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. 2012 Dec 18;1:e00117. doi: 10.7554/eLife.00117

Figure 4. CLB3 misexpression disrupts protection of centromeric cohesin.

Cyclin expression was induced after 2 hr 15 min (C) and (D), 2 hr 30 min (A), (B), (E), (F) and (H) or 3 hr (G) and (I) of sporulation. (A) Chromosomal association of Rec8-13myc was monitored by ChIP-chip in wild-type (A28716) and CUP-CLB3 (A28718) during prophase I arrest. Centromere position is identified by a black circle. (B) Centromeric Rec8 localization was monitored in spread nuclei from wild-type (A28684), CUP-CLB3 (A28685) and CUP-CLB4 (A28686) cells carrying REC8-3HA (red) and NDC10-13myc (green) (n > 40). The fraction of spread nuclei that were Rec8 positive or negative was compared to wild-type using a chi-square test (df 1): CUP-CLB4, χ2 = 0.001323, p=0.9710; CUP-CLB3, χ2 = 32.79, p<0.0001. (C) Rec8 cleavage monitored by Western blot after release from an NDT80 block (4 hr 30 min) in wild-type and CUP-CLB3 carrying both a myc-tagged REC8 allele as well as either HA-tagged REC8 or rec8-29A allele (left to right: A29957, A29959, A29961, A29963). (D) Percentage of cells with short bipolar spindles was determined at indicated times in wild-type (A22804), CUP-CLB3 (A29965), rec8-29A (A22803) and CUP-CLB3 rec8-29A (A29967) after release from an NDT80 block (4 hr 30 min) (n = 100 per time point). (E) ChIP analysis for total Rec8, p-S179 Rec8 or p-S521 Rec8 from metaphase I-arrested (cdc20-mn) wild-type (A28681), CUP-CLB3 (A28682) and Sgo1-depleted (sgo1-mn; A29994) cells. Relative occupancy at a chromosome arm site (c194) or at a centromeric site (CENV) was determined relative to a low binding region (c281). Error bars represent range (n = 2). (F) Chromosomal association of Sgo1-3V5 was monitored by ChIP-chip in wild-type (A29795) and CUP-CLB3 (A29799) cells during prophase I-arrest. Centromere position is identified by a black circle. (G), (H) Localization of Sgo1-9myc (G, green) or Rts1-13myc (H, green) relative to Ndc10-6HA (red) determined by nuclear spreads in (G) wild-type (A22868) and CUP-CLB3 (A22870) or (H) wild-type (A28329) and CUP-CLB3 (A28330) during prophase I (n > 40). For (G), the fraction of spread nuclei that display colocalized or mislocalized Sgo1 relative to Ndc10 was compared between wild-type and CUP-CLB3 using a chi-square test (df 1) χ2 = 1.554, p=0.2125. For (H), the fraction of spread nuclei that display colocalized, partial or mislocalized Rts1 relative to Ndc10 was compared between wild-type and CUP-CLB3 using a chi-square test (df 2) χ2 = 3.712, p=0.1563. (I) Localization of Sgo1-9myc (green) in binucleates relative to Ndc10-6HA (red) determined by nuclear spreads from wild-type (A22868) and CUP-CLB3 (A22870) (n > 40). The fraction of spread nuclei that were Sgo1 positive or negative was compared between wild-type and CUP-CLB3 using a chi-square test (df 1) χ2 = 23.92, p<0.0001.

DOI: http://dx.doi.org/10.7554/eLife.00117.017

Figure 4.

Figure 4—figure supplement 1. Chromosomal association of Rec8 in CUP-CLB3 cells.

Figure 4—figure supplement 1.

Wild-type (A26547) and CUP-CLB3 (A26548) cells were induced to sporulate and CuSO4 (50 μM) was added 3 hr after transfer into sporulation medium. Rec8-3HA localization (red) was determined in spread nuclei from prophase I cells. DNA is shown in blue.
DOI: http://dx.doi.org/10.7554/eLife.00117.018
Figure 4—figure supplement 2. Chromosomal association of Pds5 in CUP-CLB3 cells.

Figure 4—figure supplement 2.

Wild-type (A28681) and CUP-CLB3 (A28682) cells carrying the cdc20-mn allele were induced to sporulate and CuSO4 (50 μM) was added 2 hr 30 min after transfer into sporulation medium. Pds5 localization was determined by ChIP-chip from metaphase I-arrested cells. Black balls depict centromere positions.
DOI: http://dx.doi.org/10.7554/eLife.00117.019
Figure 4—figure supplement 3. CUP-CLB3 cells partially bypass the nuclear division delay of mam1∆ cells.

Figure 4—figure supplement 3.

Wild-type (A22678), CUP-CLB3 (A22702), mam1∆ (A31340) and mam1∆ CUP-CLB3 (A31342) cells carrying the GAL4-ER and GAL-NDT80 fusions were induced to sporulate and CuSO4 (50 μM) was added 2 hr 15 min after transfer into sporulation medium. Cells were released from the NDT80 block 4 hr 30 min after transfer into sporulation medium. The percentage of cells that had undergone one or two meiotic divisions was determined at the indicated time points (n = 100 per time point).
DOI: http://dx.doi.org/10.7554/eLife.00117.020
Figure 4—figure supplement 4. Meiotic progression of the cells analyzed for Rec8 cleavage in Figure 4C.

Figure 4—figure supplement 4.

REC8-myc/REC8-HA (A29957), REC8-myc/rec8-29A-HA (A29961), REC8-myc/REC8-HA CUP-CLB3 (A29959) and REC8-myc/rec8-29A-HA CUP-CLB3 (A29963) cells carrying the GAL4-ER and GAL-NDT80 fusions were induced to sporulate and CuSO4 (50 μM) was added 2 hr 15 min after transfer into sporulation medium. Cells were released from the NDT80 block 4 hr 30 min after transfer into sporulation medium. The percentage of cells in metaphase I (grey symbols), anaphase I (violet symbols), metaphase II (dark blue symbols) and anaphase II (green symbols) was determined at the indicated times (n = 100 per time point).
DOI: http://dx.doi.org/10.7554/eLife.00117.021
Figure 4—figure supplement 5. Analysis of Rec8 cleavage in cells used for Figure 4D.

Figure 4—figure supplement 5.

Wild-type (A22804), CUP-CLB3 (A29965), rec8-29A (A22803) and rec8-29A CUP-CLB3 (A29967) cells carrying the GAL4-ER and GAL-NDT80 fusions were induced to sporulate and CuSO4 (50 μM) was added 2 hr 15 min after transfer into sporulation medium. Cells were released from the NDT80 block 4 hr 30 min after transfer into sporulation medium. Levels of full-length Rec8, cleaved Rec8, Clb3 and Pgk1 were monitored by Western blot.
DOI: http://dx.doi.org/10.7554/eLife.00117.022
Figure 4—figure supplement 6. Meiotic progression of the cells analyzed for Rec8 cleavage in Figure 4D.

Figure 4—figure supplement 6.

Wild-type (A22804), CUP-CLB3 (A29965), rec8-29A (A22803) and rec8-29A CUP-CLB3 (A29967) cells carrying the GAL4-ER and GAL-NDT80 fusions were induced to sporulate and CuSO4 (50 μM) was added 2 hr 15 min after transfer into sporulation medium. Cells were released from the NDT80 block 4 hr 30 min after transfer into sporulation medium. The percentage of cells in metaphase I (grey symbols), anaphase I (violet symbols), metaphase II (dark blue symbols) and anaphase II (green symbols) was determined at the indicated times (n = 100 per time point).
DOI: http://dx.doi.org/10.7554/eLife.00117.023
Figure 4—figure supplement 7. Chromosomal association of Sgo1 in CUP-CLB3 cells.

Figure 4—figure supplement 7.

Wild-type (A28712) and CUP-CLB3 (A28713) cells carrying the cdc20-mn allele were induced to sporulate and CuSO4 (50 μM) was added 2 hr 30 min after transfer into sporulation medium. Sgo1-3V5 localization was determined by ChIP-chip, 7 hr after transfer into sporulation medium when cells were arrested in metaphase I. Arm peaks for Sgo1 correspond to cohesin-associated regions. The basis for Sgo1 enrichment at these sites is currently unclear. Black balls depict centromere positions.
DOI: http://dx.doi.org/10.7554/eLife.00117.024
Figure 4—figure supplement 8. Localization of Rts1 in CUP-CLB3 cells.

Figure 4—figure supplement 8.

Wild-type (A28331) and CUP-CLB3 (A28332) cells carrying the cdc20-mn allele were induced to sporulate and CuSO4 (50 μM) was added 2 hr 30 min after transfer into sporulation medium. Rts1-13myc (green) localization relative to Ndc10-6HA (red) was determined in spread nuclei from metaphase I-arrested cells (n > 40).
DOI: http://dx.doi.org/10.7554/eLife.00117.025
Figure 4—figure supplement 9. Chromosomal association of Spo13 in CUP-CLB3 cells.

Figure 4—figure supplement 9.

(Left panel) wild-type (A30856), CUP-CLB3 (A30858) and CUP-CLB4 (A30860) cells carrying the GAL4-ER and GAL-NDT80 fusions were induced to sporulate and CuSO4 (50 μM) was added 2 hr 15 min after transfer into sporulation medium. Spo13-3V5 localization was determined by ChIP from prophase I-arrested cells. Relative occupancy at a centromeric site (CEN5) relative to a low binding region (HMR) was determined. Error bars represent the range (n = 2). (Right panel) wild-type (A30743), CUP-CLB3 (A30745) and CUP-CLB4 (A30747) cells carrying the cdc20-mn allele were induced to sporulate and CuSO4 (50 μM) was added 2 hr 30 min after transfer into sporulation medium. Spo13-3V5 localization was determined by ChIP 7 hr after transfer into sporulation medium when cells were arrested in metaphase I. Relative occupancy at a centromeric site (CENV) relative to a low binding region (HMR) was determined. Error bars represent the range (n = 2).
DOI: http://dx.doi.org/10.7554/eLife.00117.026
Figure 4—figure supplement 10. Rts1 localization in binucleate CUP-CLB3 cells.

Figure 4—figure supplement 10.

Wild-type (A28329) and CUP-CLB3 (A28330) cells were induced to sporulate and CuSO4 (50 μM) was added 2 hr 30 min after transfer into sporulation medium. Rts1-13myc (green) localization relative to Ndc10-6HA (red) was determined in spread nuclei from binucleates (n > 40). Using a chi-square test (df 2) the fraction of spread nuclei that display strong, weak or negative Rts1 with respect to Ndc10 was compared between wild-type and CUP-CLB3 χ2 = 54.49, p<0.0001.
DOI: http://dx.doi.org/10.7554/eLife.00117.027
Figure 4—figure supplement 11. Analysis of Rts1 localization in Rec8 phosphomimetic mutants.

Figure 4—figure supplement 11.

Wild-type (A29645) and rec8-S136D S179D S197D T209D (A29647) cells were induced to sporulate and Rec8-3HA/rec8-4D-3HA or Rts1-3V5 localization relative to Ndc10-13myc was determined in spread nuclei from binucleates (n > 40). Characterization of rec8-S136D S179D S197D T209D has been described in Katis et al. (2010). Note that strains carrying this allele fail to maintain centromeric cohesin beyond metaphase I (bottom panel). These binucleates also have weak Rts1 staining (top panel), suggesting that Rts1 maintenance at centromeric regions in anaphase I depends on cohesin. For top panel, using a chi-square test (df 2) the fraction of spread nuclei that display strong, weak or negative Rts1 with respect to Ndc10 was compared between wild-type and CUP-CLB3 χ2 = 18.02, p=0.0001. For bottom panel, using a chi-square test (df 2) the fraction of spread nuclei that display strong, weak or negative Rec8 with respect to Ndc10 was compared between wild-type and CUP-CLB3 χ2 = 121.2, p<0.0001.
DOI: http://dx.doi.org/10.7554/eLife.00117.028