Abstract
Condensin is an integral component of the mitotic chromosome condensation machinery, which ensures orderly segregation of chromosomes during cell division. In metazoans, condensin exists as two complexes, condensin I and II. It is not yet clear what roles these complexes may play outside mitosis, and so we have examined their behaviour both in normal interphase and in premature chromosome condensation (PCC). We find that a small fraction of condensin I is retained in interphase nuclei, and our data suggests that this interphase nuclear condensin I is active in both gene regulation and chromosome condensation. Furthermore, live cell imaging demonstrates condensin II dramatically increases on G1 nuclei following completion of mitosis. Our PCC studies show condensins I and II and topoisomerase II localise to the chromosome axis in G1-PCC and G2/M-PCC, while KIF4 binding is altered. Individually, condensins I and II are dispensable for PCC. However, when both are knocked out, G1-PCC chromatids are less well structured. Our results define new roles for the condensins during interphase and provide new information about the mechanism of PCC.
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Abbreviations
- 3D FISH:
-
Three-dimensional fluorescent in situ hybridization
- Cal A:
-
Calyculin A
- CENP-O:
-
Centromere protein O
- Dox:
-
Doxycycline
- DE:
-
Differential expression
- EdU:
-
5-Ethynyl-2'-deoxyuridine
- IF:
-
Immunofluorescence
- NEBD:
-
Nuclear envelope breakdown
- PCC:
-
Premature chromosome condensation
- PFA:
-
Paraformaldehyde
- SBP:
-
Streptavidin-binding peptide
- Topo IIα:
-
Topoisomerase IIα
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Acknowledgments
The authors thank Dr. Matthew Burton for providing flow cytometry and microscopy technical support and Dr. Kathryn Marshall for careful reading of the manuscript.
This work was supported by NHMRC project grant GNT1030358 and GNT1047009 and by the Victorian Government’s Operational Infrastructure Support Program.
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Online Resource 1
A small pool of condensin I is retained in the interphase nuclei of chicken DT40 cells. a The slide in Fig. 2a was examined by confocal microscopy (Zeiss LSM780). Representative images are shown, demonstrating that some CAP-H-GFP-SBP is present in the interphase nuclei. Line profiles across the cell were plotted and are shown at the right. Scale bar represents 2 μm. b Asynchronous CAP-H-GFP-SBP cells were fixed with 4 % PFA and co-stained with mouse anti-SBP and rabbit anti-CAP-H antibodies. DNA was stained with DAPI and the cells were examined by confocal microscopy (Zeiss LSM 780). Line profiles across the cell were plotted and are shown at the right. Scale bar represents 10 μm. Inserts are higher magnification of selected cell. c Asynchronous CAP-H-GFP cells were fixed with PFA and stained with Lamin B1 antibodies. Optical sections (0.2 μm) were imaged and analysed by Imaris 8.1.2. Orthogonal (XY, XZ and YZ) views of CAP-H-GFP and Lamin B1 show clearly that CAP-H-GFP is present inside the nuclear envelope. Line profiles across the cell were plotted and are shown at the top right. Scale bar represents 5 μm. d Ponceau staining of the blot in Fig. 2d is shown as a loading control. (PDF 432 kb)
Online Resource 2
Determination of cell cycle stages in live cell imaging. a CAP-H-GFP H2B-RFP and CAP-D3-GFP H2B-RFP cells (n = 7) were subjected to live cell imaging for 15 h with 2-min intervals. Cell size during the timecourse was determined by measuring the cell diameter (μm) using the polygon function in SoftWoRx 4.1 and plotted as a function of time. CAP-H-GFP and CAP-D3-GFP cellular localisations were monitored and this is reflected in the background colour of the graph. Cell cycle stages were determined accordingly. b, c CAP-H-GFP H2B-RFP cells were subjected to live cell imaging for 15 h with 2-min intervals. Normalised CAP-H-GFP intensity was plotted with time according to the description in Fig. 1a, T = 0 min was set as a completion of anaphase. Note the level of CAP-H in the nucleus drops over time. Representative images in different cell cycle stages are shown in b. H2B-RFP intensity during the timecourses were normalised to anaphase single cell H2B-RFP intensity at T = 0 min, respectively. Normalised H2B-RFP intensity was plotted with time in c. Cell cycle stages were determined based on the change of normalised H2B-RFP intensity (i.e. double H2B) and DIC morphology. Error bars represent standard error of the mean (SEM), n = 15. (PDF 589 kb)
Online Resource 3
Complete depletion of CAP-H. a CAP-H KO cells were grown in the absence (CAP-HON) or presence (CAP-HOFF) of dox for 36 h in order to deplete CAP-H. Cells were collected for Western blotting to detect CAP-H and α-tubulin, and FACS analysis. b CAP-HON and CAP-HOFF (+ dox 36 h) cells were collected for Annexin V staining and subjected for FACS analysis. c CAP-HON and CAP-HOFF (+ dox 36 h) cells were collected and fixed with 4 % PFA. Immunofluorescent staining was used to visualise γ-H2AX (red). DNA was stained with DAPI (blue). CAP-HON cells treated with Adriamycin 6 h were used as a positive control for γ-H2AX staining. Representative images are shown. Three different experiments were performed. γ-H2AX foci were counted and scored as 0, 1, 2, 3, 4 and ≥5 foci from 100 cells in each experiment. The average percentage of cells with 0, 1, 2, 3, 4 and ≥5 γ-H2AX foci is shown in the right. (PDF 682 kb)
Online Resource 4
Functional studies of CAP-H in interphase. a Human homologs of 693 DT40 genes that are significantly misregulated in CAP-H KO were analysed with Ingenuity Pathway Analysis (IPA). The top five canonical pathways are shown at the left. The top five diseases and biological functions are shown at the right. b Significant DE genes in this study that are involved in the prostate cancer signalling pathway are shown in red. c 116 significantly DE genes with CAP-H enrichment were analysed with DAVID Bioinformatics Resources 6.7. The top nine GO terms are shown. (PDF 380 kb)
Online Resource 5
Heat map of 116 misregulated genes directly due to CAP-H binding removal. Heat map representing 116 DE genes with CAP-H enrichment in comparison between CAP-HON and CAP-HOFF samples displays significant gene expression change. Each gene is represented by a row of coloured boxes, and each replicate is represented by a column. Red indicates gene expression up-regulation, blue indicates gene expression down-regulation. (PDF 178 kb)
Online Resource 6
Condensins I and II in the G1-PCC cells. a G1 synchronised CAP-D3-GFP-SBP cells were treated with 50 nM Cal A for 1 h. Cells were collected, fixed with methanol/acetic acid (3:1) and co-stained with mouse anti-SBP and rabbit anti-CAP-H antibodies to detect CAP-D3-GFP-SBP (green) and CAP-H (red). DNA was stained with DAPI (blue). Representative G1-PCC chromosomes are shown. Scale bar represents 5 μm. b CAP-D3-GFP H2B-RFP cells were subjected to live cell imaging. NEBD times (min) were plotted according to cell sizes (cell diameter <9 μm or >9 μm) at t = 0 min with p = 6.72E-13 as described in Fig. 6a. Error bars represent 95 % confidence intervals (CI), n = 30. (PDF 149 kb)
Online Resource 7
Chromosome shattering in the PCC cells. a WT H2B-GFP cells with and without Cal A treatment were subjected to live cell imaging for 7 h at 2-min intervals. Live cell imaging was started within 7 min of Cal A addition. At t = 0 min, G1 cells (cell diameters <9 μm, based on Online Resource 2a), G2/M cells, S cells (cell diameters >9 μm, based on Online Resource 2a) and anaphase cells were selected for monitoring. b Live cell imaging using H2B-GFP of G1-PCC, S-PCC and anaphase-PCC cells. Note the shattering in the final frame for each cell. c, d CAP-H-GFP-SBP cells were treated as described in Fig. 4c, collected and fixed with PFA. Immunofluorescent staining was used to visualise (c) γ-H2AX (red) or (d) RAD51 (red). DNA was stained with DAPI (blue). CAP-H-GFP-SBP cells blocked with hydroxyurea (HU) for 16 h and then released were used as a positive control for RAD51 staining. Representative images are shown. (PDF 1332 kb)
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Zhang, T., Paulson, J.R., Bakhrebah, M. et al. Condensin I and II behaviour in interphase nuclei and cells undergoing premature chromosome condensation. Chromosome Res 24, 243–269 (2016). https://doi.org/10.1007/s10577-016-9519-7
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DOI: https://doi.org/10.1007/s10577-016-9519-7