G1-Cyclin2 (Cln2) promotes chromosome hypercondensation in eco1/ctf7 rad61 null cells during hyperthermic stress in Saccharomyces cerevisiae

Abstract

Eco1/Ctf7 is a highly conserved acetyltransferase that activates cohesin complexes and is critical for sister chromatid cohesion, chromosome condensation, DNA damage repair, nucleolar integrity, and gene transcription. Mutations in the human homolog of ECO1 (ESCO2/EFO2), or in genes that encode cohesin subunits, result in severe developmental abnormalities and intellectual disabilities referred to as Roberts syndrome and Cornelia de Lange syndrome, respectively. In yeast, deletion of ECO1 results in cell inviability. Codeletion of RAD61 (WAPL in humans), however, produces viable yeast cells. These eco1 rad61 double mutants, however, exhibit a severe temperature-sensitive growth defect, suggesting that Eco1 or cohesins respond to hyperthermic stress through a mechanism that occurs independent of Rad61. Here, we report that deletion of the G1 cyclin CLN2 rescues the temperature-sensitive lethality otherwise exhibited by eco1 rad61 mutant cells, such that the triple mutant cells exhibit robust growth over a broad range of temperatures. While Cln1, Cln2, and Cln3 are functionally redundant G1 cyclins, neither CLN1 nor CLN3 deletions rescue the temperature-sensitive growth defects otherwise exhibited by eco1 rad61 double mutants. We further provide evidence that CLN2 deletion rescues hyperthermic growth defects independent of START and impacts the state of chromosome condensation. These findings reveal novel roles for Cln2 that are unique among the G1 cyclin family and appear critical for cohesin regulation during hyperthermic stress.

Keywords: Cornelia de Lange syndrome (CdLS); Eco1/ESCO2; G1 Cyclin/Cln2; Roberts syndrome (RBS); chromosome condensation; cohesinopathies; sister chromatid cohesion.

Fig. 1.

Identification of the G1 cyclin, Cln2, as a regulator of Eco1-Rad61 pathways. a) eco1Δ rad61Δ double mutant cells are viable within a narrow temperature range. Ten-fold serial dilutions of wildtype, rad61Δ and eco1^ctf7-203 single mutant cells, and eco1Δ rad61Δ double mutant cells at the indicated temperatures. b) CLN2 deletion, and truncated Cln2^1-221 (CLN2^1-221Δ), both suppress eco1Δ rad61Δ double mutant cells growth defects. Ten-fold serial dilutions of wildtype cells, 2 independent isolates of eco1Δ rad61Δ cln2Δ triple mutant cells, and 2 independent isolates of eco1Δ rad61Δ cln2^1-221 cells grown at the indicated temperatures.

Fig. 2.

The role of Cln2 in Eco1-Rad61 functions is unique from Cln1 and Cln3 G1 cyclins. a) CLN3 deletion does not rescue eco1Δ rad61Δ cell growth defects under thermic stress. Ten-fold serial dilutions of parental eco1Δ rad61Δ double mutant cells and 3 independent isolates of eco1Δ rad61Δ cln3Δ triple mutant cells grown at the indicated temperatures. b) CLN1 deletion does not rescue eco1Δ rad61Δ cell growth defects under thermic stress. Ten-fold serial dilutions of parental eco1Δ rad61Δ double mutant cells and 3 independent isolates of eco1Δ rad61Δ cln1Δ triple mutant cells grown at the indicated temperatures.

Fig. 3.

Deletion of CLN2 suppresses eco1 mutant cell ts growth, similar to but distinct from deletion of RAD61. Ten-fold serial dilutions of wildtype cells, eco1^ctf7-203 mutant cells, and 3 independent isolates of eco1^ctf7-203 cln2Δ double mutant cells grown at the indicated temperatures.

Fig. 4.

Elevated expression of PCNA (POL30) does not suppress eco1Δ rad61Δ mutant cell growth defects during thermic stress. The growth, 2 temperatures, of 2 independent isolates of eco1Δ rad61Δ double mutant cells transformed with vector alone compared to 3 independent isolates of eco1Δ rad61Δ double mutant cells transformed with vector that directs elevated POL30 expression.

Fig. 5.

Deletion of CLN2 does not delay, but instead promotes, cell cycle progression in eco1Δ rad61Δ mutant cells. a) DNA contents assessed using flow cytometry of log phase wildtype cells, 2 independent isolates of parental eco1Δ rad61Δ double mutant cells, and 2 independent isolates of eco1Δ rad61Δ cln2Δ triple mutant before and after growth at 37°C for 3 h. b) DNA contents of the cells, described in (a), released from a mitotic arrest (NZ, nocodazole) at the permissive temperature of 30°C then shifted to 37°C. Samples harvested every 30 min were analyzed for DNA content by flow cytometry.

Fig. 6.

Deletion of CLN2 rescues the chromatin hypercondensation defect otherwise present in eco1Δ rad61Δ cells under thermic stress. a) and d) DNA content of wildtype cells, parental eco1Δ rad61Δ double mutant cells, and 2 independent isolates of eco1Δ rad61Δ cln2Δ triple mutant at either log phase (a) or synchronized in S phase using HU (d), prior to growth for 3 h at 37°C in medium supplemented with nocodazole (NZ). b) and e) Micrographs of DNA masses and rDNA obtained from preanaphase cells as described in (a) and (d), respectively. Chromosomal masses and rDNA loop structures were detected using DAPI. White arrows indicate rDNA loops, which typically reside proximal to the genomic mass. c) and f) Quantification of genome mass areas, relative to template, for cells described in (a) and (d), respectively. For graph c: wildtype (n = 38), eco1Δ rad61Δ (n = 105), eco1Δ rad61Δ cln2Δ (2 replicates: n = 237 and n = 176 to produce an n_total = 413). For graph f: wildtype (n = 117), eco1Δ rad61Δ (n = 189), eco1Δ rad61Δ cln2Δ (2 replicates: n = 381 and n = 83 to produce an n_total = 464). Chi-squared tests were subsequently used to assess the dependence of gene mutations (* indicates P-value at or below 0.05) on chromatin condensation levels. For graph c: wildtype, P-value = 0.999; eco1Δ rad61Δ, P-value = 0.0000272; eco1Δ rad61Δ cln2Δ, P-value = 0.00104. For graph f: wildtype, P-value = 0.983; eco1Δ rad61Δ, P-value = 3.74 × 10⁻¹⁴; eco1Δ rad61Δ cln2Δ, P-value = 4.45 × 10⁻¹⁴. Not shown: we also compared chromatin condensation effects across the 3 strains. The percentage of genomic masses, within a given field, that matched or exceeded the template was calculated and those values used to determine statistical significance: wildtype to eco1Δ rad61Δ, P = 0.00068; eco1Δ rad61Δ to eco1Δ rad61Δ cln2Δ (both replicates combined), P = 0.0066.

Fig. 7.

Multifaceted and opposing roles for CDK regulation of Eco1 activity. In this current study, we provide evidence that Cln2-CDK regulates chromatin condensation reactions in combination with Eco1 and Rad61. Cln2-CDK promotes chromatin hypercondensation after START (possibly during S phase) and in response to thermic stress. Also during S phase, Eco1 establishes cohesion between cohesins newly deposited (via Scc2,4) onto nascent sister chromatids after passage of the DNA replication fork. Eco1 acetylation of cohesin (stabilizing cohesin dimers) blocks Rad61-dependent cohesin dissociation activities. After S phase, M-CDKs phosphorylate Eco1, resulting in Eco1 degradation through both G2 and M phases. In response to damage, however, Eco1 expression is significantly upregulated during G2/M to promote DNA damage-induced cohesion (not shown), even as Eco1 degradation continues. Intriguingly, eco1 is synthetically lethal in combination with either cdc28 (CDK) or cak (CDK activating kinase), revealing that Eco1 and M-CDK perform complementary roles to maintain genomic integrity (which are likely to include chromosome condensation, cohesion maintenance, and transcription regulation). During G1, Eco1 is critical for regulation gene transcription and genomic architecture within the nucleus. The role of G1-CDKs in this portion of the cell cycle remain largely unknown.