Cohesin-independent segregation of sister chromatids in budding yeast

Abstract

Cohesin generates cohesion between sister chromatids, which enables chromosomes to form bipolar attachments to the mitotic spindle and segregate. Cohesin also functions in chromosome condensation, transcriptional regulation, and DNA damage repair. Here we analyze the role of acetylation in modulating cohesin functions and how it affects budding yeast viability. Previous studies show that cohesion establishment requires Eco1p-mediated acetylation of the cohesin subunit Smc3p at residue K113. Smc3p acetylation was proposed to promote establishment by merely relieving Wpl1p inhibition because deletion of WPL1 bypasses the lethality of an ECO1 deletion (eco1Δ wpl1Δ). We find that little, if any, cohesion is established in eco1Δ wpl1Δ cells, indicating that Eco1p performs a function beyond antagonizing Wpl1p. Cohesion also fails to be established when SMC3 acetyl-mimics (K113Q or K112R,K113Q) are the sole functional SMC3s in cells. These results suggest that Smc3p acetylation levels affect establishment. It is remarkable that, despite their severe cohesion defect, eco1Δ wpl1Δ and smc3-K112R,K113Q strains are viable because a cohesin-independent mechanism enables bipolar attachment and segregation. This alternative mechanism is insufficient for smc3-K113Q strain viability. Smc3-K113Q is defective for condensation, whereas eco1Δ wpl1Δ and smc3-K112R,K113Q strains are competent for condensation. We suggest that Smc3p acetylation and Wpl1p antagonistically regulate cohesin's essential role in condensation.

FIGURE 1:

The smc3-RQ allele promotes viability but the K113Q or QQ alleles do not. (A) Effect of smc3 acetyl-mimics on smc3-42 temperature sensitivity. Haploid VG3358 3B (smc3-42) alone (–) or with a second SMC3 allele, (WT) VG3377-1A, (K113Q) VG3423-1A, (QQ) VG3378-2A, or (RQ) VG3424-2A, grown at 23°C, plated at 10-fold serial dilutions on YPD, and incubated 3 d at 23, 30, 35.5, or 37°C. (B) Ability of smc3 acetyl-mimic alleles to support viability as the sole SMC3. Haploid VG3464-16C with pEU42 (SMC3 CEN URA3) and test plasmid bearing either no insert (–), SMC3 (WT), smc3K113Q (K113Q), or smc3K112R,K113Q (RQ) grown at 30°C, plated in fivefold serial dilutions on FOA or YPD, and incubated at 30°C.

FIGURE 2:

smc3 acetyl-mimics are defective in cohesion at CEN-proximal and distal loci. Cells released from G1 and then arrested in mid M phase. The percentage of cells with two GFP signals is plotted. (A) Cohesion loss at CEN-distal locus (LYS4) in smc3-42 background at mid M phase (35.5°C). Haploid VG3358-3B (smc3-42) alone or with a second SMC3 allele, (WT) VG3377-1A, (K113Q) VG3423-1A, or (RQ) VG3424-2A. (B) Cohesion loss at CEN-distal locus (LYS4) in shuffle strains at mid M phase (30°C). Strains have SMC3 (WT) VG3471-WT or (RQ) VG3471-WT as sole SMC3. (C) Cohesion loss at a CEN-proximal locus (TRP1) in smc3-42 background at mid M phase (35.5°C). Haploid VG3357-3A (smc3-42) alone or with a second SMC3 allele, (WT) VG3447-1B, (K113Q) VG3449-3B, or (RQ) VG3450-4B. (D) Time course to monitor cohesion loss at a CEN-distal locus (LYS4). Strains from A scored at 10- to 15-min intervals after G1 release into mid-M-phase arrest (35.5°C). (E) DNA content. Data were derived from two independent experiments, and error bars are SD. For cohesion assays, 100–400 cells were scored for each data point in each experiment.

FIGURE 3:

eco1Δ wpl1Δ cells are defective in cohesion at CEN-proximal and distal loci. The percentage of cells with two GFP signals is plotted. Cells released from G1 phase were arrested in mid M phase at 33°C. (A) Cohesion loss at a CEN-distal locus (LYS4) in mid M phase. Haploids VG3349-1B (WT), VG3360-3D (wpl1Δ), VG3503-1B (eco1Δ wpl1Δ), and VG3503-4A (eco1Δ wpl1Δ). (B) Cohesion loss at CEN-proximal locus (TRP1) in mid M phase. Haploids VG3460-2A (WT), VG3513-1B (wpl1Δ), VG3502-2A (eco1Δ wpl1Δ), and VG3503-4C (eco1Δ wpl1Δ). (C) Kinetics of cohesion loss at a CEN-distal locus (LYS4). Haploids VG3349-1B (WT), VG3506-5D (eco1-ts), VG3503-1B (eco1Δwpl1Δ), and VG3503-4A (eco1Δ wpl1Δ) assayed for cohesion loss after release from G1 phase. (D) FACS. Data were derived from two independent experiments, and error bars are SD. For cohesion assays,100–400 cells were scored for each data point in each experiment.

FIGURE 4:

Sister chromatids segregate in smc3-42 mutants with or without acetyl mimics. Strains from Figure 2C smc3-42 mutant (smc3) or with a second SMC3 allele, WT, K113Q, or RQ, released from G1 at 35.5°C to allow one cell cycle before rearrest in G1. Chromosome IV monitored at a CEN4-proximal locus (TRP1). (A, B) Time course assessing cohesion loss. (A) Percentage of cells with two GFP signals. (B) DNA content. (C). Chromosome IV sister chromatid disjunction scored in large-budded cells. Dotted line marks the 50% disjunction expected for random segregation. For cohesion and nondisjunction assays, 100–400 cells were scored for each data point in each experiment.

FIGURE 5:

Sister chromatids segregate in mcd1-1 mutant cells released from G1 phase at nonpermissive temperature. Haploid WT (VG3460-2A) and mcd1-1 mutant (VG3456-2C) cells released from G1 arrest at 35.5°C and allowed to complete one cell cycle. Chromosome IV was monitored at a CEN4-proximal locus (TRP1). (A, B) Time course to assess cohesion loss. (A) Percentage of cells with two GFP signals. (B) DNA content. (C). Chromosome IV sister chromatid disjunction scored in large-budded cells. Dotted line marks 50% disjunction expected for random segregation. (D) Micrographs showing the TRP1 locus (GFP) and total chromosome DNA (DAPI). For cohesion assays and nondisjunction assays, data were derived from two independent experiments, and error bars are SD. For each data point in each experiment 100–400 cells were scored.

FIGURE 6:

Random segregation of sister chromatids in mcd1-1 cells released from nocodazole arrest. WT (VG3460-2A) and mcd1-1 mutant (VG3456-2C) cells arrested in mid M phase 35.5°C using nocodazole and then released from arrest (23°C). Chromosome IV monitored at a CEN4-proximal locus (TRP1). (A) Cohesion at mid-M-phase arrest. Percentage of cells with two GFP signals plotted for G1 and mid-M-phase cells. (B, C) Large-budded cells assayed 2 h after release from nocodazole. (B) Disjunction of chromosome IV sisters. Dotted line marks 50% disjunction expected for random segregation. (C) Micrographs showing chromosomal DNA (DAPI) and CEN-proximal locus of chromosome IV sisters (GFP). For cohesion and nondisjunction assays, 200–400 cells were scored for each data point.

FIGURE 7:

smc3-RQ allele–bearing smc3-42 cells and eco1Δ wpl1Δ cells promote chromosome condensation at the rDNA locus. (A–C) Haploid strains from Figure 2A, smc3-42 parent alone (smc3) or containing a second SMC3 allele, WT, K113Q, or RQ, were synchronously arrested in mid M phase at 35.5°C using nocodazole as described in Figure 2A. The rDNA resides in the nucleolus adjacent to the bulk chromosomal DNA. (A) Micrographs of WT and smc3-42 cells subjected to FISH. Bulk chromosomal DNA (DAPI) and rDNA detected using FISH (rDNA). Arrows indicate rDNA in DAPI-stained images. (B) Quantitation of rDNA condensation by scoring loop-like rDNA structure. (C) Micrographs of RQ allele in smc3-42 cells subjected to FISH. DAPI-stained bulk chromosomal DNA shown with arrows indicating rDNA. (D) WT and eco1Δ wpl1Δ cells from Figure 3A subjected to FISH as described in A and quantified as described in B. Data were derived from two independent experiments, and error bars are SD. For condensation assays, 50–150 nuclei were scored for each data point in each experiment.

FIGURE 8:

(A) Model for regulation of cohesin's roles in cohesion and condensation. Cohesin in noncohesive form (circle). Eco1p acetylates Smc3p to remove Wpl1p inhibition to form activated cohesin (square). Activated cohesin can be used for either cohesion (oval) or condensation (diamond), depending on the activity of unidentified downstream regulators. (B) Model for cohesin-independent segregation. In early S phase, sister kinetochores form bipolar spindle attachments (left). By mid S phase, replicated regions precociously separate, but sister kinetochores retain their bipolar attachments due to unreplicated regions (middle). When replication is completed, poleward movements ensue, enabling sister segregation to opposite poles (right).