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Centromeres are dismantled by foundational meiotic proteins Spo11 and Rec8

Abstract

Meiotic processes are potentially dangerous to genome stability and could be disastrous if activated in proliferative cells. Here we show that two key meiosis-defining proteins, the topoisomerase Spo11 (which forms double-strand breaks) and the meiotic cohesin Rec8, can dismantle centromeres. This dismantlement is normally observable only in mutant cells that lack the telomere bouquet, which provides a nuclear microdomain conducive to centromere reassembly1; however, overexpression of Spo11 or Rec8 leads to levels of centromere dismantlement that cannot be countered by the bouquet. Specific nucleosome remodelling factors mediate centromere dismantlement by Spo11 and Rec8. Ectopic expression of either protein in proliferating cells leads to the loss of mitotic kinetochores in both fission yeast and human cells. Hence, while centromeric chromatin has been characterized as extraordinarily stable, Spo11 and Rec8 challenge this stability and may jeopardize kinetochores in cancers that express meiotic proteins.

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Fig. 1: Spo11 and Rec8 cause KT loss.
Fig. 2: Spo11 and Rec8 require the CHD or RSC chromatin remodellers, respectively, to dismantle meiotic KTs.
Fig. 3: Expression of Spo11 or Rec8 in proliferating cells causes KT loss.
Fig. 4: Expression of Spo11 or Rec8 reduces the overall level of centromere-bound Cnp1CenpA.
Fig. 5: Expression of SPO11 and REC8 in human cells compromises centromeres.

Data availability

Source data are provided with this paper. Reagents and any further information are available on request to the corresponding authors. Source data are provided with this paper.

Code availability

The Fiji/ImageJ macro to score for the presence of a CenpA signal on either side of a CenpB bar is available (Supplementary Software) under a GNU General Public License v3.0, and has been tested on Fiji/ImageJ 2.0.0-rc-69/1.53b.

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Acknowledgements

We thank E. L. Denchi for reagents, equipment and advice for human cell experiments; I. Cheeseman for discussion and reagents for the piggyBac transposase system; our laboratory members and M. Lichten for discussions; D. Camerini-Otero, F. Pratto and K. Brick for discussions and the Spo11 antibody; A. Nussenzweig’s laboratory for sharing equipment; and M. Diaz de la Loza for help with illustrations. This work was supported by the National Cancer Institute and the University of Colorado School of Medicine.

Author information

Authors and Affiliations

Authors

Contributions

H.H. performed all of the experiments on S. pombe, together with E.K. on Fig. 4 and Extended Data Fig. 6, and with help from J.H.C. on Fig. 2 and Extended Data Fig. 2d. E.K. performed all of the experiments in human cells. R.T. developed the algorithm for semi-automated quantification of KT loss in human cells and performed the data and statistical analysis. M.K. first observed that Spo11 is required for KT dismantlement in meiosis. H.H. and J.P.C. conceived the study. H.H., E.K. and J.P.C. designed the experiments. J.P.C., H.H., E.K. and R.T. wrote the paper.

Corresponding authors

Correspondence to Haitong Hou or Julia Promisel Cooper.

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The authors declare no competing interests.

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Peer review information Nature thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Spo11 and Rec8 cause meiotic KT loss in the absence of HC.

Quantification of KT loss phenotypes as exemplified in Fig. 1a. ac, KTs are visualized with Ndc80-2xGFP (a) or Cnp3CenpC-GFP (b, c). spo11Δ and rec8Δ rescue KT loss in clr4Δ cells while deletions in other subcomplexes25 of the meiotic recombination machinery do not (a). spo11+ deletion or the DNA binding-deficient spo11-dbd mutant rescue KT loss in the absence of Swi6HP1, while spo11-cd triggers KT loss as does wt spo11 (b). rec8+ deletion, but not rec11+ deletion, suppresses KT loss in the absence of Swi6HP1, mirroring these proteins’ effects in bqt1Δ cells (c). Total numbers of films analysed are indicated above each bar. P values are determined by two-tailed Fisher’s exact tests and indicated above brackets.

Source data

Extended Data Fig. 2 Overexpression of Spo11 or Rec8 elevates KT loss rate, and chromatin remodellers are required for meiotic KT dismantlement.

Quantification of KT loss phenotypes as in Fig. 1a. a, Expressing a tandem copy of spo11+ under the control of weaker (relative to adh1 or nmt41) adh13 promoter (spo11.OE3) does not confer KT loss in a bqt1+ setting, but increases KT loss in the bqt1Δ setting. b, c, Strong overexpression of spo11+ (spo11.OE1, controlled by nmt41 promoter) or rec8+ (rec8.OE, controlled by adh1 promoter) elevates the frequency of meiotic KT loss in bqt1Δ (b) and swi6Δ (c) settings. Total numbers of films analysed indicated above each bar. P values are determined by two-tailed Fisher’s exact tests and indicated above brackets. d, Loss of a CHD remodeller (Hrp3) or RSC component (Rsc1) rescues KT loss in the absence of swi6HP1; other chromatin remodellers have no effect. Total numbers of films analysed indicated above each bar. P values are determined by two-tailed Fisher’s exact tests and indicated above brackets.

Source data

Extended Data Fig. 3 RSC facilitates Rec8 localization to meiotic centromeres.

a, Frames of representative films monitoring endogenously GFP-tagged Rec8 through meiosis are shown. During prophase, Rec8 localizes throughout the nucleus. As cells approach MI, Rec8 begins to disappear but remains concentrated at centromeres, as seen at enhanced brightness. At the end of MI, Rec8 foci are clearly visible in the wt cell but not in the rsc1Δ cell. The frames highlighted in the yellow box are shown below; these foci subsequently disappear at MII. Scale bars, 5 μm. b, Quantification of meiotic cells harbouring zero, one or two Rec8 foci at the end of MI. Total numbers of films analysed are indicated above each bar. All rsc1Δ films show diminished Rec8-GFP at the end of MI, and most of them fail to show any detectable Rec8-GFP on at least one SPB.

Source data

Extended Data Fig. 4 RSC facilitates overexpressed Rec8 localization to meiotic centromeres, and expression of Spo11 and Rec8 in proliferating cells causes KT loss as visualized via a fluorescent array on cen1.

a, Frames of representative films monitoring overexpressed Rec8-GFP through meiosis. During prophase, Rec8 localizes throughout the nucleus. As cells approach MI, Rec8 begins to disappear but remains concentrated at centromeres. At the end of MI, Rec8 foci are clearly visible in the wt cell, but not in rsc1Δ cells. The frames highlighted in the yellow box are enlarged in the right-hand panels; these foci subsequently disappear at MII. Scale bars, 5 μm. b, Quantification of meiotic cells harbouring Rec8 foci at the end of MI. Total numbers of films analysed are indicated above each bar. All rsc1Δ films show diminished Rec8-GFP at the end of MI, and most fail to show any detectable Rec8-GFP on at least one SPB. c, Top: schematic outlining the loss of KT components on cen1 in anaphase cells. Below: images of live cells in late anaphase; the KT is visualized with Ndc80-2xGFP, cen1 via cen1-tetO/R-tomato, and the spindle via mCherry-Atb2. Arrows indicate lagging cen1 with KT loss. Lagging cen1 with intact KT is also shown (right-hand panel) but is excluded from quantification. Scale bars, 5 μm. d, Quantification of cen1 KT loss as exemplified in a. Expression of Spo11 or Rec8 increase the anaphase KT loss of cen1. Total numbers of cells analysed are indicated above each bar. P values are determined by two-tailed Fisher’s exact tests and indicated above brackets.

Source data

Extended Data Fig. 5 Expression of Spo11 or Rec8 in proliferating cells causes KT loss and cell death, directly and independently of RSC.

a, Deletion of rsc1+ does not affect centromeric localization of Rec8 in proliferating cells. Representative images of proliferating cells expression Rec8-GFP based on two independent experiments. Rec8-GFP forms foci at centromeres that co-localize with SPBs. No visible change in the intensity of these Rec8 foci is observed upon rsc1+ deletion. Scale bar, 5 μm. b, Expression of Spo11 or Rec8 increases the percentage of inviable cells. Representative images of fields of cells stained with methylene blue, which cannot be exported from inviable cells. Scale bars, 10 μm. c, Quantification of 1 representative experiment (of three performed). d, Rec8 overexpression does not affect expression of other DSR-containing genes. The levels of crs1 and mei4 transcripts are elevated by red1+ deletion but not affected by rec8+ overexpression. Total RNA was extracted and analysed by Real-time PCR with primers annealing to crs1+ and mei4+. The expression level is normalized against that of act1+, and then with that of a control wt strain. Mean expression levels and standard deviations are calculated based on 3 repeats of each genotype.

Source data

Extended Data Fig. 6 Expression of Spo11 and Rec8 reduces levels of CenpA at functioning KTs.

a, b, ChIP analysis of Cnp1CenpA at centromeric central core region. Endogenously 3xFlag-tagged Cnp1CenpA was immunoprecipitated with anti-Flag antibody and analysed by Real-Time qPCR using a primer pair annealing to the central cores of chromosomes I and III. Enrichment level is normalized against that of act1+, and then with that of a control wt strain with no tag on Cnp1CenpA. Spo11 was expressed under control of nmt41 (spo11-OE1) and cells were grown under conditions that induce nmt41 (minimal media). Mitotic Cnp1CenpA enrichment at the central core is reduced upon expression of Spo11 (a) or Rec8 (b), as is the case for cells overexpressing Spo11 under the adh1 promoter, or Rec8 under adh1 control, in rich media (Fig. 4b). The values for each biological replicate are shown as grey dots. Mean enrichment and standard deviations are shown in bars. ce, Expression of Spo11 and Rec8 confers overall reduction of KT components on properly segregating chromosomes. Dotplots of Cnp1CenpA-GFP, Swi6HP1-GFP and Ndc80-GFP intensity on segregated chromosomes (see Methods). Anaphase cells without lagging chromosomes, as exemplified in the images atop each graph, were analysed, using the same strains as in Fig. 4. Arrows indicate foci whose intensities are measured. Each dot in the plot represents a distinct centromere focus. Mean intensity and standard deviation are shown for a representative experiment of two or more performed. Spo11 or Rec8 expression reduces Cnp1CenpA and Ndc80 intensity (c, e) on properly segregated anaphase centromeres, but modestly increases Swi6HP1-GFP intensity (d). P values are determined by Kolmogorov–Smirnov tests and indicated above brackets. Scale bars, 5 μm.

Source data

Extended Data Fig. 7 Expression of Rec8 leads to HC loss on lagging cen1.

a, Top: schematic of possible cen1 and HC focus behaviours in anaphase cells; HC (represented by Swi6HP1-GFP) localizes to large centromeric foci as well as multiple small subtelomeric foci. Below: images of live cells in late anaphase; HC is visualized via Swi6HP1-GFP, cen1 via cen1-tetO/R-tomato, and microtubules via mCherry-Atb2. Arrows indicate lagging cen1 with HC loss. Lagging cen1 with intact HC is excluded from quantification. Scale bars, 5 μm. b, Quantification of cen1 HC loss as exemplified in a. Expression of Rec8 causes anaphase HC loss at cen1. Total numbers of cells are indicated above each bar. P values are determined by two-tailed Fisher’s exact tests and indicated above brackets. c, Quantification of HC loss on unsegregated cen1 during meiosis. Overexpression of Rec8 during meiosis increases the incidence of unsegregated cen1 lacking detectable Swi6HP1-GFP. Total numbers of films analysed are indicated above each bar. P values are determined by two-tailed Fisher’s exact tests and indicated above brackets.

Source data

Extended Data Fig. 8 Inducible expression of SPO11 alleles and REC8 in U2OS cells.

a, Experimental setup for inducible expression of SPO11 alleles and REC8 in U2OS cells. Schematic of Tet-ON system used for inducible expression of GFP-tagged genes of interest (GOI) (upper panel). The reversible tetracycline activator (Tet-ON 3G) is constitutively expressed under regulation of the EF1α core promoter. Addition of doxycycline (dox) induces binding of the transactivator 3G (blue circles) to the TRE3G promoter, inducing expression of the GOI. b, c, Representative images of fixed cells with or without addition of doxycycline. SPO11 (b) and REC8 (c) are C-terminally tagged with GFP and nuclei are counterstained with DAPI. Scale bars, 20μm. While SPO11/REC8 localize largely to the cytoplasm, all alleles enter the nucleus as well. d, Western blots probed with anti-SPO11 or anti-GFP antibody show no detectable expression of endogenous SPO11 (predicted ~44 kDa), but show robust induced expression of the GFP-tagged SPO11 alleles (predicted size ~82.5 kDa). Loading is controlled with an antibody against tubulin, and westerns were performed three times with identical results. e, Western blot probed with anti-GFP antibody shows expression of tagged REC8 (predicted ~100 kDa) upon doxycycline induction. Loading is controlled with anti-tubulin antibody.

Extended Data Fig. 9 U2OS cells expressing SPO11 alleles and REC8 show centromeric and mitotic defects.

a, Additional images of mitoses scored for segregation defects. Chromosomes are counterstained with DAPI. Examples of lagging chromosomes or micronuclei are indicated by yellow arrows. Scale bars, 10 μm. b, Individual channels from representative image of metaphase chromosome spreads from Fig. 5c, in grayscale. KTs are stained for CENPB (green) and CENPA (magenta); chromosomes are counterstained with DAPI. Scale bar, 10 μm. c, Data for KT loss (Fig. 5d) bootstrapped as described in Methods to generate 100 estimates of %CENPB half-bars (centromeres) without CENPA per sample. Mean ± standard deviation bars are superimposed on individual data points. P values indicated above brackets are calculated by an ordinary one-way ANOVA and Sidak’s multiple comparison test (for each genotype ± dox). The data show low variability between samples of the same genotype from different experiments, justifying pooling of data from independent experiments; statistically significant differences in means are seen upon doxycycline induction.

Source data

Extended Data Fig. 10 Working model for centromere dismantlement in meiosis.

Meiotic Spo11 and Rec8 binding to chromatin (1) destabilizes CenpA nucleosomes; (2) their dismantlement propagates outwards to surrounding pericentric heterochromatin (HC) (3). (4) Telomeres, which are in close proximity to centromeres during bouquet formation in meiotic prophase, provide a microdomain that favours centromeric reassembly.

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Uncropped images of western blots shown in this study.

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Supplementary Table 1

List of total numbers of events analyzed and numbers of repeats of each experiment.

Supplementary Table 2

List of S. pombe strains used in the study.

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This zipped file contains the Fiji/ImageJ macro for semi-automated quantification of kinetochore loss in human cells, a readme file and the licence.

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Hou, H., Kyriacou, E., Thadani, R. et al. Centromeres are dismantled by foundational meiotic proteins Spo11 and Rec8. Nature 591, 671–676 (2021). https://doi.org/10.1038/s41586-021-03279-8

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  • DOI: https://doi.org/10.1038/s41586-021-03279-8

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