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. 2011 Apr;31(8):1771-86.
doi: 10.1128/MCB.01284-10. Epub 2011 Feb 7.

Psm3 acetylation on conserved lysine residues is dispensable for viability in fission yeast but contributes to Eso1-mediated sister chromatid cohesion by antagonizing Wpl1

Amélie Feytout  1 , Sabine Vaur, Sylvie Genier, Stéphanie Vazquez, Jean-Paul Javerzat
Affiliations

Psm3 acetylation on conserved lysine residues is dispensable for viability in fission yeast but contributes to Eso1-mediated sister chromatid cohesion by antagonizing Wpl1

Amélie Feytout et al. Mol Cell Biol. 2011 Apr.

Abstract

In budding yeast and humans, cohesion establishment during S phase requires the acetyltransferase Eco1/Esco1-2, which acetylates the cohesin subunit Smc3 on two conserved lysine residues. Whether Smc3 is the sole Eco1/Esco1-2 effector and how Smc3 acetylation promotes cohesion are unknown. In fission yeast (Schizosaccharomyces pombe), as in humans, cohesin binding to G(1) chromosomes is dynamic and the unloading reaction is stimulated by Wpl1 (human ortholog, Wapl). During S phase, a subpopulation of cohesin becomes stably bound to chromatin in an Eso1 (fission yeast Eco1/Esco1-2)-dependent manner. Cohesin stabilization occurs unevenly along chromosomes. Cohesin remains largely labile at the rDNA repeats but binds mostly in the stable mode to pericentromere regions. This pattern is largely unchanged in eso1Δ wpl1Δ cells, and cohesion is unaffected, indicating that the main Eso1 role is counteracting Wpl1. A mutant of Psm3 (fission yeast Smc3) that mimics its acetylated state renders cohesin less sensitive to Wpl1-dependent unloading and partially bypasses the Eso1 requirement but cannot generate the stable mode of cohesin binding in the absence of Eso1. Conversely, nonacetylatable Psm3 reduces the stable cohesin fraction and affects cohesion in a Wpl1-dependent manner, but cells are viable. We propose that Psm3 acetylation contributes to Eso1 counteracting of Wpl1 to secure stable cohesin interaction with postreplicative chromosomes but that it is not the sole molecular event by which this occurs.

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Figures

Fig. 1.
Fig. 1.
Characterization of the cohesin fraction stably bound to postreplicative chromosomes. (A to D) A fraction of Rad21 persists on G2 chromatin after inactivation of the cohesin-loading machinery. Cycling cells were shifted to 36.5°C to shut off cohesin loading (mis4-367) while keeping the cells in G2 (cdc25-22). Chromatin-bound Rad21-9PK was measured at the indicated time points by nuclear spreading and immunofluorescence using anti-PK antibodies. (A) Images of nuclear spreads showing chromatin-bound Rad21-9PK before (cycling) and 180 min after the temperature shift. Chromatin was counterstained with DAPI (4′,6-diamidino-2-phenylindole). The arrowheads point to the nucleoli, which are located in the area of the nucleus unstained by DAPI. Bar, 2.5 μm. (B) Rad21-9PK fluorescence intensity was measured for 50 to 100 nuclei for each sample. Error bars, 95% confidence intervals of the mean. a.u., arbitrary units. (C) Analysis of the cell cycle stage confirmed that most cells (>80%) were in the G2 phase before the temperature shift and remained in G2 throughout the experiment. The positions of cells within the cell cycle were analyzed by DNA content analysis. DAPI and calcofluor staining were used to score mononucleate, binucleate, and septated cells. (D) Kinetics of Rad21 dissociation from chromatin. The data presented are the means of 10 independent experiments. The error bars represent standard deviations (SD). wt, wild type. (E) Cell survival remained high during the course of the experiment. Cells were withdrawn at the indicated time points and plated at the permissive temperature to determine the number of viable cells. (F) The major foci of cohesin in the stable fraction colocalize with Swi6. Cells were treated as in panel A, and nuclear spreads were prepared 180 min after the temperature shift, at which time only the stable cohesin fraction is retained on chromatin. Bar, 2.5 μm. (G) The stable cohesin fraction is reduced in cells lacking Swi6. The Rad21-9PK fluorescence intensity was measured for 50 to 100 nuclei for each sample. Error bars, 95% confidence intervals of the mean. (H) Rad21 ChIP assay showing the distribution of the labile and stable cohesin fractions along the chromosomes. Cells were cultured as in panel A and processed for ChIP just after (15 min) and 180 min after the temperature shift. Rad21 enrichment was measured at centromeres (imr and dg), at two chromosome arm sites (CAR1806 and CAR1979), within the rDNA gene cluster (NTS and 28S), and at telomeres (Tel). Rad21 enrichment was calculated from duplicate samples. The error bars indicate SD.
Fig. 2.
Fig. 2.
Eso1 function is dispensable when the wpl1 gene is deleted. (A) Representative example of tetrad analysis showing that eso1Δ spores do not support colony formation, whereas eso1Δ wpl1Δ spores form wild-type-size colonies. (Left) Tetrads were dissected, and spores were deposited on complete medium. Colonies were replica plated onto appropriate media to follow the segregation of eso1 and wpl1 alleles. −URA, minimum medium lacking uracil; G418, YES medium plus G418; NS, nonselective YES medium. (B) cen2 FISH on metaphase-arrested cells. Cells were arrested at metaphase by the use of the thermosensitive cut9ts mutation (anaphase-promoting complex). Fixed cells were stained for tubulin and processed for FISH using a probe mapping close to the centromere of chromosome 2 (cen2 FISH). The image shows an example of a typical metaphase cell. The mitotic spindle (red) is <2.5 μm in length, and the two cen2 FISH signals (green) are closely apposed to each other. No major defect in sister centromere cohesion was observed in wpl1Δ and wpl1Δ eso1Δ cells as opposed to the thermosensitive rad21-K1 mutant. (C to E) The stable cohesin fraction is created and is functional in eso1Δ wpl1Δ cells. Strains were cultured and processed for nuclear spreading and ChIP as in Fig. 1. (C) Kinetics of Rad21 dissociation from chromosomes. Chromatin-bound Rad21-PK was quantified from nuclear spreads. The error bars represent SD from two independent experiments. (D) Cell survival remained high throughout the duration of the experiment. (E) Cell cycle staging shows that eso1Δ wpl1Δ cells were mostly (>80%) in the G2 phase before the temperature shift and remained arrested in G2 afterward. (F) ChIP assay comparing Rad21 binding to chromosomes in eso1Δ wpl1Δ versus eso1+ wpl1+ cells from 3 h to 5 h after cohesin loading was shut off. The error bars show the SD from two quantitative PCR (qPCR) quantifications.
Fig. 3.
Fig. 3.
Eso1-dependent Psm3K106 acetylation during the cell cycle. (A) The anti-Psm3K106Ac antibody reacts with Psm3 in a K106-dependent manner in total extracts (left) and GFP immunoprecipitates (middle). (Right) Psm3 detection by anti-Psm3K106Ac antibodies is dependent on Eso1, but not on Wpl1. (B and C) Psm3K106 acetylation during the cell cycle. (B) Cells bearing the thermosensitive mutation cdc10-129 were shifted to the restrictive temperature to induce G1 arrest. Samples were taken at regular time intervals after the temperature shift to monitor Psm3K106Ac on total protein extracts as the cells progressed from an asynchronous population (time zero) to a homogeneous G1 arrest. Flow cytometry analysis of the DNA content (right) showed that Psm3K106Ac started to decrease as the G1 peak appeared. The drift in the G1 peak to the right at later time points was due to an increase in the mitochondrial DNA content as the cells elongated (47). (C) Cells were arrested in G1 by nitrogen deprivation and released synchronously into the cell cycle. Psm3K106Ac probed from total protein extracts was weakest in cells with a G1 DNA content and began to rise at the time of DNA replication. Anti-Psm3 antibodies were used to adjust protein loading so that similar amounts of Psm3 were present in all samples.
Fig. 4.
Fig. 4.
Nonacetylatable mutants are viable but generate a cohesion defect. (A) Nonacetylatable psm3 mutants display a wild-type growth rate. Serial dilutions of cells were spotted on YES medium and incubated at the indicated temperatures. (B) cen2 FISH on metaphase-arrested cells revealed a wpl1-dependent defect in sister centromere cohesion in the psm3RR mutant. The cells were arrested at metaphase by the use of the thermosensitive cut9ts mutation (anaphase-promoting complex). (C to E) Tetrad analysis showing the segregation of psm3 nonacetylatable alleles. (Left) Tetrads were dissected, and spores were deposited on complete medium. Colonies were replica plated onto appropriate media to follow the segregation of the markers. −URA, miminum medium lacking uracil; G418, YES medium containing G418; NAT, YES medium containing nourseothricin. YES plates also contained the vital stain phloxin B as an indicator of cell lethality within the colony. Representative tetrads are shown. The green boxes show that the nonacetylatable psm3 alleles are neutral in an eso1Δ wpl1Δ background. The red boxes show that eso1Δ combined with a nonacetylatable psm3 allele does not form viable progeny. (F and G) Serial dilutions of cells were spotted on YES medium and incubated at the indicated temperatures. (F) Acetyl-mimicking psm3 mutants are viable and display a wild-type growth rate. (G) Acetyl-mimicking psm3 alleles can bypass the eso1 requirement. The comparison of colony growth rates shows the additive effect of the amino acid substitutions.
Fig. 5.
Fig. 5.
Effects of nonacetylatable Psm3 on stable cohesin binding to chromosomes and long-term cohesion. (A to D) Strains were cultured and processed for nuclear spreading and ChIP as in Fig. 1. (A) Kinetics of Rad21 dissociation from chromatin. Chromatin-bound Rad21-PK was quantified from nuclear spreads. The error bars represent SD from two independent experiments. (B) Cell survival during the course of the experiment. (C) Cell cycle staging of cells used for the ChIP assay before and after the temperature shift. (D) ChIP assay showing the amount of Rad21 bound to pericentromeric regions (imr and dg) just after (15 min) and 3 h after the temperature shift. Rad21 enrichment was calculated from duplicate samples. The error bars represent SD. (E to G) Nonacetylatable Psm3 does not affect long-term cohesion. (E) Cells were grown to saturation in EMM2-YE medium to arrest them in G2. (F) Samples were taken at the indicated time points to determine cell survival. (G) After 6 days, the cells were transferred into fresh medium, and chromosome segregation was observed during the first division after reentry into the cell cycle. Anaphase cells (spindle length, >5 μm) were scored for lagging chromatin (red) and/or cen2-GFP missegregation (white). Bar = 5 μm.
Fig. 6.
Fig. 6.
Psm3K106Ac is not detectable in the eso1-H17 mutant strain at the permissive temperature. (A) Cells were cultured at 25°C, arrested in early S phase by adding 12 mM hydroxyurea (HU), and released into the cell cycle. Samples were taken at the indicated time points (min). Psm3-GFP was immunoprecipitated from total protein extracts and probed with anti-Psm3K106Ac antibodies. Equal amounts of Psm3-GFP were verified by probing with anti-GFP antibodies. (B) Psm3-GFP immunopurified from the wt was serially diluted with Psm3-GFP immunopurified from eso1Δ wpl1Δ cells and probed with anti-Psm3K106Ac and anti-GFP antibodies as in panel A. (C) DNA content analysis before and after release from HU arrest. (D) eso1-H17 is still viable and thermosensitive for growth in a psm3RR background. Serial dilutions of cells were spotted on YES medium and incubated at the indicated temperatures. (E) Analysis of the cohesin complex with anti-acetyl antibodies. The cohesin complex was immunoprecipitated from native protein extracts by anti-GFP antibodies and probed with the indicated antibodies. Budding yeast SMC3-PK was immunopurified from the indicated strains (8) and used as a control for anti-acetyl antibodies. (F to I) The stable cohesin fraction is reduced in the eso1-H17 mutant at the permissive temperature. The strains contain the cdc25-22 and mis4-367 alleles and were cultured and processed for nuclear spreading and ChIP as in Fig. 1. (F) Chromatin-bound Rad21-PK was quantified from nuclear spreads before (T0) and after (T150 and T180 min) the temperature shift. The error bars represent SD from duplicate samples. (G) Cell survival during the course of the experiment. (H) ChIP assay showing the amount of Rad21 bound to pericentromeric regions (imr and dg) just after (15 min) and 3 h after the temperature shift. Rad21 enrichment was calculated from duplicate samples. The error bars represent SD. (I) Fluorescence-activated cell sorter (FACS) and cell cycle staging of cells before and after the shift to 36.5°C.
Fig. 7.
Fig. 7.
Acetyl-mimicking forms of Psm3 by themselves are not sufficient for sustained Rad21 binding to chromatin. All strains contain the cdc25-22 and mis4-367 mutations and were cultured and processed as in Fig. 1. (A and B) Kinetics of Rad21 dissociation from chromatin. Chromatin-bound Rad21 was measured from nuclear spreads. The error bars represent SD from at least two independent experiments, except for the wpl1Δ controls, which were done once. The data are presented in two separate graphs for clarity. (C) Cell survival was determined at the indicated time points.
Fig. 8.
Fig. 8.
The acetylation status of Psm3 modulates Wpl1-dependent cohesin removal from chromatin. All strains contain the cdc25-22 and mis4-367 mutations and were cultured and processed as in Fig. 1. (A to C) Chromatin-bound Rad21 was measured from nuclear spreads. The error bars represent SD from 3 to 5 independent experiments. (D to F) Kinetics of Rad21 dissociation from chromatin. Each data set (A to C) was normalized respective to its time zero value (arbitrarily set at 100).
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