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 (RBS) and Cornelia de Lange Syndrome (CdLS), respectively. In yeast, deletion of ECO1 results in cell inviability. Co-deletion 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.

Fdo1, Fkh1, Fkh2 and the Swi6-Mbp1 MBF complex regulate Mcd1 levels to impact eco1 rad61 cell growth in Saccharomyces cerevisiae

“…Defects in cohesin regulation are also tightly correlated with several forms of cancer (80)(81)(82). Despite the importance of cohesin functions, little is known regarding the regulation of cohesins early during the cell cycle, but recent findings confirm that MCD1 expression may be a critical component (83,84). In this study, we identify FDO1 as a novel regulator of MCD1 expression.…”

“…Eco1 (which is essential only during S phase) and Pds5 (an auxiliary cohesin subunit required for cohesion from S phase to anaphase onset) support cohesin functions through independent mechanisms (35,41,42,53). In a remarkable convergence of studies, however, deletion of CLN2 was found to promote the viability of both eco1Δ rad61Δ at 37°C and pds5 elg1 cells (83,84). The latter study provided evidence that CLN2 deletion increased Mcd1 levels, such that simply elevating Mcd1 protein levels was sufficient to render viable pds5 elg1 double mutant cells (83).…”

“…Given that CLN2 is common to both eco1Δ rad61Δ and pds5 elg1 cells as a bypass suppressor (83,84) protein alone is sufficient to rescue eco1Δ rad61Δ cell temperature sensitivity, we generated an MCD1 high-copy (2µ TRP1) plasmid. We validated this reagent by confirming that it indeed rescued the ts-growth of mcd1-1 mutated cells (Figure 2A).…”

“…Previously, a genome-wide suppressor screen identified the G1 cyclin Cln2 as a novel regulator of eco1Δ rad61Δ mutant cells (84). To further identify novel genes that rescue eco1Δ rad61Δ temperature sensitivity, a second revertant (YBS1638) was backcrossed 2 times to wildtype cells and multiple non-ts eco1Δ rad61Δ segregants pooled together prior to whole genome sequencing as previously described (84).…”

“…Previously, a genome-wide suppressor screen identified the G1 cyclin Cln2 as a novel regulator of eco1Δ rad61Δ mutant cells (84). To further identify novel genes that rescue eco1Δ rad61Δ temperature sensitivity, a second revertant (YBS1638) was backcrossed 2 times to wildtype cells and multiple non-ts eco1Δ rad61Δ segregants pooled together prior to whole genome sequencing as previously described (84). Mutations that were common across all segregants (present at a frequency of 1.0) were prioritized as revertant gene candidates that rescued the temperature sensitivity otherwise exhibited by eco1Δ rad61Δ cells.…”