Displacement and re-accumulation of centromeric cohesin during transient pre-anaphase centromere splitting

Abstract

The ring-shaped cohesin complex links sister chromatids until their timely segregation during mitosis. Cohesin is enriched at centromeres where it provides the cohesive counterforce to bipolar tension produced by the mitotic spindle. As a consequence of spindle tension, centromeric sequences transiently split in pre-anaphase cells, in some organisms up to several micrometers. This 'centromere breathing' presents a paradox, how sister sequences separate where cohesin is most enriched. We now show that in the budding yeast Saccharomyces cerevisiae, cohesin binding diminishes over centromeric sequences that split during breathing. We see no evidence for cohesin translocation to surrounding sequences, suggesting that cohesin is removed from centromeres during breathing. Two pools of cohesin can be distinguished. Cohesin loaded before DNA replication, which has established sister chromatid cohesion, disappears during breathing. In contrast, cohesin loaded after DNA replication is partly retained. As sister centromeres re-associate after transient separation, cohesin is reloaded in a manner independent of the canonical cohesin loader Scc2/Scc4. Efficient centromere re-association requires the cohesion establishment factor Eco1, suggesting that re-establishment of sister chromatid cohesion contributes to the dynamic behaviour of centromeres in mitosis. These findings provide new insights into cohesin behaviour at centromeres.

Fig. 1

Prominent centromeric cohesin enrichment in the absence of spindle forces. Cells of strain Y379 (MATa MET3-CDC20 SCC1-HA6) were synchronized in G1 with α-factor and released into metaphase arrest by Cdc20 depletion in the presence (−Cdc20 + noc) or absence of (−Cdc20) nocodazole. Cultures were processed for chromatin immunoprecipitation against the HA epitope-tagged cohesin subunit Scc1. Enrichment of DNA fragments in the immunoprecipitate relative to a whole genome sample is shown over an approximately 110 kb-wide region surrounding centromere 6. Each bar represents the average of 16 oligonucleotide probes within adjacent 300 bp windows. The y-axis scale is log₂. Grey signals represent significant binding as described (Katou et al. 2003). Blue bars above and below the midline are genes transcribed from left to right and opposite, respectively. The centromere is depicted in dark red, origins of replication are in red, tRNA genes in yellow and Ty elements in green. The cohesin signal over Ty elements is not interpretable in this analysis due to their repetitive nature in the genome

Fig. 2

Cohesin removal from centromere 6 during pre-anaphase splitting. Cells of strain Y3138 (MATa scc2-4 MET3-CDC20 SCC1-HA6 GAL1-CDC14 CEN5(1.4 kb)tetOs tetR-GFP) were synchronized in G1 with α-factor and released into HU arrest in early S-phase before Scc2 was inactivated by temperature shift. Cells were released into metaphase arrest by Cdc20 depletion in the presence of nocodazole (state a). Nocodazole was then washed out to allow spindle assembly and centromere splitting (state b), and Cdc14 expression was induced by galactose addition to stabilise split centromeres (state c). Cohesin distribution around centromere 6 and splitting of the GFP-marked centromere 5 were analysed

Fig. 3

Cohesin removal from all centromeres in response to centromere splitting. As in Fig. 2, but the cohesin immunoprecipitate was hybridised to a microarray covering all of the budding yeast genome. States a, no centromere breathing in the presence of nocodazole and c, permanently split centromeres after nocodazole washout and Cdc14 induction are presented. Cohesin distribution over a region of approximately 30 kb surrounding each centromere is shown. Tick marks along the x-axis represent 5 kb intervals. Origins of replication are indicated at a selection of chromosomes. The green line to the left of centromere 5 indicates the location of the tetO repeats

Fig. 4

Differential behaviour of pre- and post-replicatively loaded cohesin. Cells of strain Y3267 (MATa MET3-CDC20 SCC1-Pk9 GAL1-SCC1) were synchronized in G1 with α-factor and released into HU arrest in early S-phase. From there cells were released into metaphase arrest by Cdc20 depletion in the presence of nocodazole. Loading of ectopic, HA epitope-tagged cohesin was induced by induction of the GAL1 promoter for 1 h either during the time in the HU arrest (HU-loaded), or metaphase arrest (noc-loaded). Nocodazole was then washed out to allow spindle assembly and centromere breathing. Cohesin association around centromere 6 is shown before (+noc) and after nocodazole wash out (−noc)

Fig. 5

a Scc2-independent cohesin re-accumulation during sister centromere re-association. Cells of strain Y3138 (MATa scc2-4 MET3-CDC20 SCC1-HA6 GAL1-CDC14 CEN5(1.4 kb)tetOs tetR-GFP) were synchronized in G1 with α-factor and released into HU arrest in early S-phase before Scc2 was inactivated by temperature shift. From there, cells were released into metaphase arrest by Cdc20 depletion. Centromere breathing was then stopped and centromere re-association facilitated by addition of nocodazole. Cohesin distribution around chromosome 6 and splitting of the GFP-marked centromere 5 were analysed before and after nocodazole addition. b Eco1, and therefore most likely re-establishment of sister chromatid cohesion, contributes to centromere re-association. Cells of strain Y763 (MATa MET3-CDC20 SCC1-HA6 CEN5(1.4 kb)tetOs tetR-GFP) and Y762 (MATa eco1-1 MET3-CDC20 SCC1-HA6 CEN5(1.4 kb)tetOs tetR-GFP) were synchronized in metaphase by Cdc20 depletion at permissive temperature. Eco1 was either inactivated by temperature shift to 35°C or retained at permissive temperature for 1 h before addition of nocodazole to the cultures. Efficient disruption of the spindle by nocodazole in all cells was confirmed by immunostaining (not shown), and splitting of the GFP-marked centromeres was analysed before and 1 h after nocodozole addition