Establishment and maintenance of sister chromatid cohesion in fission yeast by a unique mechanism - PubMed (original) (raw)

Establishment and maintenance of sister chromatid cohesion in fission yeast by a unique mechanism

K Tanaka et al. EMBO J. 2001.

Abstract

During S phase, chromatid cohesion is established only between nascent sisters and with faithful pairing along their entire region, but how this is ensured is unknown. Here we report that sister chromatid cohesion is formed and maintained by a unique mechanism. In fission yeast, Eso1p, functioning in close coupling to DNA replication, establishes sister chromatid cohesion whereas the newly identified Cohesin-associated protein Pds5p hinders the establishment of cohesion until counteracted by Eso1p, yet stabilizes cohesion once it is established. Eso1p interacts physically with Pds5p via its Ctf7p/Eco1p-homologous domain.

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Figures

Fig. 1. Isolation of the pds5+ gene. (A) Suppression of _eso1_ts mutant by the pds5+ gene. The _eso1_ts cells stably transfected with the indicated plasmids were incubated on MM plates at the indicated temperatures. pAL-pds5+ contains an insert of the initially isolated 6.5 kb _Bgl_II fragment. pAL-X is the pALSK+ vector with no insert and used as a negative control. (B) Restriction map of the pds5+ gene. The white and black arrows indicate the direction and extent of the pds5+ ORF and pds5+ cDNA, respectively. The structures of the _Pst_I–_Xba_I (Δpds5-1) and the _Ban_III–_Ban_III (Δpds5-2) fragments used for the disruption of pds5+ gene are also shown. The pds5+ sequence data have been submitted to the DDBJ/EMBL/GenBank databases under accessionNo. AB067651.

Fig. 2. Δpds5 cells lose colony-forming ability during G2 arrest. (A) Δpds5 cells lose colony-forming ability (referred to as viability) after entry into stationary phase. Logarithmically proliferating wild-type, Δpds5-1 and Δpds5-1 + pds5+ (Δpds5-1 with pds5+ integration) cells were cultured to confluence in MM+YE. Cell aliquots were taken at the times indicated and determined for their cell number (left) and viability (right). (B and C) Δpds5 cells lose viability during stationary phase entry only from G2. Wild-type and Δpds5-1 cells were grown to mid-log phase in MM and inoculated in plain MM (MM), MM-C (–C), MM-S (–S), MM-N (–N) or MM+YE (+YE) at a density of 2 × 106 cells/ml. Both wild-type and Δpds5 cells reached confluence within 15 h. After 55 h incubation, cell aliquots were taken and determined for the cell cycle profile (B) and viability (C). (D) Δpds5 cells also lose viability during cell cycle arrest at G2. Wild-type and Δpds5-1 cells (left), _cdc25_ts and _cdc25_ts Δpds5-1 cells (second), _cdc10_ts and _cdc10_ts Δpds5-1 cells (third), or _cdc22_ts and _cdc22_ts Δpds5-1 cells (right) were grown at 25°C to mid-log phase in MM and then cultured at 36°C. Cells were sampled at indicated times and determined for viability.

Fig. 3. Sister chromatids prematurely separate in G2-arrested Δpds5 cells. (A) G2-arrested Δpds5 cells are defective in chromosome segregation upon re-entry in the cell cycle. Δpds5-1 cells were inoculated in MM+YE at a density of 2 × 106 cells/ml and cultured for 70 h to be allowed to enter stationary phase from G2. Cells were then stimulated to re-enter the cell cycle by re-inoculation into fresh YE medium. Cell aliquots were fixed at indicated times and stained with DAPI. Bar, 10 µm. (B) Sister chromatids are separated in G2-arrested Δpds5 cells. cen1–GFP (referred to as wild-type) and Δpds5-1 cen1–GFP (referred to as Δpds5-1) cells were inoculated in MM-S at a density of 2 × 106 cells/ml. Cells divided once to twice in this medium and mostly arrested in G2 within 5 h. After 20 h incubation, cells were examined for the presence of two split cen1–GFP signals under the fluorescence microscope. Bar, 10 µm. (C) Sister chromatids prematurely separate during G2 arrest induced by Cdc25p inactivation. _cdc25_ts cen1–GFP (referred to as _cdc25_ts) and _cdc25_ts Δpds5-1 cen1–GFP (referred to as _cdc25_ts Δpds5-1) cells were grown to mid-log phase in MM at 25°C and then shifted to 36°C, with sampling cells at indicated times to determine cell viability (left) and the presence of two split cen1–GFP signals (right). For the cen1–GFP examination, cells were fixed with methanol at –80°C. (D) Cut2p is present in G2-arrested Δpds5 cells. cut2HA+ (referred to as wild type) and cut2HA+ Δpds5-1 (referred to as Δpds5-1) cells were inoculated in MM+YE and cultured. Cells were taken at the times indicated and determined for the level of Cut2p, cell number and viability. Cdc2 and Pef1 kinases (Tanaka and Okayama, 2000) detected by anti-PSTAIR mAb were loading controls.

Fig. 4. Pds5p is closely associated with Cohesin. (A) Pds5p is localized as punctate stains in the nucleus throughout the cell cycle. pds5GFP+ cells were grown to mid-log phase in MM and directly stained with HOE to visualize DNA. Bar, 10 µm. (B) Pds5p is constitutively present throughout the cell cycle. The _cdc25_ts pds5Myc+ cells were synchronized at late G2 by temperature shift-up and shift-down. Their cell cycle progression was monitored for two generations. Cells were taken at indicated times, and determined for the septation index and the level of Pds5p. α-tubulin was used as a loading control. (C) Punctate Pds5p signals are seen on M phase chromosomes, but not on those at the metaphase-to-anaphase transition. nda3-KM311 pds5GFP+ cells were grown to mid-log phase in MM at 34°C, and then shifted to the non-permissive temperature of 18°C for 8 h to induce prometaphase arrest. Cells were then released from the arrest by a shift back to the permissive temperature and directly stained with HOE to visualize DNA. Images of four independent cells corresponding to metaphase, early anaphase, late anaphase and telophase are shown. Bar, 10 µm. (D) Pds5p colocalizes with Rad21p. Pds5p and Rad21p were detected by indirect immunofluorescence analysis. DNA was stained with DAPI (referred to as DNA). The Pds5p and Rad21p images were merged in the bottom panel (referred to as Merge). Images of two independent cells are shown. Bar, 5 µm. (E) Association of Pds5p with the centromere regions. Exponentially growing rad21GFP+ and pds5GFP+ cells in MM were subjected to CHIP analysis. Representative PCRs of whole-cell extracts (WCE) and immunoprecipitates (IP) are shown. The percentage of immunoprecipitation (IP%) is indicated on the bottom graph. Schematic representation of chromosome I and the primers used are shown at the top. (F) Punctate nuclear localization of Pds5p depends on intact Rad21p. _rad21_ts pds5GFP+ and _eso1_ts pds5GFP+ cells were grown to mid-log phase in MM at 25°C, then cultured at 34°C for 3.5 h and examined by fluorescent microscopy followed by direct staining with HOE to visualize DNA. Bar, 10 µm. (G) Punctate nuclear localization of Rad21p is independent of Pds5p. pds5GFP+ (referred to as wild type) and _rad21_ts pds5GFP+ (referred to as _rad21_ts) cells, or rad21GFP+ (referred to as wild type) and Δpds5-1 rad21GFP+ (referred to as Δpds5-1) cells were grown to mid-log phase in MM at 25°C and directly examined by fluorescent microscopy. Bar, 10 µm. (H) Chromatin localization of Pds5p depends on intact Rad21p. Negative control without GFP tagging gene (referred to as no tag), pds5GFP+ (referred to as wild type), _rad21_ts pds5GFP+ (referred to as _rad21_ts) and _eso1_ts pds5GFP+ (referred to as _eso1_ts) cells grown at 25°C or cultured at 34°C for 3.5 h were subjected to CHIP analysis. (I) Chromatin localization of Rad21p is independent of Pds5p. Negative control without GFP tagging gene (referred to as no tag), rad21GFP+ (referred to as wild type) and Δpds5-1 rad21GFP+ (referred to as Δpds5-1) cells grown at 25°C were subjected to CHIP analysis. (J) Pds5p associates with Rad21p. Extracts of pds5Myc+ rad21HA+ and pds5Myc+cells were immunoprecipitated with an anti-HA antibody, and the original cell extracts and precipitates (from 15 times more extracts) were analyzed by western blotting. Pds5p and Rad21p were detected with anti-Myc and anti-HA antibodies, respectively. (K) Cells lacking Pds5p are synthetically lethal with the _rad21_ts mutation. The _rad21_ts, _rad21_ts _eso1_ts and _rad21_ts Δpds5-1 cells transfected with pREP81-rad21+ were inoculated on both an MM plate (–thiamine) and a plate containing 10 µM thiamine (+thiamine) and incubated for 6 days at 23°C. The _rad21_ts _eso1_ts cells are a positive control for synthetic lethality (K.Tanaka et al., 2000).

Fig. 5. Eso1p is dispensable for cohesion establishment in the absence of Pds5p. (A) Deletion of pds5+ suppresses the lethality of Δeso1 cells. Spores from Δeso1/eso1+ pds5+/pds5+ diploid cells (left) or Δeso1/eso1+ Δpds5-1/pds5+ diploid cells (right) were tetrad-dissected on YE plates and incubated at 30°C for 4 days. (B) Deletion of pds5+ allows Δeso1 cells to grow at 36°C as well as fully suppressing the temperature-sensitive lethality of the _eso1_ts cells. Approximately 104, 103, 102 and 10 wild-type, Δpds5-1, _eso1_ts, _Δpds5-1 eso1_ts, _Δpds5-1 eso1_ts + pds5+ (_Δpds5-1 eso1_ts integrated with the pds5+ gene), Δpds5-1 Δeso1, Δpds5-2, _Δpds5-2 eso1_ts and Δpds5-2 Δeso1 cells were spotted on two YE plates and incubated at 25 and 36°C, respectively. The _Δpds5-1 eso1_ts + pds5+ strain generated a few colonies that could grow even at 36°C. They were ‘pop out’ revertants in which the integrated pds5+ gene was spontaneously lost from chromosome by homologous recombination. (C) Δpds5 Δeso1 cells are phenotypically indistinguishable from Δpds5 cells. Logarithmically proliferating wild-type, Δpds5-1 and Δpds5-1 Δeso1 cells were cultured in MM+YE. Cells were taken at the times indicated and determined for cell number (left) and viability (right). (D) Δpds5 Δeso1 cells perform proper chromosome segregation. Wild-type, Δpds5-1 and Δpds5-1 Δeso1 cells were grown to mid-log phase in YE medium, fixed and stained with DAPI. Bar, 10 µm. (E) Sister chromatid cohesion is established in Δpds5 Δeso1 cells. cen1–GFP (referred to as wild type), _eso1_ts cen1–GFP (referred to as _eso1_ts), Δpds5-1 cen1–GFP (referred to as Δpds5-1) and Δpds5-1 Δeso1 cen1–GFP (referred to as Δpds5-1 Δeso1) cells were grown to mid-log phase at 25°C in MM and examined directly for the presence of two split cen1–GFP signals. The _eso1_ts cen1–GFP cells cultured at 34°C for 3 h were used as a control for unestablished sister chromatid cohesion.

Fig. 6. Δpds5 cells are sensitive to DNA damage. (A) Δpds5 cells are sensitive to MMS but not to bleomycin. Approximately 104, 103, 102 and 10 _mis4_ts, _rad21_ts, wild-type, _eso1_ts, Δpds5-1 and Δpds5-1 Δeso1 cells were spotted on YE plates containing none (referred to as control), bleomycin [0.000025% (w/v)] or MMS [0.004% (v/v)] and incubated at 27°C for 3 days (control) or 4 days (others). (B) Δpds5 cells are sensitive to UV irradiation. Wild-type, _eso1_ts, Δpds5-1, _Δpds5-1 eso1_ts and Δpds5-1 Δeso1 cells were plated on YE, irradiated with various doses of UV and incubated at 27°C for 7 days.

Fig. 7. Physical interaction between Eso1p and Pds5p. (A) Schematic representation of eso1+ genes used in this experiment. (B) Two independent yeast transformants containing the indicated two-hybrid plasmids were grown on the nitrocellulose filters at 30°C and their β-galactosidase activities were assayed. Filters were incubated with X-gal for 2 h (left) or overnight (right) at 30°C. Blue color indicates interaction. X is a vector plasmid with no insert. The pair p53 (pVA3) and SV40 large T-antigen (pTD1) represents a standard positive control. (C) Yeast strains containing the indicated plasmids were grown at indicated temperatures and their β-galactosidase activities were assayed.

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Establishment and maintenance of sister chromatid cohesion in fission yeast by a unique mechanism - PubMed (original) (raw)