Sororin cooperates with the acetyltransferase Eco2 to ensure DNA replication-dependent sister chromatid cohesion

Abstract

Sister chromatids are held together, from the time they are made during S phase until they are pulled apart just before cell division, by a protein complex called cohesin. The mechanistic details by which sister chromatid cohesion is established and maintained have remained elusive, particularly in vertebrate systems. Sororin, a protein that interacts with the cohesin complex, is essential for cohesion in vertebrates, but how it participates in the process is unknown. Here we demonstrate that sororin recruitment depends on active DNA replication and that sororin loading onto chromosomes depends upon another essential cohesion factor, the acetyltransferase Eco2. We find that Eco2, like sororin, is a substrate of the anaphase-promoting complex (APC), which ensures that protein levels remain low before S phase. These findings demonstrate that sororin and Eco2 work together to form a unique regulatory module that limits cohesion to cells with replicated chromatin and support a model in which cohesion in vertebrates is not fully established until the G2 phase of the cell cycle.

Fig. 1.

Regulation of sororin recruitment to chromatin by the cohesin complex and DNA replication. (A) Sororin depends on cohesin for association with chromatin. Nuclear assembly reactions were set up in extract that had been depleted of cohesin (ΔSmc3), sororin (Δsororin), or mock-depleted (mock), and chromatin was isolated at the indicated time points and assayed for cohesin (Smc3) and sororin by immunoblot. Control: background band used as loading control. (B) Sororin recruitment requires DNA replication. Sperm nuclei were added to interphase extract supplemented with geminin, p27, aphidicolin, or a buffer control (mock). At the indicated times, chromatin was isolated and analyzed by immunoblot for the presence of cohesin (Smc3) and sororin. (C) DNA replication assay of the same samples shown in B. All three inhibitors effectively blocked DNA replication. Coomassie stained histones were used as a loading control (Lower).

Fig. 2.

The sororin-binding site remains after DNA replication is complete. (A) Experimental scheme to test whether sororin binds chromatin only during DNA replication or after replication is complete. Aliquots of a nuclear assembly reaction in sororin-depleted extract were supplemented with in vitro-translated sororin at indicated times after the start of the reaction (t = 0, 30, 60, 90, or 120′, Upper). Following sororin addition, chromatin fractions were isolated immediately (0′) and at 7.5 and 15′ and assayed by immunoblot for cohesin (Smc3) and sororin (B). (C) DNA replication was assessed in extract depleted for sororin (as used in B) and mock-depleted extract. (D) Chromatin was collected from all reactions shown in (B) at 150′ after the start of the experiment and analyzed for cohesin (Smc3) and sororin by immunoblot. Ongoing replication is not required for sororin recruitment. (E) Nuclei were added to a sororin-depleted extract and the reaction was allowed to proceed for 60′. Aphidicolin (or DMSO control) was added. After 10 min, in vitro-translated sororin was added to both extracts and the levels of chromatin-associated Smc3 and sororin were assessed by immunoblot. (F) DNA replication levels over a 30-min period from the time of sororin addition.

Fig. 3.

Eco2 is required for recruitment of sororin to chromatin. (A) Nuclei were added to interphase extracts that were mock depleted (mock) or immunodepleted of Eco1 (ΔEco1), Eco2 (ΔEco2), Eco1 and Eco2 together (ΔEco1/2), cohesin (ΔSmc3), or sororin (ΔSor). At the indicated times, chromatin fractions were isolated and analyzed by immunoblot for the presence of topoisomerase (loading control), Smc3, Eco2, and sororin. (B) Protein levels in total extract. Depleted extracts used in A were analyzed by immunoblot for topoisomerase II, Smc3, or Eco2 (Eco1 was undetectable in this experiment, and sororin was obscured by background bands in total extract). (C) Rescue of sororin recruitment with recombinant Eco2 interphase extract was depleted of Eco2 and either left unsupplemented (−) or supplemented with recombinant 6His–Eco2 at 0, 60, or 120′ as indicated (Upper). Chromatin-bound fractions were collected at both 90 and 150′ after sperm addition and analyzed by immunoblot for the presence of Smc3, Eco2, and sororin. Also shown are immunoblots for Eco2 and Smc3 in total extracts (extract) (Upper). Coomassie-stained histones were used as loading controls. (D) Eco2 recruitment to chromatin independent of replication interphase extracts were supplemented with geminin, p27, or aphidicolin as in Fig. 1. Chromatin-associated proteins were analyzed at the indicated times after the addition of sperm nuclei.

Fig. 4.

Eco2 is a substrate of Cdh1-activated APC. (A) Radiolabeled in vitro-translated proteins (indicated at Left: cyclin B, sororin, xEco2, xEco1, and xEco2^KEN) were incubated in interphase extract (IE), extract supplemented with cyclin B (+CycB), or supplemented with Cdh1 protein (+Cdh1). Aliquots were removed at the indicated times (t = 0, 45, 90, and 135′), separated by SDS/PAGE, and the gel was dried and exposed in a phosphor storage cassette. The phosphorimages are shown. Loading controls are shown in Fig. S6. (B) APC-dependent degradation of endogenous Eco2 protein interphase extract was analyzed by immunoblot for Smc3, Eco2, and sororin at the indicated times following the addition of buffer alone (IE), recombinant cyclin B (+CycB), or Cdh1 (+Cdh1).

Fig. 5.

Models for regulation of cohesion by sororin and Eco2 cohesin (green clamps) associates with chromatin (blue) before DNA replication. DNA replication either promotes activation of the Eco2 acetyltransferase (orange) directly, causing it to modify the cohesin complex, or causes a change in the cohesin complex (indicated as opening of the complex), which makes it a good Eco2 substrate. Once the cohesin complex is modified by Eco2 it can be bound by sororin (yellow) (A). Eco2 added after DNA replication is able to bind chromatin, but unable to modify cohesin and generate the sororin-binding site (B).