocodazole-induced G2/M-arrest after a synchronous S-phase at 35°C. (<b>E</b>) Chromosomal localization of Smc6-FLAG cells arrested in telophase due to non-functional Cdc15 mitotic exit network kinase. Samples were prepared as in (D) but released from G1-arrest in nocodazole-free medium. Panel details as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004680#pgen-1004680-g002" target="_blank">Figure 2</a> with the addition of vertical dashed lines showing that Smc6-FLAG and Nse4-FLAG co-localize with Scc1 in between convergently oriented genes in the G2/M-arrest. (<b>F</b>–<b>H</b>) Scatterplots comparing Smc6-FLAG enrichment with full chromosome length (<b>F</b>), chromosome arm length (<b>G</b>), or the distance from the centromere to the nearest telomere (i. e. the length of the shorter chromosome arm) (<b>H</b>). Enrichment values represent Smc6-FLAG enrichment in 100 kb regions spanning the centromeres (<b>F</b> and <b>H</b>) or in 50 kb regions to the left or right of the centromere (<b>G</b>). Enrichment values from ChIP-seq performed on a control strain lacking tagged proteins were subtracted for each region (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004680#pgen.1004680.s004" target="_blank">Figure S4</a>). Correlation values are indicated in each graph. Chromosome 12 was excluded from the scatterplots due to the unknown length of the rDNA present on that chromosome.</p

The chromosomal association of Smc6 does not depend on replication fork stalling.

<p>(<b>A</b>) ChIP-seq analysis of FLAG-tagged DNA polymerase II subunit Dpb3 in G2/M-arrested wild-type and <i>top2-4</i> cells. Compare to the enrichment of Smc6-FLAG seen at specific loci in this region in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004680#pgen-1004680-g005" target="_blank">Figure 5A</a> (<b>B</b>) ChIP-on-chip analysis of chromosome association of FLAG-tagged Dpb3 (upper panel) and BrdU-incorporation (lower panel) in HU-arrested S-phase cells. (<b>C</b>) Chromosome arm association of Smc6-FLAG in G2/M-arrested <i>rrm3Δ</i> cells. G2/M-samples were collected after a synchronous S-phase at 35°C, nonpermissive for the <i>top2-4</i> allele (<b>A</b>). For analysis in S-phase, cells were arrested in G1 and subsequently released into media containing BrdU and HU. Samples were collected 1 hour after the release (<b>B</b>). Analysis was performed using anti-FLAG (<b>A</b>, upper panel in <b>B</b>, and <b>C</b>), or anti-BrdU antibodies (lower panel in <b>B</b>). A region spanning 100–200 kb from the left telomere of chromosome 7 is shown, panel details are described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004680#pgen-1004680-g002" target="_blank">Figure 2</a>.</p

Chromosome-bound Smc5/6 predicts the degree of missegregation after Top2 inactivation.

<p>(<b>A</b>–<b>C</b>) Segregation of a fluorescently labeled region located ∼995 kb from the centromere of chromosome 4 (<b>A</b>), and ∼350 kb from centromere of chromosome 4 (<b>B</b>), and 5 (<b>C</b>), in wild-type and <i>top2-4</i> cells. The level of Smc6 enrichment in the 350 kb region between the centromere and the tetracycline operators on chromosome 4 and 5, calculated as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004680#pgen-1004680-g004" target="_blank">Figure 4</a>, is indicated in the schematic maps of the chromosomes above each panel. Cells were first arrested in G1 at permissive temperature for <i>top2-4</i> (23°C), and thereafter either incubated at the restrictive temperature 35°C during 30 minutes before release at 35°C, or released into a G2/M-arrest at 23°C. In the G2/M-arrest the temperature was raised to 35°C for 1 hour before release at the high temperature. Segregation was subsequently scored as in (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004680#pgen-1004680-g011" target="_blank">Figure 11A</a>) in both cell populations.</p

Smc5/6 accumulates on chromosomes after Top2 inhibition, and co-localizes with cohesin in both wild-type and <i>top2-4</i> cells.

<p>(<b>A–D</b>) Chromosomal association of (<b>A</b>, <b>C</b> and <b>D</b>) Smc6-FLAG and (<b>B</b>) Scc1-FLAG in indicated strains as determined by (<b>A–C</b>) ChIP-seq and (<b>D</b>) ChIP-qPCR. All cells were arrested in G2/M after a synchronous S-phase at 35°C before sample preparation. The ChIP-seq maps displays a chromosomal region spanning 100–200 kb from the left telomere of chromosome 7. Panel details and statistical analysis are described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004680#pgen-1004680-g002" target="_blank">Figure 2</a>. In (D), results for Smc6-FLAG in wild-type cells are identical to those displayed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004680#pgen-1004680-g002" target="_blank">Figure 2C</a>, and shown for comparison. (<b>E</b>) Overlap of Smc6 and Scc1 binding sites on chromosome arms. For annotation of Smc6 and Scc1 binding sites, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004680#s4" target="_blank">Material and Methods</a>.</p

Chromosomal regions where Smc5/6 accumulates after Top2 inhibition show no sign of persistent replication or recombination intermediates.

<p>(<b>A</b>) Chromosomal localization of Smc6-FLAG as determined by ChIP-seq at two loci, <i>UBP10-MRPL19</i> and <i>MPP10-YJR003C</i>, showing abundant Smc6-FLAG binding in <i>top2-4</i>. The lowest panel shows a ChIP-seq map from a control experiment performed on cells lacking FLAG-tagged proteins. Panel details and cellular growth conditions are as described in the legend of <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004680#pgen-1004680-g002" target="_blank">Figure 2</a>. In the top panel describing genomic features, arrows and chromosomal positions denote the restriction sites for <i>PstI</i>, used to produce the analyzed fragments. (<b>B</b>) Two-dimensional gel electrophoresis of <i>UBP10-MRPL19</i> (<i>left</i>) and <i>MPP10-YJR003C</i> (<i>right</i>) in wild-type and <i>top2-4</i> cells. Cell cycle progression monitored by fluorescence-activated cell sorting (FACS) is shown below and time-points of sample preparation are indicated. Membranes were first probed against <i>UBP10-MRPL19</i> (<i>left</i>), then stripped and re-probed against <i>MPP10-YJR003C</i> (<i>right</i>), leaving some residual signal from <i>UBP10-MRPL19</i> in the <i>MPP10-YJR003C</i> blots (white arrows). (<b>C</b>) Two-dimensional gel electrophoresis after DNA isolation using CTAB-extraction to preserve X-shaped molecules, of <i>ARS305</i> (<i>left</i>) and <i>UBP10-MRPL19</i> (<i>right</i>) in wild-type and <i>top2-4</i> cells. The <i>ARS305</i> containing fragment was produced by digestion using <i>EcoRI</i> and <i>HindIII</i>, and is a positive control for X-shaped molecule isolation (white arrowheads). Cell cycle progression monitored by FACS is shown below and indicates the time-points of sample preparation.</p

The chromosomal association of Smc5/6 depends on sister chromatid cohesion.

<p>(<b>A</b>–<b>C</b>) Chromosomal association of (<b>A</b> and <b>C</b>) Smc6-FLAG and (<b>B</b>) Scc1-FLAG in indicated strains as determined by (<b>A</b> and <b>B</b>) ChIP-seq and (<b>C</b>) ChIP-qPCR. All cells were arrested in G2/M after a synchronous S-phase at 35°C before sample preparation. Note that the false positive signal in denoted (*) is mostly absent in <i>SCC1-FLAG</i> cells. Figure details and statistical analysis are described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004680#pgen-1004680-g002" target="_blank">Figure 2</a>. In (C), results for Smc6-FLAG in wild-type cells are identical to those displayed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004680#pgen-1004680-g002" target="_blank">Figure 2C</a>, and shown for comparison.</p