Rad21 remains chromatin bound when cohesin loading is inactivated in S-phase. Cells bearing rad21-HA and the thermosensitive mutations mis4–367 or ssl3–29 were arrested in early S-phase by hydroxyurea (HU) treatment at 25°C and then shifted to 37°C for 2 h in the presence of HU. (A) Rad21-HA association with chromatin was monitored on chromosome spreads by immunofluorescence before and after the shift to 37°C. Scale bar, 5 μm. (B) Rad21-HA fluorescence was measured in 50–100 nuclei. The error bars show the confidence interval of the mean with α=0.05. (C) DNA content analysis and septation index (SI) show that cells remained arrested with close to 1C DNA content throughout the experiment.

Inactivation of the cohesin-loading complex in G1 causes loss of cohesin from centromeres and chromosome arms. Rad21-HA association with chromosomes was determined by ChIP before and after Mis4 or Ssl3 inactivation as in Figure 1. Rad21-HA enrichment was monitored at centromeres (A), and three cohesin-binding sites along chromosome 2 (B–D, the numbering refers to their coordinates in kb). The error bars show the s.d. calculated from at least two independent experiments. (E) DNA content analysis and septation index (SI) confirm the arrest in G1. (F) Rec8 accumulation at centromeres in G1 cells relies on functional Mis4. Strains bearing Rec8-GFP in a mis4–367 or wt background were arrested in G1 by nitrogen starvation at 25°C to induce Rec8-GFP accumulation at centromeres and then shifted to 37°C for 2 h. Scale bar, 5 μm.

The loading machinery is required for sustained cohesin binding to chromatin in G1. Cells bearing thermosensitive mutations in genes encoding the cohesin-loading complex mis4 and ssl3 were arrested in G1 at permissive temperature by overexpressing a C-terminal Res1 fragment, under the control of the inducible nmt1 promoter (Ayte et al, 1995). Cells were then shifted to 37°C for 2 h to inactivate cohesin loading. (A) Rad21-HA chromatin association was monitored on chromosome spreads by immunofluorescence at 25°C and after shift to 37°C. DNA was stained with 4′-6-diamidino-2-phenylindole (DAPI). Scale bar, 5 μm. (B) Quantification of Rad21-HA fluorescence intensity. A total of 50–100 nuclei were analyzed for each sample. The error bars show the confidence interval of the mean with α=0.05. (C) DNA content analysis and septation index (SI) show that cells remained arrested in G1 throughout the course of the experiment.

Fission yeast Wapl regulates cohesin dynamics in G1. (A) Cells were arrested in G1 at 20°C by Res1 C-terminal overexpression and then shifted to 37°C for 2 h. Rad21-GFP association with chromatin was monitored on chromosome spreads by immunofluorescence using anti-GFP antibodies. Scale bar, 5 μm. (B) Rad21-GFP fluorescence intensity was measured for 50–100 nuclei per sample. The error bars show the confidence interval of the mean with α=0.05. (C) Strains bearing rec8-GFP were arrested in G1 by nitrogen starvation at 25°C to induce Rec8-GFP accumulation at centromeres and then shifted to 37°C for 90 min. Samples were taken every 30 min and examined for Rec8-GFP fluorescence. Scale bar, 5 μm. (D) Proportion of cells with a dot of Rec8-GFP after inactivation of Mis4. More than 150 cells were examined for each sample. (E) Quantification of Rec8-GFP fluorescence before and 90 min after the temperature shift. More than 150 cells were analyzed for each sample. The error bars show the confidence interval of the mean with α=0.05. (F) Relative Rec8-GFP fluorescence intensity after shift to 37°C. Rec8-GFP fluorescence for each strain is normalized to its value at the time of temperature shift.

Rad21 stabilization occurs independent of DNA replication. (A) Wt and ssl3–29 cells bearing rad21-HA were arrested in early S-phase by hydroxyurea (HU) treatment at 25°C and then shifted to 37°C for 3 h in the presence of HU. Rad21-HA binding to chromosomes was assessed by ChIP and hybridization of the chromatin immunoprecipitate to oligonucleotide tiling arrays covering chromosomes 2 and 3. The data sets from the two strains were merged to facilitate comparison. Intergenic regions containing replication origins known to fire in HU (Heichinger et al, 2006) are depicted as black boxes. Four regions of chromosome 2 are shown as an example. (B) Determination of the replication status of selected loci in the HU arrest. Cells were arrested in G1 by nitrogen starvation and released into the cell cycle at 25°C in the presence of HU and BrdU. After 2 h at 37°C (HU37), HU was washed out and cells were allowed to resume replication for 1 h in the presence of BrdU (REL37). Replicated DNA was immunoprecipitated using anti-BrdU antibodies and quantified by real-time PCR. The extent of DNA replication in the HU arrest at 37°C is given by the ratio HU37/REL37.

The contribution of Eso1 to cohesin stabilization on chromosomes. (A) Eso1 is not required for sustained Rad21 binding to chromatin in HU-arrested cells. HU was added to cycling cells at 25°C and the culture was immediately shifted to 32°C to induce the early S-phase arrest at 32°C, a restrictive temperature for eso1-H17. Cells were then shifted to 37°C for 2 h to inactivate mis4–367 in the presence of HU. Rad21-HA association with chromatin was monitored on chromosome spreads by immunofluorescence before and after the shift to 37°C. Rad21-HA fluorescence was measured in 50–100 nuclei. The error bars show the confidence interval of the mean with α=0.05. DNA content analysis and septation index (%) show that cells remained arrested in early S-phase throughout the experiment. (B) Cohesin stability on chromosomes is compromised after passage through an unperturbed S-phase without Eso1. Cells were arrested in G1 by nitrogen starvation (25°C−N) and released to pass through S-phase at 32°C, a restrictive temperature for eso1-H17. At 3.75 h after release, when cells had completed S-phase (32°C+N), the temperature was raised to 37°C for 1.5 h to inactivate Mis4 and probe the stability of cohesin on chromosomes in G2 (37°C+N). Samples were analyzed before and after temperature shift to 37°C as in (A). Scale bar, 5 μm. The quantification is based on two independent experiments with the error bars indicating the s.d. of the means. The fluorescence for each sample is normalized to the value of the mis4–367 sample before the 37°C shift.