Pds5 regulators segregate cohesion and condensation pathways in Saccharomyces cerevisiae

Abstract

Cohesins are required both for the tethering together of sister chromatids (termed cohesion) and subsequent condensation into discrete structures-processes fundamental for faithful chromosome segregation into daughter cells. Differentiating between cohesin roles in cohesion and condensation would provide an important advance in studying chromatin metabolism. Pds5 is a cohesin-associated factor that is essential for both cohesion maintenance and condensation. Recent studies revealed that ELG1 deletion suppresses the temperature sensitivity of pds5 mutant cells. However, the mechanisms through which Elg1 may regulate cohesion and condensation remain unknown. Here, we report that ELG1 deletion from pds5-1 mutant cells results in a significant rescue of cohesion, but not condensation, defects. Based on evidence that Elg1 unloads the DNA replication clamp PCNA from DNA, we tested whether PCNA overexpression would similarly rescue pds5-1 mutant cell cohesion defects. The results indeed reveal that elevated levels of PCNA rescue pds5-1 temperature sensitivity and cohesion defects, but do not rescue pds5-1 mutant cell condensation defects. In contrast, RAD61 deletion rescues the condensation defect, but importantly, neither the temperature sensitivity nor cohesion defects exhibited by pds5-1 mutant cells. In combination, these findings reveal that cohesion and condensation are separable pathways and regulated in nonredundant mechanisms. These results are discussed in terms of a new model through which cohesion and condensation are spatially regulated.

Keywords: Elg1 and PCNA; Rad61/WAPL; chromosome condensation; cohesin; sister chromatid cohesion.

Fig. 1.

ELG1 deletion promotes Pds5 function. (A) Percent viability of yeast strains at 23 °C or 37 °C during G1, S and M phase arrests. Statistically significant differences (*) based on P < 0.05 (#, P = 0.053). (B) Micrographs of sister chromatid foci (GFP) relative to DNA (DAPI) and Pds1. (C) Percent of preanaphase cells with precocious sister chromatid separation (#, P = 0.057). (D) DNA content of cells arrested in G1 at 23 °C, then shifted to 37° and arrested preanaphase. (E) Percent of preanaphase cells with precocious sister chromatid separation.

Fig. 2.

ELG1 deletion fails to rescue pds5-1 condensation defects. (A) Micrographs reveal changes in rDNA condensation as detected by Net1-GFP (GFP) and DNA counterstained with DAPI. (B) Percent of cells that contain condensed (Lines) or uncondensed (Puffs) rDNA chromatin detected using Net1-GFP (*/**, statistical differences between wild-type and pds5-1 mutant cells and also between wild-type and pds5-1 elg1 double mutant cells).

Fig. 3.

PCNA promotes Pds5 function in cohesion. (A) Serial dilutions of cells harboring either vector or vector directing overexpression of PCNA (POL30). (B) Micrographs of sister chromatids (GFP), DNA (DAPI), and Pds1. (C) Percent of cells that exhibit precocious separated sister chromatids quantified as described in Fig. 1C.

Fig. 4.

Pds5 role in condensation appears independent of PCNA. (A) Micrographs of cells harboring either vector alone or vector directing PCNA overexpression and assessed for rDNA chromatin as described in Fig. 2A. (B) Percent of cells exhibiting condensed (Lines) or uncondensed (Puffs) rDNA structures as described in Fig. 2B.

Fig. 5.

RAD61 deletion suppresses the condensation defect of pds5-1 mutant cells. (A) Micrographs of rDNA chromatin structure and DNA as described in Fig. 2A. (B) Percent of cells exhibiting condensed (Lines) or uncondensed (Puffs) rDNA structures as described in Fig. 2B. (C) Serial dilutions of cells performed as described in Fig. 3A. (D) Percent of preanaphase cells with precocious sister chromatid separation as described in Fig. 1.

Fig. 6.

Model of Pds5-dependent cohesion and condensation. (A) pds5-1 mutant cells exhibit both cohesion and condensation defects with cohesins retaining their acetylation state (31). (B) Elevated PCNA retention onto DNA (ELG1 deletion) rescues the cohesion establishment (but not condensation) defect otherwise present in pds5-1 mutant cells. (C) Elevated retention of an as yet unidentified factor (green star) in RAD61 deletion strains rescues the condensation (but not cohesion) defect otherwise present in pds5-1 mutant cells.