Condensin II initiates sister chromatid resolution during S phase

Abstract

Condensins I and II are multisubunit complexes that play essential yet distinct functions in chromosome condensation and segregation in mitosis. Unlike condensin I, condensin II localizes to the nucleus during interphase, but it remains poorly understood what functions condensin II might have before mitotic entry. Here, we report that condensin II changes its chromatin-binding property during S phase. Remarkably, advanced premature chromosome condensation (PCC) assays enabled us to visualize condensin II forming "sister axes" in replicated regions of chromosomes in S phase cells. Depletion of condensin II compromised PCC-driven sister chromatid resolution during S phase. Moreover, fluorescence in situ hybridization assays revealed that condensin II, but not condensin I, promotes disjoining duplicated chromosomal loci during S phase. Application of mild replicative stress partially impaired this process and further exacerbated phenotypes arising from condensin II depletion. Our results suggest that condensin II initiates structural reorganization of duplicated chromosomes during S phase to prepare for their proper condensation and segregation in mitosis.

Figure 1.

Condensin II changes its chromatin-binding property during S phase. (A) HeLa cells were synchronized by means of double thymidine block and release. Cells were fixed at the time points indicated after release and immunolabeled with anti–CAP-H2 (extraction −). Alternatively, the same set of cells was treated with a detergent-containing buffer before fixation and processed for immunofluorescence labeling (extraction +). Bar, 100 µm. (B) Asynchronously grown HeLa cells were pulse labeled with EdU, fixed, and immunolabeled with anti–topo IIα and anti–CAP-H2 (extraction −). Alternatively, the same set of cells was treated with a detergent-containing buffer before fixation and processed in the same way (extraction +). Quantitative imaging analyses were performed using CELAVIEW RS100. The first row shows DAPI-stained images and profiles of total intensity of DNA per nucleus (x axis) and cells counted (y axis). The second row shows EdU-labeled images and scattergrams of DNA intensity and max intensity of EdU. The third and fourth rows show topo IIα– and CAP-H2–labeled images and scattergrams of DNA intensity and total nuclear intensity of topo IIα and CAP-H2. In the right panels of the third and fourth rows (EdU +), the data of EdU-positive cells were extracted from the pentagonal areas in the second row, and plotted in the same way. In the scattergrams of EdU-positive cells, regression lines were drawn in red. Bar, 100 µm.

Figure 2.

Advanced PCC assays show that condensin II forms sister axes in replicated regions of chromosomes in S phase cells. (A) Asynchronously grown lymphoblastoid cells were pulse labeled with EdU and then treated with calyculin A. The cells were subjected to a hypotonic treatment, fixed with Carnoy’s solution, and spread onto glass slides. The cell cycle stages were judged by EdU-labeling patterns and chromosome morphology. Shown here are representative images of cells at different stages, along with their closeups. Bars, 5 µm. (B) Asynchronously grown lymphoblastoid cells were pulse labeled with EdU and chased for 3 h before treatment with calyculin A. Shown here is a representative image of late S-PCC. Bars, 5 µm. (C) Lymphoblastoid cells were subjected to the same treatment as A and processed for immunolabeling with antibodies against CAP-G and -H2 (see Materials and methods). As a control, metaphase chromosome spreads were prepared in the same way from a culture that had been treated with colcemid (bottom). Cartoons depict the localization of condensins I and II in PCC products at the different stages. Bar, 5 µm. (D) The intensity of fluorescence signals was measured using the Polygon tool of the DeltaVision, and normalized to the intensity of DAPI. Plotted here are the relative intensities of the G2-PCC chromatids to the G1-PCC chromatids (for CAP-G and -H2). The mean and SD were calculated from the data from three independent experiments. The total numbers examined were described in the figure.

Figure 3.

Condensin II plays a key role in PCC-driven sister chromatid resolution in late S phase cells. (A) Experimental protocol for calyculin A–induced PCC from siRNA-treated HeLa cells. After two rounds of siRNA transfection, the cells were pulse labeled with EdU and then treated with calyculin A (Cal). (B) Cells were processed as in Fig. 2 B. Shown here are representative images of late S-PCC cells from populations mock depleted or depleted of SMC2, CAP-G, or CAP-G2. Bars, 5 µm. (C) HeLa cells (mock depleted or depleted of CAP-G or -G2) were pulse labeled with EdU and treated with calyculin A as described in A. The cells were fixed and immunolabled with antibodies against CAP-G and -H2, as described in Fig. 2 C. Shown here are representative images of late S-PCC cells, along with their merged closeups. Bars, 5 µm. Cartoons depict late S-PCC chromosomes observed under the different conditions. (D) Plotted here are the frequencies of resolution defects in late S-PCC products observed under the three conditions. This quantitative evaluation was completed once. For additional information, see Fig. S5 (A–C).

Figure 4.

FISH assays reveal that condensin II promotes disjoining of duplicated chromosomal loci during S phase. (A) Experimental protocol for enriching late S phase cells depleted of condensin subunits (see Fig. S2 E for cohesin depletion). Cells were pulse labeled with EdU before harvest. (B) After a hypotonic treatment, the cells were fixed with Carnoy’s solution and spread onto glass slides. EdU-positive cells were classified into early, middle, or late S on the basis of the EdU-labeling pattern. Representative images of cells at each stage are shown. Bar, 10 µm. (C) Plotted here are cell cycle stages that were judged on the basis of the EdU-labeling pattern and cell morphology. The data shown are from a single representative experiment out of two repeats. (D) FISH assays of a chromosomal locus whose sister distance is susceptible to condensin II depletion. HeLa cells mock depleted or depleted of CAP-G or -G2 were prepared as described in B and hybridized with the BAC clone 145C4. Cells in late S phase were selected as judged by the EdU-labeling pattern, and the distance between sister FISH signals was measured. Shown here are representative images of late S cells from the three different cultures, along with their merged closeups. FISH signals are indicated by the arrows. Bars: (white) 10 µm; (black) 1 µm. (E) Plots of the measured distance between sister FISH signals hybridized with 145C4. Under each condition, >120 pairs of sister FISH signals from late S cells were analyzed. For reasons of convenience, all values judged to be <0.2 µm, the limit of optical resolution, were plotted as 0.1 µm (as indicated by the shadowed areas). The red bar indicates the median value. (F) Plots of the measured distance between sister FISH signals hybridized with 10A17. Under each condition, >130 pairs of sister FISH signals from late S cells were analyzed. Each assessment shown in E and F was completed once. Detailed statistical data for FISH analyses are shown in Tables S1 and S2.

Figure 5.

Application of mild replicative stress partially impairs sister chromatid resolution during S phase. (A) Experimental protocol for enriching late S and G2 phase cells from synchronized cultures treated with aphidicolin (APH) and/or calyculin A (Cal). HeLa cells were released in a medium with or without 0.1 µg/ml aphidicolin and pulse labeled with EdU before being treated with calyculin A. (B) Cells released for 8 (−APH release) or 13 h (+APH release) were fixed with Carnoy’s solution and spread onto glass slides. Late S cells were selected on the basis of EdU-labeling patterns. The morphology of PCC products was classified into three types, and representative images are shown. Bar, 10 µm. (C) Plotted here are the frequencies of the three types of morphology observed in late S-PCC cells. (D) Cells were incubated for an extended period (9 h, −APH release; 15 h, +APH release; see A) and fixed and treated as in B. G2 cells with no EdU signals were selected, and the frequencies of the three types of morphology observed are plotted. Each experiment shown in C and D was completed once. (E) Experimental protocol for enriching late S phase cells from synchronized cultures treated with siRNAs and aphidicolin. Cells were released in the absence or presence of aphidicolin and pulse-labeled with EdU before harvest. See Fig. S2 E for Rad21 depletion. (F) HeLa cells mock depleted or depleted of CAP-G, CAP-G2, or Rad21 were prepared, hybridized with the BAC clone 285H13, and analyzed as described in Fig. 4 D. Shown here are representative images of late S cells from the mock-depleted culture released in a medium with or without aphidicolin. FISH signals are indicated by the arrows. Bars: (white) 10 µm; (black) 1 µm. (G) Plots of the measured distance between sister FISH signals hybridized with 285H13. In each culture, >110 pairs of sister FISH signals from late S cells were analyzed. This assessment was completed once. Detailed statistical data are shown in Table S3.

Figure 6.

Application of mild replicative stress to condensin II–depleted cells exacerbates their defects in chromosome architecture and segregation. (A) Experimental protocol for enriching metaphase cells from siRNA-treated synchronized cultures. Cells were harvested at the time points indicated by the arrows after colcemid treatment. (B) Metaphase chromosome spreads were prepared and immunolabeled with antibodies against CAP-G and -H2. Bar, 5 µm. (C) The morphology of metaphase chromosomes observed were classified into three types and plotted for each condition tested. The data shown are from a single representative experiment out of two repeats. For additional information, see Fig. S5 (D and E). (D) Experimental protocol for enriching mitotic cells from siRNA-treated synchronized cultures. No colcemid treatment was applied in this protocol. (E) HeLa cells treated as described in A were fixed on coverslips and immunolabeled with anti-H3K9me3 and a CREST serum. Shown here are representative images of cells displaying anaphase or anaphase-like chromosome morphology: normal segregation, chromosome bridges, and cotton candy chromosomes. Bar, 10 µm. (F) Defects in chromosome segregation were classified into five categories and plotted for each condition. The data shown are from a single representative experiment out of three repeats.

Figure 7.

Models for the action of condensin II from S phase through mitosis. (top) Condensin II associates with chromatin to initiate sister chromatid resolution during S phase. It is assumed here that the activity of condensin II increases gradually over the period of S and G2 phases and rises quickly in prophase. Unlike condensin II, condensin I associates with chromosomes only after NEBD in prometaphase. (bottom) Condensin II (red circles) associates with replicated regions of chromosomes and initiates a “tug-of-war” with cohesin (blue circles) as early as in S phase, which persists until metaphase. Replication forks are indicated by orange triangles.