Condensin positioning at telomeres by shelterin proteins drives sister-telomere disjunction in anaphase
- CNRS - Laboratory of Biology and Modelling of the Cell, France
- ENS de Lyon, Université Lyon, site Jacques Monod, France
- MCD, Centre de Biologie Intégrative, Université de Toulouse, CNRS, UPS, France
- CNRS, INSERM, Aix Marseille Université, Institut Paoli-Calmettes, CRCM, Equipe labellisée par la Ligue Nationale contre le Cancer, France
- CBI, MCD-UMR5077, CNRS, Chromatin Dynamics Team, France
Figures
Figure 1 with 1 supplement
Fission yeast condensin is enriched at telomeres during metaphase and anaphase.
(A) The telomere and sub-telomere of the right arm of chromosome 2 (Tel2R) as an example of chromosome end sequence in fission yeast. Sub-telomeric elements (STE), the heterochromatic gene tlh2, the domain bound by Taz1 (orange) (Kanoh et al., 2005) and primers for ChIP-qPCR (blue arrows) are shown. (B) Cnd2-GFP ChIP-qPCR from cells synchronized at G2/M (time post-release 0 min) and upon their release in mitosis (time post-release 7 min and 15 min). Left panel: Cell cycle stages determined by scoring the accumulation of Cnd2-GFP in the nucleus (metaphase) and by DAPI staining (anaphase). Right panel: ChIP-qPCR results, cdc22, exg1, and gly05 loci, being high or low condensin binding sites which are used as controls. Shown are the averages and standard deviations (SD) from three independent biological and technical replicates. (C–D) Cnd2-GFP calibrated ChIP-sequencing (ChIP-seq) in metaphase arrests at 36°C. (C) Left panel: Mitotic indexes of the two independent biological and technical replicates used. Right panel: Genome browser views of replicate #1. The second is shown in Figure 1—figure supplement 1. (D) Metagene profiles of all condensin binding sites along chromosome arms from replicates #1 and #2; TSS (transcription start site), TES (transcription end site). (E) Western blot showing Cnd2-GFP steady-state level in indicated cells arrested in metaphase for 3 hr at 36°C. Tubulin (Tub.) serves as loading control. Statistical analysis was performed using Mann-Whitney non-parametric test with p<0.001 considered significant.
-
Figure 1—source data 1
Raw data of Figure 1E.
Raw data of the western blot shown in Figure 1E.
- https://cdn.elifesciences.org/articles/89812/elife-89812-fig1-data1-v1.zip
Figure 1—figure supplement 1
Fission yeast condensin is enriched at telomeres during metaphase and anaphase.
(A) Telomeric plasmid pNSU70 (Sugawara, 1988) aligned against the S. pombe genome (ASM294v2) using Blast shows a best hit with cosmid SPBCT2R1 that corresponds to the right end of chromosome 2 (nucleotides 4500619–4539800 in the ASM294v2 genome). SPBCT2R1 was aligned with pNSU70 using Clustal Omega (Madeira et al., 2019) and the nucleotides 4137–7223 of pNSU70 were added to the sequence of chromosome 2 at position 4539800 to create a TEL2R-extended version of the genome, which is available under the accession number GSE196149. (B) Principle of the base per base normalization method applied to ChIPs to assess the occupancy of Cnd2-GFP from S. pombe (S.p.) at repeated DNA elements using chromatin from Saccharomyces cerevisiae (S.c.) cells expressing SMC3-GFP for internal calibration. (C) Results of the base per base normalization method applied to calibrated ChIP-sequencing (ChIP-seq) data to correct for biases in coverage in the IP and Total (T) fractions. Calibrated read counts obtained in the IP and Total (T) fractions, and their base per base ratios (IP/T) obtained at centromere 2 (cen 2), composed of a central core (cc) flanked by repetitive heterochromatic outer repeats (otr) and at rDNA repeats are shown. The single-copy gene exg1 serves as a non-repetitive control. (D) Calibrated read counts and base per base IP/T ratios obtained at TEL2R for two biological and technical replicates. Data from replicate #1 are also shown in Figure 1C. (E) Results from calibrated ChIP-seq against Cnd2-GFP in metaphase arrests WT and cut3-477 condensin mutant. Shown are the base per base ratios and metagene profiles obtained from two biological and technical replicates. Mitotic indexes of cell cultures were: WT (77% ± 6%), cut3-477 (70% ± 5%).
Figure 2 with 1 supplement
Condensin takes part in telomere disjunction during anaphase in a decatenation-independent manner.
(A) Left panel: WT or cut14-208 condensin mutant cells shifted to the restrictive temperature of 36°C for 3 hr were fixed with formaldehyde and directly imaged. Telomeres were visualized via Taz1-GFP (green), kinetochores/centromeres via Mis6-RFP (red), and spindle pole bodies (SPBs) via Cdc11-CFP (blue). Right panel: Number of telomeric foci according to the distance between SPBs at 36°C (n>90 cells for each strain). The data shown are from a single representative experiment out of three repeats. (B) Same procedure as in (A) applied to the cut3-477 condensin mutant. (C) Genomic DNA from the indicated strains cultured at 32°C was digested with ApaI and southern blotted using a telomeric probe (green), as represented by the gray bar. The relative gain or loss of telomeric DNA compare to WT is indicated. (D) Cells expressing Taz1-GFP and Cdc11-CFP, cultured at 25°C, were shifted to 19°C (restrictive temperature of top2-250) or 36°C (restrictive temperature of cut3-477), further incubated for 3 hr and fixed with formaldehyde. DNA was stained with DAPI, chromosome and telomere separation in anaphase (distance between the SPBs >5 µm) were assessed. Shown are averages and SD obtained from three independent experiments (n=100 cells for each condition). (E) Left panel: Live imaging of telomere separation according to the length of the mitotic spindle (distance between the SPBs) in top2-250 cells undergoing mitosis at 25°C or after a shift to the restrictive temperature of 19°C using fast microfluidic temperature control. Right panel: Number of telomeric foci according to the distance between SPBs at 25°C or 19°C in the top2-250 mutant. Shown is a representative experiment out of three replicates with n>70 cells, each. Statistical analysis was performed using Mann-Whitney non-parametric test with p<0.001 considered significant.
-
Figure 2—source data 1
Raw data of Figure 2C.
The first part of the gel from the left has been cut out for Figure 2C.
- https://cdn.elifesciences.org/articles/89812/elife-89812-fig2-data1-v1.zip
Figure 2—figure supplement 1
Condensin takes part in telomere disjunction during anaphase in a decatenation-independent manner.
(A) Left panel: Condensin mutant cells cut14-208 grown at the permissive temperature (25°C) or shifted to the restrictive temperature of 36°C for 3 hr were fixed with formaldehyde and stained with calcofluor to reveal the septum. Telomeres were visualized via Taz1-GFP (green), kinetochores/centromeres via the colocalization of Mis6-RFP (red) and Ndc80-GFP (green), and spindle pole bodies (SPBs) via Cdc11-CFP (blue). Right panel: Number of telomeric foci according to the distance between SPBs at 25°C and 36°C in the cut14-208 mutant (n>90 cells for each strain). The data shown are from a single representative experiment out of three repeats. (B) Left panel: WT or cut14-208 condensin mutant cells shifted to the restrictive temperature of 36°C for 3 hr were fixed with formaldehyde and directly imaged. Sister-telomeres 1L (Tel1-GFP, green), sister centromeres 3L (imr3-tdTomato, red), and the nucleolus (Gar1-CFP) were visualized. Right panel: Number of telomeric foci according to the distance between sister-centromeres 3L at 36°C (n>43 cells for each strain). The data shown are from a single representative experiment out of three repeats. (C) Kymograph representation of kinetochore dynamics in live top2-250 mutant cells at permissive temperature (25°C) or restrictive temperature (18°C). Kinetochores are visualized via Ndc80-GFP (green) and SPBs via Cdc11-CFP (red). Right panel: Quantification of the percentage of top2-250 anaphase cells showing lagging centromeres. Error bars indicate SD obtained from three independent experiments (n=100 for each condition). Statistical analysis was performed using Mann-Whitney non-parametric test with p<0.001 considered significant.
Figure 3 with 1 supplement
Condensin deficiency increases contact frequencies between telomeres in metaphase.
(A) Mitotic indexes of cell cultures used for Hi-C. (B) Hi-C contact probability matrix at 25 kb resolution of wild-type metaphase arrests at 33°C. Contacts between telomeres (arrows) and centromeres (circles) are indicated. (C) Median contact probabilities as a function of distance along chromosomes for wild-type and cut14-208 metaphases at 33°C. (D) Differential Hi-C contact map between wild-type and cut14-208. (E) Measurements of aggregated contact frequencies at high resolution (5 kb) over the ends of chromosomes in metaphase arrests at 33°C. Boxes indicate the median, first and third quartiles, whiskers the minimum and maximum, and notches represent the 95% confidence interval for each median. Data points are shown as gray circles. The significance in contact frequencies was confirmed statistically by Mann-Whitney-Wilcoxon test between cut14-208 and wild-type conditions.
Figure 3—figure supplement 1
Condensin deficiency increases contact frequencies between telomeres in metaphase.
Data from the biological and technical replicate of those shown in Figure 3. (A) Mitotic indexes of cell cultures used for Hi-C. (B) Hi-C contact probability matrix at 25 kb resolution of wild-type cells arrested in metaphase at 33°C. Contacts between telomeres (arrows) and centromeres (circles) are indicated. (C) Median contact probabilities as a function of distance along the chromosomes for wild-type and cut14-208 cells arrested in metaphase at 33°C. (D) Hi-C difference map at 25 kb resolution comparing cut14-208 and wild-type cells. (E) Aggregation of contact frequencies at chromosome ends in wild-type and cut14-208 mutant cells in metaphase at 33°C. Boxes indicate the median, first and third quartiles, whiskers extend to minimum and maximum values and notches represent the 95% confidence interval for each median. Data points are shown as gray circles.
Figure 4 with 1 supplement
Condensin enrichment at telomeres results from positive and negative interplays with telomeric factors.
(A) Cnd2-GFP calibrated ChIP-qPCR from cells arrested in metaphase at 30°C. Shown are averages and standard deviations (SD) of mitotic indexes and ChIP-qPCRs for three biological and technical replicates. cnt1 is the kinetochore domain of cen1, exg1, gas1, and cnd1 are high or low occupancy binding sites on chromosome arms. (B–C) Cnd2-GFP occupancy assessed at non-telomeric Taz1 islands (isl) in the same samples as in Figure 1C, Figure 1—figure supplement 1D, and A, respectively. (D–E) Cnd2-GFP calibrated ChIP-sequencing (ChIP-seq) in metaphase arrests. (D) Left panel: Mitotic indexes of the two independent biological and technical replicates used. Right panel: Genome browser views of replicate #1. The second replicate is shown in Figure 4. (E) Metagene profiles of all condensin binding sites along chromosome arms from replicates #1 and #2; TSS (transcription start site), TES (transcription end site). Statistical analysis was performed using Mann-Whitney non-parametric test with p<0.001 considered significant.
Figure 4—figure supplement 1
Taz1 specifically enriches condensin at telomeres.
(A) Cnd2-GFP calibrated ChIP-qPCR shown in Figure 1C were normalized with respect to their corresponding IP/T ratios of budding yeast SMC3-GFP at CEN4. cnt1 is the kinetochore domain of cen1 while exg1, gas1, and cnd1 loci are high or low occupancy binding sites on chromosome arms. Shown are the average and standard deviations from n=3 biological and technical replicates. (B) Cnd2-GFP occupancy at non-telomeric Taz1 islands in wild-type or cut14-208 mutant cells arrested in metaphase, as determined by calibrated ChIP-sequencing (ChIP-seq) (n=2 biological and technical replicates). (C) Summary of the ChIP-seq results obtained at several representative Taz1 islands (Zofall et al., 2016). (D) Cnd2-GFP calibrated ChIP-qPCR from indicated metaphase arrests. Left panel: Mitotic indexes of cell cultures used for ChIP. Right panel: Results of calibrated ChIP-qPCR. Shown are averages and SD from six independent biological and technical replicates. (E) Left panel: Genome browser views of Cnd2-GFP calibrated ChIP-seq in indicated metaphase arrests, with two independent biological and technical replicates (#). Replicate #1 is shown in Figure 4. Right panel: Metagene profile of all condensin binding sites along chromosome arms from replicate #2. TSS (transcription start site), TES (transcription end site). Statistical analysis was performed using Mann-Whitney non-parametric test with p<0.001 considered significant.
Figure 5
Condensin level at telomeres is a limiting parameter for their disjunction during anaphase.
(A) Cells were grown at 25°C or shifted to 32°C for 3 hr, fixed with formaldehyde and stained with DAPI to reveal DNA. Left panel: Example of anaphase cells showing chromatin bridges and non-disjoined telomeres in late anaphase in the cut3-477 taz1∆ double mutant. Right panel: Telomere non-disjunction events were scored in anaphase cells. Shown are averages and standard deviation from three independent biological and technical replicates with n=100 cells, each. (B) Cells of indicated genotypes were serially diluted 1/5 and spotted onto YES plates at indicated temperatures for 7 (18°C), 3 (25°C), and 2 (32°C and 34°C) days. (C) Left panel: cut3-477 or cut3-477 mit1∆ mutant cells shifted to the restrictive temperature of 36°C for 3 hr were fixed with formaldehyde and directly imaged. Telomeres were visualized via Taz1-GFP (green) and spindle pole bodies (SPBs) via Cdc11-CFP (blue). Right panel: Number of telomeric foci according to the distance between SPBs at 36°C (n>90 cells for each strain). The data shown are from a single representative experiment out of three repeats.Statistical analysis was performed using Mann-Whitney non-parametric test with p<0.001 considered significant.
Figure 6 with 1 supplement
Condensin counteracts cohesin at telomeres.
(A) Top panel: WT, rad21-K1 or rad21-K1 cut3-477 cells were shifted to the restrictive temperature of 36°C for 3 hr, fixed with formaldehyde and directly imaged. Telomeres were visualized via Taz1-GFP (green) and spindle pole bodies (SPBs) via Cdc11-CFP (blue). Lower panels: Number of telomeric foci according to the distance between SPBs at 36°C (n>90 cells for each strain). The data shown are from a single representative experiment out of three repeats. (B) Psm3-GFP calibrated ChIP-qPCR from cells synchronized in G2/M and shifted at 36°C to inactivate condensin during the G2 block (time 0 min) and upon their release in anaphase (time 30 min). Left panel: Cell cycle stages determined by DAPI staining. Right panel: ChIP-qPCR results. cendh1, kgd1, and lvs1 are cohesin binding sites, while exg1, gas1, and rRNA37 are condensin binding sites. Percentage of IP with Psm3-GFP have been normalized using S. cerevisiae (S.c.) CEN4 locus. Shown are the averages and standard deviations from three independent biological and technical replicates. Statistical analysis was performed using Mann-Whitney non-parametric test with p<0.001 considered significant.
Figure 6—figure supplement 1
Condensin counteracts cohesin at telomeres.
(A) cut3-477 and cut3-477 rad21-K1 mutants were grown at permissive temperature and small, early G2 cells were purified using a lactose gradient. After synchronization, the entire cell population was in G2 (0% of cells in mitosis or cytokinesis). Purified early G2 cells were shifted to the restrictive temperature of 36°C and telomeric foci were scored according to the distance between spindle pole bodies (SPBs) (n>80 cells for each strain). The data shown are from a single representative experiment out of three repeats. (B) Psm3-GFP calibrated ChIP-qPCR from cells synchronized at G2/M (time 0 min; top panel) and upon their release in anaphase (time 30 min; bottom panel). cendh1, kgd1, and lvs1 loci are cohesin binding sites, while exg1, gas1, and rRNA37 loci are condensin binding sites. Percentage of IP with Psm3-GFP has been normalized using S. cerevisiae (S.c.) CARIV locus. Shown are the averages and standard deviations (SD) from three independent biological and technical replicates. Statistical analysis was performed using Mann-Whitney non-parametric test with p<0.001 considered significant.
Figure 7
Model for condensin-driven sister-telomere disjunction.
Loop extruding condensin accumulates against a barrier formed by arrays of Taz1 proteins bound to telomeric repeats, allowing condensin-mediated DNA translocation to pass a threshold beyond which the ties between sister-telomeres such as cohesin would be eliminated. See Discussion for details.
Additional files
-
Supplementary file 1
Strain list used in this study.
The strain number, genotype, and figures corresponding to the use of these strains are indicated.
- https://cdn.elifesciences.org/articles/89812/elife-89812-supp1-v1.docx
-
Supplementary file 2
Primers used for qPCR.
The forward and reverse primers used in this study are indicated.
- https://cdn.elifesciences.org/articles/89812/elife-89812-supp3-v1.docx
-
Supplementary file 3
Antibodies used in this study.
- https://cdn.elifesciences.org/articles/89812/elife-89812-supp6-v1.docx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/89812/elife-89812-mdarchecklist1-v1.pdf
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Condensin positioning at telomeres by shelterin proteins drives sister-telomere disjunction in anaphase
eLife 12:RP89812.
https://doi.org/10.7554/eLife.89812.3
