A novel mechanism for the establishment of sister chromatid cohesion by the ECO1 acetyltransferase

Abstract

Cohesin complex mediates cohesion between sister chromatids, which promotes high-fidelity chromosome segregation. Eco1p acetylates the cohesin subunit Smc3p during S phase to establish cohesion. The current model posits that this Eco1p-mediated acetylation promotes establishment by abrogating the ability of Wpl1p to destabilize cohesin binding to chromosomes. Here we present data from budding yeast that is incompatible with this Wpl1p-centric model. Two independent in vivo assays show that a wpl1∆ fails to suppress cohesion defects of eco1∆ cells. Moreover, a wpl1∆ also fails to suppress cohesion defects engendered by blocking just the essential Eco1p acetylation sites on Smc3p (K112, K113). Thus removing WPL1 inhibition is insufficient for generating cohesion without ECO1 activity. To elucidate how ECO1 promotes cohesion, we conducted a genetic screen and identified a cohesion activator mutation in the SMC3 head domain (D1189H). Smc3-D1189H partially restores cohesion in eco1∆ wpl1∆ or eco1 mutant cells but robustly restores cohesion in cells blocked for Smc3p K112 K113 acetylation. These data support two important conclusions. First, acetylation of the K112 K113 region by Eco1p promotes cohesion establishment by altering Smc3p head function independent of its ability to antagonize Wpl1p. Second, Eco1p targets other than Smc3p K112 K113 are necessary for efficient establishment.

FIGURE 1:

eco1∆ wpl1∆ cells lack segregation-competent cohesion. (A) Cartoon depicting the structure of the cohesin complex. Smc3p (red) interacts with Smc1p (orange) at the hinge and head domains. Mcd1p (green) binds to both the Smc1p and Smc3p heads. Scc3p (brown) binds to Mcd1p. Two Walker A/B ATPases (yellow balls) are in the heads. (B) Regimen used to assess segregation-competent segregation in cells. (C) Cohesion loss at CEN-proximal TRP1 locus at mid–M phase arrest. Haploid WT (VG3460-2A), eco1∆ wpl1∆ (VG3502-1C), and wpl1∆ (VG3513-1B) strains were arrested in G1 using αFactor at 23°C and then released and rearrested in mid–M phase at 23°C using nocodazole (Materials and Methods). The percentage of cells with two GFP spots (separated sister chromatids) is plotted (top) along with DNA content (bottom). The lack of G1 cells with two GFP spots demonstrates absence of preexisting aneuploidy. The green dot above the graph shows the position of LacO arrays relative to the centromere. (D, E) Assay for segregation-competent cohesion. Haploid WT (VG3460-2A) and eco1∆ wpl1∆ (VG3502-1C) cells were treated as in B. (D) Assessment of cell-cycle progression after release from mid–M (nocodazole) arrest. Percentage of large-budded cells remaining at 1, 2, and 3 h after release from mid–M phase arrest (top) and DNA content (bottom). (E) Segregation of chromosome IV sister chromatids after release from mid–M phase. Proper segregation in large-budded cells 2 h after release (left). Proper segregation in unbudded cells 3 h after release (right). Random segregation is 50% and is marked by a dotted red line. Data for C–E are from two independent experiments; 100–300 cells scored for each data point.

FIGURE 2:

Genetic screen identifies the smc3-D1189H mutation as a suppressor of eco1∆ wpl1∆ strain sensitivity to benomyl and camptothecin. (A) Rationale for screen. Haploid WT (VG3349-1B) and eco1∆ wpl1∆ (VG3503-4A) were grown to saturation at 23°C, plated at 10-fold serial dilution on YPD alone or containing 12.5 μg/ml BEN, and then incubated at 23°C for 3 d. Left, eco1∆ wpl1∆ cell viability on YPD and schematic showing sister segregation via early S-phase attachments despite failure to establish cohesion. Right, eco1∆ wpl1∆ cell inviability on BEN and schematic showing benomyl-induced loss of S-phase spindle attachments and consequent missegregation and inviability. (B) Cartoon showing localization of the D1189 residue in the SMC3 head domain. (C) Cross drug resistance of the smc3-D1189H suppressor. Haploid WT (3460-2A) and three eco1∆ wpl1∆ background strains, parent SMC3 (VG3502-1A), D1189H suppressor 1 (Sup #1), and rebuilt smc3-D1189H (VG3547-3B), were grown and plated as described in A onto YPD alone or containing 12.5 μg/ml BEN or 10 μg/ml CPT and incubated for 3 d at 23°C. Strains below the red line are eco1∆ wpl1∆ background. (D) Schematic showing evolutionary conservation of budding yeast smc3-D1189 residue (red letter). The black line above the sequence shows the conserved DE residues of the Smc3p Walker B box.

FIGURE 3:

smc3-D1189H partially restores sister chromatid cohesion in eco1∆ wpl1∆ cells. (A) Cohesion loss at a CEN-proximal TRP1 locus. Haploid WT (VG3460-2A), eco1∆ wpl1∆ (VG3502-1C), and smc3-D1189H eco1∆ wpl1∆ (VG3547-3B) were arrested in mid–M phase as described in Figure 1C. The percentage of cells with two GFP signals (sister separation) is plotted. The lack of G1 cells with two GFP spots demonstrates absence of preexisting aneuploidy. Data are from four independent experiments; 100–300 cells were scored for each data point in each experiment. (B) Assay for segregation-competent cohesion. Haploid smc3-D1189H eco1∆ wpl1∆ (VG3549-7A) and wpl1∆ (VG3513-1B) cells were treated as depicted in Figure 1B and then assayed for chromosome segregation after release from mid–M arrest. Proper segregation of chromosome IV sister chromatids in large-budded cells 2 h after release (left) and in unbudded cells 3 h after release (middle) and DNA content (right). Random segregation will be 50% and is marked by a dotted red line. Data were generated simultaneously with strains in Figure 1, C–E, in two independent experiments in which 100–300 cells were scored for each data point. (C) Kinetics of cohesion loss at a CEN-proximal TRP1 locus. Strains in A released from G1 and arrested in mid–M phase as described in Figure 1C. The percentage of cells with two GFP spots is plotted (left) along with DNA content (right). Gray box shows S phase. Between 100 and 300 were cells scored for each data point. (D, E) Cohesion loss at a CEN-distal LYS4 locus. Haploid wild-type (VG3349-1B), eco1∆ wpl1∆ (VG3503-4A), and smc3-D1189H eco1∆ wpl1∆ (VG3549-7A) were arrested in mid–M phase arrest as described in Figure 1C. (D) Cohesion loss at mid–M phase arrest. The percentage of cells with two GFP spots is plotted (left) along with DNA content (right). Data were derived from two independent experiments, and 100–300 cells were scored for each data point. (E) Time course to assess kinetics of cohesion loss. The percentage of cells with two GFP spots is plotted (left) along with DNA content (right). Gray box shows S phase. Between 100 and 300 were cells scored for each data point. The green dot above the graph shows the position of LacO arrays relative to the centromere.

FIGURE 4:

smc3-D1189H cohesin is fully functional in a WT background. (A) Assessing the drug sensitivity of smc3-D1189H cells. Haploid WT (VG3599-9C), smc3-D1189H (VG3600-13C), wpl1∆ (VG3604-4C), and smc3-D1189H wpl1∆ (VG3605-5D) were grown and plated onto YPD alone or containing BEN (12.5 μg/ml) or CPT (15 μg/ml) as described in Figure 2A and then incubated at 23°C for 3 d for YPD and CPT and 4 d for BEN. (B) Cohesion loss in mid–M phase cells. WT and smc3-D1189H cells were released from G1 and arrested in mid–M phase as described in Figure 1C. Left, cohesion loss at CEN-proximal TRP1 locus in haploid WT (VG3599-9C) and smc3-D1189H (VG3600-13C) cells. Right, cohesion loss at CEN-distal LYS4 locus in haploid WT (VG3557-2A) and smc3-D1189H (VG3558-2D) strains. Cohesion loss is the percentage of cells with two GFP spots. Data were derived from two independent experiments. Between 100 and 300 cells were scored for each data point in each experiment. (C) ChIP of Mcd1p in mid–M phase–arrested cells. Haploid WT (VG3599-9C) and smc3-D1189H (VG3600-13C) mid–M phase–arrested cells from B were subjected to ChIP using αMcd1p antibodies (top) and DNA content determined (bottom). Mcd1p binding was assessed by quantitative PCR. Data are presented as percentage of total DNA assayed using the same primer pairs at each site. Left, chromosome III pericentric CARC1. Seven primer pairs used to assay Mcd1p binding at loci spanning an ∼2.6-kb region including CARC1 of chromosome III. Right, chromosome XII CEN-distal CARL1. Seven primer pairs used to assay Mcd1p binding at loci spanning an ∼4.5-kb region including CARL1 of chromosome XII. WT (gray line, gray squares) and smc3-D1189H (black line, open circles).

FIGURE 5:

Smc3-D1189H fails to suppress the characteristic wpl1∆ defects of reduced cohesin binding and partial cohesion loss. (A–C) Haploid wild-type (WT; VG3349-1B), eco1∆ wpl1∆ (VG3503-4A), and smc3-D1189H eco1∆ wpl1∆ (VG3549-7A) cells were arrested in mid–M phase as described in Figure 1C. (A) Chromosome spreads of mid–M phase cells. WT (top), eco1∆ wpl1∆ (middle), and smc3-D1189H eco1∆ wpl1∆ (bottom). Cells were processed to detect chromosomal DNA (4′,6-diamidino-2-phenylindole) and cohesin using αMcd1p antibodies. (B) ChIP of Mcd1p in mid–M phase cells. Cells were fixed and processed for ChIP using αMcd1p antibodies. WT (gray line, gray diamonds), eco1∆ wpl1∆ (red line, red circles), and smc3-D1189H eco1∆ wpl1∆ (black line, gray triangles). Mcd1p binding was assessed as described in Figure 4C. Top, chromosome III pericentric CARC1. Bottom, chromosome XII CEN-distal CARL. (C) Effect of smc3-D1189H on cohesion loss in a wpl1∆ background. Haploid WT, wpl1∆, and smc3-D1189H wpl1∆ cells were arrested in mid–M phase as described in Figure 1C. Left, cohesion loss at CEN-proximal TRP1 locus assayed in haploid WT (VG3460-2A), wpl1∆ (VG3604-4C), and smc3-D1189H wpl1∆ (VG3605-5D) strains. Right, cohesion loss at CEN-distal LYS4 locus assessed in haploid WT (VG3349-1B), wpl1∆ (VG3626-2E), and smc3-D1189H wpl1∆ (VG3627-3C) strains. Bottom, DNA content. The percentage of cells with two GFP signals (sister separation) is plotted. Data were derived from two independent experiments; 100–300 cells were scored for each data point in each experiment.

FIGURE 6:

smc3-D1189H robustly suppresses the cohesion defect of the smc3-K112R, K113R (RR) mutation. (A) Plasmid shuffle to assess viability of the chimeric smc3-RR-D1189H allele. Haploid shuffle strain VG3464-16C bearing plasmid pEU42 (SMC3 CEN URA3) and a second SMC3 “test allele,” SMC3, smc3-D1189H, smc3-RR, or chimeric smc3RR-D1189H, was grown and plated as described in Figure 2A onto URA–dropout or FOA-containing media. Plates were incubated 3 d at 23°C. (B) Assessment of drug sensitivity. Haploid SMC3 (MB45-1A), smc3-D1189H (MB46-1A), or chimeric smc3-RR-D1189H (MB47-1A) strains grown and plated as described in Figure 2A onto YPD, BEN, and CPT and incubated at 30°C for 2 d. (C). Cohesion loss of in smc3-RR cells. Haploids bearing SMC3-AID and a second SMC3 allele, either WT or smc3-RR, were depleted for SMC3-AID from G1 through mid–M phase arrest. Left, cohesion loss at CEN-proximal TRP1 locus assessed in haploid SMC3 SMC3-AID (MB84-1A) and smc3-RR SMC3-AID (MB83-1A) strains. Right, cohesion loss at CEN-distal LYS4 locus assessed in haploid SMC3 SMC3-AID (MB81-1A) and smc3-RR SMC3-AID (MB79-1A) strains. The percentage of cells with two GFP spots (sister separation) is plotted. (D) Cohesion loss of smc3-RR-D1189H in mid–M phase–arrested cells. Haploid SMC3, smc3-D1189H and chimeric smc3-RR-D1189H were arrested in mid–M phase as described in Figure 1B. Left, cohesion loss at CEN-proximal TRP1 locus assessed in haploid wild-type (SMC3; MB65-1A), smc3-D1189H (MB66-1A), or chimeric smc3-RR-D1189H (MB67-1A) strain. Right, cohesion loss at CEN-distal LYS4 locus assessed in haploid strains from B. The percentage of cells with two GFP spots (sister separation) is plotted. (E, F) Assessing smc3-D1189H ability to suppress Eco1p depletion. (E) Viability after Eco1p depletion. Haploid WT (VG3620-4C), ECO1-AID (VG3662-1D), and smc3-D1189H ECO1-AID (VG3663-2E) were grown and plated as described in Figure 2A onto YPD alone or containing auxin (750 μM) and then incubated at 23°C for 2 d. (F) Cohesion loss after auxin-mediated Eco1p depletion from G1 cells through mid–M phase arrest. Left, cohesion loss at CEN-proximal TRP1 locus assayed in haploid strains WT (VG3460-2A), ECO1-AID2 (VG3659-1A) and smc3-D1189H ECO1-AID2 (VG3663-2E) strains. Right, cohesion loss at CEN-distal LYS4 locus assessed in haploid WT (VG3620-4C), ECO1-AID2 (VG3646-1A) and smc3-D1189H ECO1-AID2 (VG3650-1E) strains. The percentage of cells with two GFP signals (sister separation) is plotted. (C, D, F) Data were derived from two independent experiments; 100–300 cells were scored for each data point in each experiment.

FIGURE 7:

A wpl1∆ suppresses the inviability of smc3-RR–bearing cells but not the cohesion defect. (A) Plasmid shuffle to assess viability of smc3-RR wpl1∆ cells. Haploid wpl1∆ SMC3 shuffle strain (VG3578-1A) bearing pEU42 (SMC3 CEN URA3) and a second integrated test SMC3 allele—WT (SMC3 wpl1∆), smc3-D1189H (smc3-D1189H wpl1∆), or smc3-RR (smc3-RR wpl1∆)—were grown and plated as described in Figure 2A onto URA–dropout or FOA-containing media. Plates were incubated 2 d at 30°C. (B) Assessment of smc3-RR wpl1∆ strain drug sensitivity. Haploid SMC3 wpl1∆ (MB48-1A), smc3-D1189H wpl1∆ (VG3627-3C), and smc3-RR wpl1∆ (MB50-1A) strains were grown and plated as described in Figure 2A onto YPD alone or containing BEN (10 μg/ml) or CPT (10 μg/ml) and incubated at 23°C for 3 d for YPD and 4 d for BEN and CPT plates. (C) Cohesion loss at CEN-proximal locus LYS4. Haploid wild-type (WT; VG3627-3C), wpl1∆ (SMC3 wpl1∆; MB48-1A), and smc3-RR wpl1∆ (MB50-1A) strains arrested in mid–M phase as described in Figure 1B. Cells were scored for cohesion loss (top) and DNA content (bottom). Data are derived from two independent experiments; 100–300 cells were scored for each data point in each experiment.

FIGURE 8:

Regulatory and structural model for Eco1p-mediated regulation of cohesin. (A) Model of cohesin regulation. Eco1p-mediated acetylation of Smc3p and other targets play a Wpl1p-independent role to promote cohesion establishment (arrow 1) and overcome Wpl1p inhibition of condensation and cohesion (T-bar 2). Cohesin in the noncohesive form (red circles) and a cohesive form (blue ovals) are shown. (B) smc3-D1189 modeled on Smc1p crystal structure. Shown are the head domains of Smc3p (gray) and Smc1p (blue) bound as a dimer to Mcd1p N-terminus and C-terminus (green). Indicated by arrows are the D1189 residue (red) at the bottom of Smc3p, the ATP molecule in the Smc3p Walker A/B box (yellow), and the Smc3-K112, K113 residues (salmon colored).