Abstract
In addition to inter-chromatid cohesion, mitotic and meiotic chromatids must have three physical properties: compaction into ‘threads’ roughly co-linear with their DNA sequence, intra-chromatid cohesion determining their rigidity, and a mechanism to promote sister chromatid disentanglement. A fundamental issue in chromosome biology is whether a single molecular process accounts for all three features. There is universal agreement that a pair of Smc–kleisin complexes called condensin I and II facilitate sister chromatid disentanglement, but whether they also confer thread formation or longitudinal rigidity is either controversial or has never been directly addressed respectively. We show here that condensin II (beta-kleisin) has an essential role in all three processes during meiosis I in mouse oocytes and that its function overlaps with that of condensin I (gamma-kleisin), which is otherwise redundant. Pre-assembled meiotic bivalents unravel when condensin is inactivated by TEV cleavage, proving that it actually holds chromatin fibres together.
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Acknowledgements
We thank Micron microscopy facility and especially I. Dobbie for his technical support; all the staff of the BSB facility; B. Novák and all the members of the K.N. laboratory for discussions and comments on the manuscript. This work was supported by the Wellcome Trust (Grant Ref 091859/Z/10/Z), ERC grant (Proposal No 294401) and MRC (Grant ID 84673).
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M.H. designed, performed and analysed the experiments. J.G. provided technical advice for oocytes manipulation. J.M. realized all the genotyping. J.L. provided reagents. T.H. provided reagents. K.N. designed and analysed the experiments and coordinated the work.
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Integrated supplementary information
Supplementary Figure 1
(A) Ncaph1 is not essential during meiosis I. Oocytes from Ncaph1f/f females (Wild type Ncaph1) or Ncaph1f/f Tg(ZP3Cre) females (ΔNcaph1) were injected at GV stage with mRNA coding for H2B-mCherry to mark the whole chromosomes in red and TALE-mClover_MajSat to mark the pericentric repeats in green. Meiosis I progression was followed by live cell confocal imaging. Maximum intensity z projection images of the major time points are shown. The phenotype was observed in all oocytes analysed (24 oocytes from 3 females in 3 experiments). Scale bar: 10 μm. (B) Segregation defects inΔNcaph2 oocytes Snapshot 16 h post GVBD of one of the 18% of ΔNcaph2 oocytes that go through polar body extrusion. Phenotype observed in all the 18% of ΔNcaph2 oocytes that extrude a polar body. Scale bar, 10 μm.
Supplementary Figure 2 Deletion of Ncaph1 doesn’t impede on Ncaph2 loading.
Chromosomes from oocytes isolated from Ncaph1f/fTg(ZP3Cre) or Ncaph2f/f Tg(ZP3Cre) females were analysed by chromosome spreading 7 h (meiosis I) or 16 h (meiosis II) after GVBD. Immunofluorescence was performed using antibodies directed against NCAPH2 and NCAPH1 on ΔNcaph1 (A) and ΔNcaph2 oocytes (B) respectively. The DNA was stained with DAPI. Oocytes from females Ncaph1f/f or Ncaph2f/f were used as control (Wild type). The phenotypes were observed in all oocytes analysed analysed (More than 10 oocytes from 2 females in 2 experiments). Scale bar, 5 μm.
Supplementary Figure 3 Topoisomerase 2 alpha localization on ΔNcaph1 and ΔNcaph2 chromosome spreads.
(A) Chromosomes from oocytes isolated from Ncaph1f/f Tg(ZP3Cre) females were analysed by chromosome spreading 17 h (meiosis II) after GVBD. Immunofluorescence was performed using antibodies directed against Topoisomerase 2 Alpha. The DNA was stained with DAPI. Oocytes from Ncaph1f/f females were used as control (Wild type). The phenotype was observed in all oocytes analysed (14 oocytes from 2 females in 2 experiments). Scale bar: 5 μm. (B) Chromosomes from oocytes isolated from Ncaph2f/f Tg(ZP3Cre) females were analysed by chromosome spreading 7 h (meiosis I) or 17 h (meiosis II) after GVBD. Immunofluorescence was performed using antibodies directed against Topoisomerase 2 Alpha. The DNA was stained with DAPI. Oocytes from Ncaph2f/f females were used as control (Wild type). The phenotype was observed in all oocytes analysed (More than 5 oocytes from 2 females in 2 experimetns) Scale bar: 5 μm.
Supplementary Figure 4 GVBD kinetics of ΔNcaph1ΔNcaph2 oocytes.
Oocytes from Ncaph1f/f Ncaph2f/f and Ncaph1f/fNcaph2f/f Tg(ZP3Cre) females were isolated in M16 supplemented with IBMX. The kinetics starts as soon as the oocytes are washed in M16 to resume meiosis. The two groups of oocytes were followed by Live cell confocal microscopy to evaluate the time of GVBD. Results from two independent experiments performed using oocytes from one female of each genotype per experiment. The total number of oocytes are Ncaph1f/f Ncaph2f/f: n = 11 and Ncaph1f/f Ncaph2f/f Tg(ZP3Cre): n = 15. Error bars represent mean ± s.d.
Supplementary Figure 5 Chromosome spread analysis of NCAPH2-eGFP rescued Ncaph2f/f Tg(ZP3Cre) oocytes.
Oocytes from Ncaph2f/f Tg(ZP3Cre) females were injected at GV stage with Ncaph2-eGFP encoding RNA and analysed by chromosome spreading at 7 h (Meiosis I) and 16 h (Meiosis II) post GVBD using an antibody directed against eGFP. The phenotype was observed in all oocytes analysed (9 oocytes from 2 females in 2 experiments). Scale bar: 5 μm.
Supplementary information
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Supplementary Information (PDF 1327 kb)
Meiosis I in Ncaph1 wild type oocytes.
Oocytes from Ncaph1f/f females were isolated in M16 supplemented with IBMX and injected at GV stage by mRNA encoding mCherry tagged H2B histone and TALE-mClover_MajSat. After two hours of incubation to allow the expression of the injected mRNA, IBXM was washed out and oocytes followed by live cell confocal microscopy. The time (h:min:s:ms) is indicated (top right). (MOV 25068 kb)
Meiosis I in oocytes deleted for Ncaph1.
Oocytes from Ncaph1f/f Tg(ZP3Cre) females were isolated in M16 supplemented with IBMX and injected at GV stage by mRNA encoding mCherry tagged H2B histone and TALE-mClover_MajSat. After two hours of incubation to allow the expression of the injected mRNA, IBXM was washed out and oocytes followed by live cell confocal microscopy. The time (h:min:s:ms) is indicated (top right). (MOV 26065 kb)
Meiosis I in Ncaph2 wild type oocytes.
Oocytes from Ncaph2f/f females were isolated in M16 supplemented with IBMX and injected at GV stage by mRNA encoding mCherry tagged H2B histone and TALE-mClover_MajSat. After two hours of incubation to allow the expression of the injected mRNA, IBXM was washed out and oocytes followed by live cell confocal microscopy. The time (h:min:s:ms) is indicated (top right). (MOV 15781 kb)
Meiosis I in oocytes deleted for Ncaph2.
Oocytes from Ncaph2f/f Tg(ZP3Cre) females were isolated in M16 supplemented with IBMX and injected at GV stage by mRNA encoding mCherry tagged H2B histone and TALE-mClover_MajSat. After two hours of incubation to allow the expression of the injected mRNA, IBXM was washed out and oocytes followed by live cell confocal microscopy. The time (h:min:s:ms) is indicated (top right). (MOV 34800 kb)
Meiosis I in oocytes Rec8Tev/Tev wild type Ncaph2.
Oocytes from Ncaph2f/f Rec8Tev/Tev females were isolated in M16 supplemented with IBMX and injected at GV stage by mRNA encoding mCherry tagged H2B histone, eGFP-Tubulin and TEV protease. After two hours of incubation to allow the expression of the injected mRNA, IBXM was washed out and oocytes followed by live cell confocal microscopy. The time (h:min:s:ms) is indicated (top right). (MOV 11414 kb)
Meiosis I in oocytes Rec8Tev/TevΔit Ncaph2.
Oocytes from Ncaph2f/f Tg(ZP3Cre) Rec8Tev/Tev females were isolated in M16 supplemented with IBMX and injected at GV stage by mRNA encoding mCherry tagged H2B histone, eGFP-Tubulin and TEV protease. After two hours of incubation to allow the expression of the injected mRNA, IBXM was washed out and oocytes followed by live cell confocal microscopy. The time (h:min:s:ms) is indicated (top right). (MOV 8065 kb)
Meiosis I in wild type Ncaph1 and 2 oocytes.
Oocytes from Ncaph1f/f Ncaph2f/f females were isolated in M16 supplemented with IBMX and injected at GV stage by mRNA encoding mCherry tagged H2B histone and TALE-mClover_MajSat. After two hours of incubation to allow the expression of the injected mRNA, IBXM was washed out and oocytes followed by live cell confocal microscopy. The time (h:min:s:ms) is indicated (top right). (MOV 16524 kb)
Meiosis I in oocytes deleted for Ncaph1 and Ncaph2.
Oocytes from Ncaph1f/f Ncaph2f/f Tg(ZP3Cre) females were isolated in M16 supplemented with IBMX and injected at GV stage by mRNA encoding mCherry tagged H2B histone and TALE-mClover_MajSat. After two hours of incubation to allow the expression of the injected mRNA, IBXM was washed out and oocytes followed by live cell confocal microscopy. The time (h:min:s:ms) is indicated (top right). (MOV 9072 kb)
In metaphase I, the chromosome shape is not altered by TEV protease in Δ Ncaph2 oocytes rescued by wild type Ncaph2.
Oocytes from Ncaph2f/f Tg(ZP3Cre) females were isolated in M16 supplemented with IBMX and injected at GV stage by mRNA encoding mCherry tagged H2B histone, TALE-mClover_MajSat, WT-NCAPH2 and MAD2. After two hours of incubation to allow the expression of the injected mRNA, IBXM was washed out and oocytes incubated overnight. Metaphase I arrested oocytes were then injected for a second time, with mRNA encoding TEV protease and followed by live cell confocal microscopy. The time (h:min:s:ms) is indicated (top right). (MOV 3023 kb)
In metaphase I, TEV protease induces the rapid stretching of chromosomes in Δ Ncaph2 oocytes rescued by TEV-Ncaph2.
Oocytes from Ncaph2f/f Tg(ZP3Cre) females were isolated in M16 supplemented with IBMX and injected at GV stage by mRNA encoding mCherry tagged H2B histone, TALE-mClover_MajSat, TEV-NCAPH2 and MAD2. After two hours of incubation to allow the expression of the injected mRNA, IBXM was washed out and oocytes incubated overnight. Metaphase I arrested oocytes were then injected for a second time, with mRNA encoding TEV protease and followed by live cell confocal microscopy. The time (h:min:s:ms) is indicated (top right). (MOV 3353 kb)
In metaphase I, the chromosome shape is not altered by TEV protease in ΔNcaph1Δ Ncaph2 oocytes rescued by wild type Ncaph2.
Oocytes from Ncaph1f/f Ncaph2f/f Tg(ZP3Cre) females were isolated in M16 supplemented with IBMX and injected at GV stage by mRNA encoding mCherry tagged H2B histone, TALE-mClover_MajSat, WT-NCAPH2 and MAD2. After two hours of incubation to allow the expression of the injected mRNA, IBXM was washed out and oocytes incubated overnight. Metaphase I arrested oocytes were then injected for a second time, with mRNA encoding TEV protease and followed by live cell confocal microscopy. The time (h:min:s:ms) is indicated (top right). (MOV 3547 kb)
In metaphase I, TEV protease induces the rapid stretching of chromosomes in ΔNcaph1ΔNcaph2 oocytes rescued by TEV-Ncaph2.
Oocytes from Ncaph1f/f Ncaph2f/f Tg(ZP3Cre) females were isolated in M16 supplemented with IBMX and injected at GV stage by mRNA encoding mCherry tagged H2B histone, TALE-mClover_MajSat, TEV-NCAPH2 and MAD2. After two hours of incubation to allow the expression of the injected mRNA, IBXM was washed out and oocytes incubated overnight. Metaphase I arrested oocytes were then injected for a second time, with mRNA encoding TEV protease and followed by live cell confocal microscopy. The time (h:min:s:ms) is indicated (top right). (MOV 4862 kb)
In metaphase II, the chromosome morphology is not altered by TEV protease in ΔNcaph2 oocytes rescued by wild type Ncaph2.
Oocytes from Ncaph2f/f Tg(ZP3Cre) females were isolated in M16 supplemented with IBMX and injected at GV stage by mRNA encoding mCherry tagged H2B histone, TALE-mClover_MajSat and WT-NCAPH2. After two hours of incubation to allow the expression of the injected mRNA, IBXM was washed out and oocytes incubated overnight. Metaphase II arrested oocytes were then injected for a second time, with mRNA encoding TEV protease and followed by live cell confocal microscopy. The time (h:min:s:ms) is indicated (top right). (MOV 2962 kb)
In metaphase II, the chromosome morphology is not altered by TEV protease in ΔNcaph2 oocytes rescued by TEV-Ncaph2.
Oocytes from Ncaph2f/f Tg(ZP3Cre) females were isolated in M16 supplemented with IBMX and injected at GV stage by mRNA encoding mCherry tagged H2B histone, TALE-mClover_MajSat and TEV-NCAPH2. After two hours of incubation to allow the expression of the injected mRNA, IBXM was washed out and oocytes incubated overnight. Metaphase II arrested oocytes were then injected for a second time, with mRNA encoding TEV protease and followed by live cell confocal microscopy. The time (h:min:s:ms) is indicated (top right). (MOV 2064 kb)
In metaphase I, the co-injection of wild type Ncaph2 does not rescue the unravelling of the chromosomes induced by the TEV protease.
Oocytes from Ncaph2f/f Tg(ZP3Cre) females were isolated in M16 supplemented with IBMX and injected at GV stage by mRNA encoding mCherry tagged H2B histone, TALE-mClover_MajSat, TEV-NCAPH2 and MAD2. After two hours of incubation to allow the expression of the injected mRNA, IBXM was washed out and oocytes incubated overnight. Metaphase I arrested oocytes were then injected for a second time, with mRNA encoding TEV protease and WT-NCAPH2 and followed by live cell confocal microscopy. The time (h:min:s:ms) is indicated (top right). (MOV 3021 kb)
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Houlard, M., Godwin, J., Metson, J. et al. Condensin confers the longitudinal rigidity of chromosomes. Nat Cell Biol 17, 771–781 (2015). https://doi.org/10.1038/ncb3167
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DOI: https://doi.org/10.1038/ncb3167
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