Cohesin acetylation promotes sister chromatid cohesion only in association with the replication machinery

Abstract

Acetylation of the Smc3 subunit of cohesin is essential to establish functional cohesion between sister chromatids. Smc3 acetylation is catalyzed by members of the Eco family of acetyltransferases, although the mechanism by which acetylation is regulated and how it promotes cohesion are largely unknown. In vertebrates, the cohesin complex binds to chromatin during mitotic exit and is converted to a functional form during or shortly after DNA replication. The conserved proliferating cell nuclear antigen-interacting protein box motif in yeast Eco1 is required for function, and cohesin is acetylated during the S phase. This has led to the notion that acetylation of cohesin is stimulated by interaction of Eco1 with the replication machinery. Here we show that in vertebrates Smc3 acetylation occurs independently of DNA replication. Smc3 is readily acetylated before replication is initiated and after DNA replication is complete. However, we also show that functional acetylation occurs only in association with the replication machinery: disruption of the interaction between XEco2 and proliferating cell nuclear antigen prevents cohesion establishment while having little impact on the overall levels of Smc3 acetylation. These results demonstrate that Smc3 acetylation can occur throughout interphase but that only acetylation in association with the replication fork promotes sister chromatid cohesion. These data reveal how the generation of cohesion is limited to the appropriate time and place during the cell cycle and provide insight into the mechanism by which acetylation ensures cohesion.

FIGURE 1.

Recombinant XEco2 rescues sister chromatid cohesion when added early to an Eco2-depleted extract. A, XEco2 acetylates Smc3 in Xenopus egg extract. Interphase extract was mock depleted (ΔM); depleted of XEco1 (ΔE1), XEco2 (ΔE2), or both (ΔE1,E2); and supplemented as indicated with recombinant XEco2 (rE2). Sperm nuclei were added to the reactions, and at the indicated times the chromatin fractions were isolated and assayed by immunoblot for the proteins indicated at left. Smc3, total Smc3; Smc3^Ac, acetylated Smc3. Coomassie-stained histones were used as loading controls. Endogenous XEco1 was undetectable in this extract. B, total extract. Total extract samples from the experiment shown in A were analyzed for the indicated proteins. The six-histidine tag causes recombinant Eco2 to migrate more slowly than the endogenous protein. XEco1 was undetectable in this extract. Orc2 immunoblot was used as a loading control. C, Smc3 acetylation occurs only in the presence of chromatin. Extract was either mock supplemented (lanes 1, 4, and 7) or supplemented with exogenous rEco2 to approximately two and three times endogenous levels (indicated by black bars). In the absence of sperm nuclei (lanes 1–3), acetylated Smc3 was undetectable. In extracts supplemented with sperm nuclei, acetylated Smc3 was detectable, both in whole extract (lanes 4–6) and on chromatin (lanes 7–9). D, chromosomes assembled in the presence and absence of Eco2. Extract was mock depleted (ΔMock) or depleted of XEco2 protein (ΔEco2). Sperm nuclei were added at t = 0, and the extracts were cycled through interphase to allow DNA replication and then induced to enter mitosis by the addition of CSF extract (depleted or mock-depleted) at 150 min. Recombinant (rEco2) was added to the depleted reactions either at t = 0 (before DNA replication) or at 120 min (after DNA replication) as indicated. Buffer was added to the mock depleted extract at t = 0 (dark blue bars in E) and the depleted extract (red bars in E) as controls. Mitotic chromosomes were spun onto coverslips stained with DAPI. Shown are DAPI stain (DAPI), anti-centromere protein A (Cenp-A) immunofluorescence, and a merged image. Shown at right (C-A+) are 2.5× enlargements of centromere protein A staining from centromere regions indicated by the boxes in the Cenp-A panels. Scale bar, 5 μm. E, cohesion assay. Histogram showing the distribution of measurements between sister chromatids in experiment presented in A. The mean ± S.D. of intersister distances for each sample is shown in parentheses. The bar colors correspond to the font colors in D.

FIGURE 2.

XEco1 rescues acetylation but not cohesion. A, recombinant XEco1 mediates Smc3 acetylation in egg extract was either mock depleted (mock), or depleted of XEco2 (ΔXEco2) and supplemented with buffer, recombinant XEco2 (+rEco2), or recombinant XEco1 protein (+rEco1). Chromatin was isolated at the indicated times and assayed by immunoblot for the proteins named at left. B, immunoblot analysis of total extract samples from the experiment shown in A. C, cohesion assay. Chromosomes were assembled in mock depleted extract or extract depleted of XEco2 supplemented with buffer or rEco1 or rEco2 as in A. The means ± S.D. of intersister distances are reported as a measure of sister chromatid cohesion.

FIGURE 3.

XEco2 mediates acetylation of Smc3 when replication is largely complete. A, strategy used for time of addition experiment with recombinant XEco2 Gray bars indicate mock-depleted extract, whereas white bars indicate XEco2-depleted extract. Black arrowheads indicate time at which rEco2 was added to individual samples, and hatched areas show duration of presence of recombinant protein. Samples were collected at times indicated by open arrowheads. The numbers refer to the lane numbers in B. B, chromatin-associated proteins from the experiment illustrated in A. Chromatin was isolated from the samples illustrated in A and assayed by immunoblot for the indicated proteins. C, acetylation of Smc3 occurs in the absence of DNA replication. Interphase extract was mock treated or treated to inhibit DNA replication by the addition of recombinant geminin (Gem, 500 nm), recombinant p27 (200 nm), aphidicolin (50 μg/ml), or actinomycin D (10 μg/ml). Samples were collected at the indicated times, chromatin was isolated, and loading of cohesin (Smc1, upper bands; Smc3, lower bands), Sororin, XEco2, and Pds5, as well as Smc3 acetylation, was assessed by immunoblot. Coomassie-stained histones were used as a loading control. D, replication assays showing effective inhibition of DNA replication. Reactions identical to those shown in C were supplemented with radiolabeled dATP, and samples were collected at the indicated times and assayed for DNA replication. The data indicate that all inhibitors effectively inhibited DNA replication compared with controls. E, DNA damage signaling does not correlate with Smc3 acetylation. Extracts treated as indicated with inhibitors of DNA replication were assayed for phospho-Chk1, as well as total Chk1 and Smc1 as controls by immunoblot. Both aphidicolin and actinomycin D stimulated Chk1 phosphorylation, which was inhibited by addition of caffeine, an inhibitor of ATR kinase (45). Addition of p27 to the extract did not induce damage signaling.

FIGURE 4.

Acetylation of Smc3 in somatic cells. A, Smc3 is acetylated in G₁ in HeLa cells. HeLa cells were synchronized by thymidine arrest followed by brief nocodazole treatment. The cells were washed into media with or without thymidine (2 mm), and samples were collected at the indicated time points and analyzed for the indicated proteins. WCE, whole cell extract. Chromatin-associated samples were also assessed. Smc3 acetylation (arrowhead) was unaffected by treatment with thymidine to inhibit DNA replication. Mcm4 loaded onto chromatin in G₁, whereas PCNA levels rose during the S phase. B, flow cytometry data illustrating entry into S phase. Cells treated as in A were fixed and stained with propidium iodide to allow measurement of DNA content. Entry into the S phase, as illustrated by the increase in DNA content per cell, began between 6 and 8 h after nocodazole release and was inhibited by thymidine treatment (some time points were excluded for clarity). noc, cells collected immediately after nocodazole washout (t = 0).

FIGURE 5.

Interaction of XEco2 with PCNA is not required for Smc3 acetylation. A, XEco2 recruitment to chromatin does not require PCNA. Interphase extract was mock treated or supplemented (as in Fig. 3) with p27 or aphidicolin. Chromatin-associated XEco2 and PCNA were assayed by immunoblot. Orc2 immunoblot and Coomassie-stained histones were used as loading controls. B, characterization of a PIP box mutant of XEco2. Equal amounts of rEco2^WT or rEco2^PIP were incubated in XEco2-depleted extract and then immunoprecipitated with either anti-XEco2 antibody or normal rabbit IgG. The immunoprecipitated samples were tested by immunoblot for the presence of XEco2 and PCNA (IP). Input samples were also assayed to confirm that the starting levels of the recombinant proteins were comparable. C, the PIP box mutant of XEco2 rescues Smc3 acetylation but not Sororin loading. XEco2-depleted interphase extract was supplemented with buffer, rEco2^WT, or rEco2^PIP, and sperm nuclei were added. Chromatin was isolated at the indicated times and assayed by immunoblot for the proteins indicated at left. D, total extract samples for experiment in C. E, the PIP box mutant of XEco2 binds chromatin in the absence of DNA replication. Extract was either mock-depleted or depleted of XEco2 and supplemented with rEco2^PIP as indicated. The nuclei we added and the samples were assayed for chromatin-associated proteins at the indicated time points by immunoblot analysis. Coomassie-stained histones were used as a loading control.

FIGURE 6.

XEco2 recruitment to chromatin is mediated by two highly conserved motifs in the N-terminal domain. A, alignment of Eco2/Esco2 protein sequences from a number of vertebrate species. Shown are two short stretches in the N terminus that are very well conserved among vertebrates. We refer to these motifs as PLERK and GAAFF based on highly conserved amino acids within them. For full alignment please see Fig. S1. The numbers refer to the amino acid numbers in the Xenopus protein; the black bar indicates the amino acids deleted in the mutants analyzed in B. B, conserved motifs in the N terminus of XEco2 mediate chromatin binding and Smc3 acetylation by XEco2. Egg extract was either mock-depleted or XEco2 depleted and supplemented with recombinant wild-type XEco2 protein (rE2^WT) or proteins with deletions of the PLERK motif (rE2ΔP), GAAFF motif (rE2ΔG) or both (rE2ΔPG). C, immunoblot of total extract samples from experiment in B.

FIGURE 7.

The XEco2 PIP box is required for cohesion establishment. A, chromosomes assembled in extract containing the PIP box mutant of XEco2. Chromosomes were assembled in vitro (as in Fig. 1) in mock depleted or XEco2-depleted extract that had been supplemented with buffer, rEco2^WT, or rEco2^PIP prior to sperm addition, as indicated. Following mitotic entry, chromosomes were spun onto coverslips, immunostained with anti-centromere protein A, and counterstained with DAPI. Representative images of chromosomes from each treatment are shown. Scale bar, 10 μm. B, cohesion assay. The distance between sister chromatids was measured in the samples described in A and is reported in the histogram. The means ± S.D. of intersister distances are shown in parentheses. The bar colors correspond to the font colors in A. C, model for Smc3 acetylation and cohesion establishment. Cohesin (blue circles) is loaded onto chromatin (dark blue lines) in telophase by the activity of the Scc2/4 loading complex. XEco2 (red boxes) associates with chromatin prior to the onset of DNA replication. Cohesin acetylation can occur independently of DNA replication, either before DNA replication or after replication is complete (orange stars) but is not functional for cohesion establishment or Sororin recruitment. Acetylation in the context of interaction between XEco2 and PCNA (red star) ensures cohesion establishment. The cohesin complex itself may be altered during association with the replication fork (indicated by stretching of the blue cohesin circles) in a manner that renders it functional for cohesion and allows Sororin binding.