Pds5 promotes and protects cohesin acetylation

Abstract

Cohesin's Smc1 and Smc3 subunits form V-shaped heterodimers, the nucleotide binding domains (NBDs) of which bind the C- and N-terminal domains, respectively, of the α-kleisin subunit, forming a large tripartite ring within in which sister DNAs are entrapped, and thereby held together (sister chromatid cohesion). During replication, establishment of stable cohesion is dependent on Eco1-mediated acetylation of Smc3's NBD, which is thought to prevent dissociation of α-kleisin from Smc3, thereby locking shut a "DNA exit gate." How Scc3 and Pds5, regulatory subunits bound to α-kleisin, regulate cohesion establishment and maintenance is poorly understood. We show here that by binding to α-kleisin adjacent to its Smc3 nucleotide binding N-terminal domain, Pds5 not only promotes cohesin's release from chromatin but also mediates de novo acetylation of Smc3 by Eco1 during S phase and subsequently prevents de-acetylation by the deacetylase Hos1/HDAC8. By first promoting cohesin's release from chromosomes and subsequently creating and guarding the chemical modification responsible for blocking release, Pds5 enables chromosomal cohesin to switch during S phase from a state of high turnover to one capable of tenaciously holding sister chromatids together for extended periods of time, a duality that has hitherto complicated analysis of this versatile cohesin subunit.

Keywords: cell; gene.

Fig. 1.

Cohesin’s α-kleisin subunit recruits Pds5 via its distinct domain adjacent to the N-terminal α-helix. (A) Scc1(V137G L138G) and Scc1(V137K) mutations cause cell lethality. Different Scc1 mutants are expressed in cells whose endogenous Scc1 is under control of the GAL promoter (K9514, K16779, K16832, K17132, K17120, K17133, K17121, and K17119). Lethality was determined by growing cells on YEP+glucose plates at 30 °C. (B) Coimmunoprecipitation showing that Scc1V137K but not L89K abolishes Pds5 interaction (K8069, K16867, K17297, and K17289). (C) Live-cell imaging showing nuclear localization of ectopically expressed GFP-tagged wild-type Scc1 (Left, K18038) and Scc1V137K (Right, K17370) in G₂/M cells. (D) Western blotting showing the ectopic wild-type Scc1 (K17356) and Scc1V137K (K17357) protein levels in Pds5-GFP diploid cells. (E) Distribution of Pds5-GFP in cells expressing ectopic wild-type Scc1 (K17356) or V137K (K17357) at the leu locus after switching off endogenous GAL1-SCC1. (F) Wild-type Scc1 (K18846), but not Scc1V137K (K18847), recruits pericentric Wapl-GFP after endogenous GAL1-SCC1 is switched off. (G) Scc1 with photocross-linking ρ-BPA substitution at positions M127, D130 and T133 cross-links to Pds5 (K17830, K17827, K17828, and K17829).

Fig. 2.

The association of Pds5 and α-kleisin is required for Smc3 acetylation. (A) Depletion of Pds5 through auxin-dependent degradation pathway. Cells Pds5 tagged with auxin-degron (K18421) were pheromone arrested in G₁ for 1.5 h (t = 0 min) and 3 mM Indole-3-acetic acid sodium salt (auxin) was added or not for another 1 h. Proteins were extracted to monitor the level of Pds5. (B) Pds5 is required for Smc3 acetylation. Cells (K18421) were arrested in G₁ and Pds5 was depleted as described in A, and then released to S phase. (C) Pds5-binding mutant Scc1V137K is defective in Smc3 acetylation. Immunoprecipitation of Scc1 was performed on strains K10905, K17234, and K18326.

Fig. 3.

Cohesin dissociation from DNA does not trigger Smc3 deactylation. Release of cohesin from chromatin via Scc1 (B) but not Smc3 cleavage trigger de-acetylation (A). Strains K20018, K20019, K20021, and K20022 were grown in medium lacking tryptophan [2% (wt/vol) glucose] and then transferred in YEP medium containing 2% raffinose for 5 h. Subsequently, cells were arrested in metaphase by nocodazole for 2 h and then 2% galactose was added to induce the expression of TEV. Western blots against the indicated proteins were performed on whole-cell extracts. Full pictures of A are shown in SI Appendix, Fig S12C. An asterisk indicates a-specific bands.

Fig. 4.

Pds5 protects acetylated Smc3. Inactivation of pds5-101 but not pds5-99 ts mutants causes Hos1-dependent de-acetylation in G₂/M cells. (A)Yeast strains K12757, K17780, K17778, and K17774 were arrested with nocodazole at 25 °C (90 min) and then incubated at 35 °C. Proteins were extracted at indicated time points, and the levels of the indicated proteins were monitored. (B) Inactivation of pds5-101 but not pds5-99 ts mutants in G₂/M causes loss of cohesion maintenance. Cells growing in synthetic medium lacking methionine were arrested at 25 °C in G₁. Cells were then released in YEPD medium supplemented with 2 mM methionine to induce metaphase. Subsequently, the cultures were incubated at 35 °C. Graphs showing percentage of cells with single or double (split) GFP dots and the average split dots distance were shown in K15024, K15073, and K20119 strains.

Fig. 5.

The absence of Hos1 partially suppresses the loss of G₂/M cohesion in the pds5 mutant. Yeast strains K15024, K15073, and K20054 were arrested in metaphase at 25 °C (as shown in Fig. 4). Then, the cultures were incubated at 35 °C. Sister chromatid cohesion was monitored by fluorescence microscopy. (A) Distance between split dots. (B) Increased population with split dots after shifting to 35 °C. (C) Mean distance of split dots.