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Cohesion between sister chromatids by the cohesin protein complex ensures accurate chromosome segregation and enables recombinational DNA repair. Sister chromatid cohesion is promoted by acetylation of the SMC3 subunit of cohesin by the ESCO2 acetyltransferase, inhibiting cohesin release from chromatin. The interaction of ESCO2 with the DNA replication machinery, in part through PCNA-interacting protein (PIP) motifs in ESCO2, is required for full cohesion establishment. Recent reports have suggested that Cul4-dependent degradation regulates the level of ESCO2 protein following replication. To follow up on these observations, we have characterized ESCO2 stability in Xenopus egg extracts, a cell-free system that recapitulates cohesion establishment in vitro. We found that ESCO2 was stable during DNA replication in this system. Indeed, further challenging the system by inducing DNA damage signaling or increasing the number of nuclei undergoing DNA replication had no significant impact on the stability of ESCO2. In transgenic somatic cell lines, we also did not see evidence of GFP-ESCO2 degradation during S phase of the cell cycle using both flow cytometry and live-cell imaging. We conclude that ESCO2 is stable during DNA replication in both embryonic and somatic cells.
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Fig. 1
ESCO2 protein level remains constant during DNA replication. a Approach. CSF-arrested extract was induced to enter interphase by the addition of calcium (CaCl2). Thirty minutes later, sperm nuclei (2300/μl, final) were added and aliquots were collected at the indicated times. b Representative fluorescence images of nuclei sampled right after addition into extracts (0 min) and after a 120 min incubation. Nuclear morphology associated with mitotic exit at bottom right was observed after addition of calcium. DAPI stain marks nuclei. c Immunoblot analysis. Reaction samples were analyzed by immunoblot for the indicated proteins. Cyclo: Cyclohexamide was added where indicated to prevent protein translation. Solid outlines denote membrane fragments that were processed separately. Dotted lines denote where gel images were cropped. ESCO2 and Cdt1 were analyzed on the same membrane fragment. β-tubulin served as a loading control. The foot of the gel was collected and stained with Coomassie as an additional loading control. d Quantification of results. The representative experiment shown in c was repeated three times and the results were plotted as a fraction of the remaining signal for the indicated proteins normalized to the β-tubulin signal for each sample. Error bars = SD
Fig. 2
ESCO2 stability is unaffected by changes in the nucleus:cytoplasm ratio. a Immunoblot analysis. Interphase extract was supplemented with the indicated concentrations of sperm nuclei (2000, 4000, or 8000 sperm nuclei/μl). Samples were collected at the indicated time points after the addition of sperm nuclei and were processed for immunoblot as in Fig. 1c. Solid outlines denote membrane fragments that were processed separately. b Quantification of results. The representative experiment shown in a was repeated three times and the results were plotted as a fraction of the remaining signal for the indicated proteins normalized to the β-tubulin signal for each sample. Error bars = SD
Fig. 3
ESCO2 is stable during DNA damage signaling. a Immunoblot analysis. Reactions were assembled as in Fig. 1 with the indicated modifications. UV: sperm were UV treated before they were added to the extract. Aphid: the DNA replication inhibitor aphidicolin was added before the addition of nuclei. p27: Recombinant p27 protein was added to the extract before the addition of nuclei. Samples were collected at the indicated times and probed by immunoblot for the indicated proteins. Antibody specific for phosphorylated Chk1 kinase (pChk1) was used to confirm DNA damage signaling. Solid outlines denote membrane fragments that were processed separately. Dotted lines denote where blot images were cropped. b The representative experiment shown in a was repeated three times and the results were plotted as a fraction of remaining signal for the indicated proteins for each sample. Error bars = SD
Fig. 4
ESCO2 levels in cultured somatic cells. a Flow cytometry. Live non-extracted HeLa cells expressing GFP-ESCO2 were collected and analyzed for GFP levels and DNA content. Parental and uninduced GFP-ESCO2 (no doxycycine added) cell lines are also shown. n ≧ 5500. b Quantification of GFP-ESCO2 levels during cell cycle progression. The flow cytometry experiment shown in a was repeated three times and are shown together as a SuperPlot (Lord et al. 2020). Mean values from each replicate, normalized to their G1 mean value, are plotted together. NI, normalized intensity. Error bars = SEM. ns, not significant; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. c Time-lapse imaging. Shown are selected frames from time-lapse imaging of HeLa cells expressing GFP-ESCO2. Intervals were chosen to highlight details of cell division. Time elapsed (in minutes) since metaphase is indicated by numbers in black font. The complete movie is shown in Supplementary Video 1. d Quantification of GFP signal. Schematic at left shows how GFP fluorescence intensity was scored across a complete cell division cycle, from an initial metaphase (M1) to metaphase 2 (M2) for each daughter cell, and graphed in aggregate at right. n = 20 M1 cells, 40 M2 cells analyzed. e Immunoblot analysis. The cell line used in a–d was treated either with doxycycline to induce transgene expression or siRNA to deplete the endogenous protein, and cell lysates were analyzed by immunoblot for the indicated proteins. Solid outlines denote membrane fragments that were processed separately. SMC3, a subunit of cohesin, and β-tubulin served as loading controls. A replicate blot was prepared and probed for GFP. Phosphorylated H3 (pH3), a marker of mitotic cells, confirmed increased mitotic index in cells depleted of ESCO2 seen previously (Alomer et al. 2017)
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