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Chromosome segregation errors generate a diverse spectrum of simple and complex genomic rearrangements

Nature Genetics volume 51, pages 705–715 (2019)Cite this article

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Abstract

Cancer genomes are frequently characterized by numerical and structural chromosomal abnormalities. Here we integrated a centromere-specific inactivation approach with selection for a conditionally essential gene, a strategy termed CEN-SELECT, to systematically interrogate the structural landscape of mis-segregated chromosomes. We show that single-chromosome mis-segregation into a micronucleus can directly trigger a broad spectrum of genomic rearrangement types. Cytogenetic profiling revealed that mis-segregated chromosomes exhibit 120-fold-higher susceptibility to developing seven major categories of structural aberrations, including translocations, insertions, deletions, and complex reassembly through chromothripsis coupled to classical non-homologous end joining. Whole-genome sequencing of clonally propagated rearrangements identified random patterns of clustered breakpoints with copy-number alterations resulting in interspersed gene deletions and extrachromosomal DNA amplification events. We conclude that individual chromosome segregation errors during mitotic cell division are sufficient to drive extensive structural variations that recapitulate genomic features commonly associated with human disease.

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Fig. 1: A centromere-inactivation and chromosome-selection system (CEN-SELECT) identifies cells harboring previously mis-segregated chromosomes.
Fig. 2: Mis-segregated chromosomes acquire a broad spectrum of structural genomic rearrangement types.
Fig. 3: Systematic classification of the structural rearrangement landscape.
Fig. 4: Chromosome rearrangements develop with high frequency and specificity through classical NHEJ repair.
Fig. 5: Isolation and propagation of single-cell-derived clones with genetically stable derivative chromosomes.
Fig. 6: Whole-genome sequencing reveals complex rearrangements that include the hallmark signatures of chromothripsis.
Fig. 7: Gene disruption and extrachromosomal DNA amplification from chromosome mis-segregation-induced rearrangements.

Data availability

The data that support the findings of this study are available from the corresponding authors upon request. Whole-genome-sequencing data have been deposited in the European Genome-phenome Archive under accession number EGAD00001004163.

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Acknowledgements

We thank H. Skaletsky for advice on targeting the Y chromosome, P. Rao and T. Senaratne for assistance with initial cytogenetic experiments, P. Mischel for helpful discussions, J. Santini and M. Erb for assistance with super-resolution imaging, the UCSD School of Medicine Microscopy Core (grant no. P30 NS047101) and A. Shiau for shared use of equipment, and the UCSD Animal Care Program for irradiator use. This work was supported by the US National Institutes of Health (grant no. K99 CA218871 to P.L. and no. R35 GM122476 to D.W.C.); the Wellcome Trust (grant no. WT088340MA to P.J.C. and no. 110104/Z/15/Z to S.B.); Hope Funds for Cancer Research (grant no. HFCR-14-06-06 to P.L.); the Swiss National Science Foundation (grant no. P2SKP3-171753 and no. P400PB-180790 to S.F.B.); St. Baldricks Foundation (Robert J. Arceci Innovation Award to S.B.); the NIHR UCLH Biomedical Research Centre, the UCL Experimental Cancer Centre, and the RNOH NHS Trust (to A.M.F., who is an NIHR Senior Investigator); and the Howard Hughes Medical Institute (to D.C.P.). D.W.C. receives salary support from the Ludwig Institute for Cancer Research.

Author information

Author notes

  1. Peter Ly

    Present address: Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, USA

Authors and Affiliations

  1. Ludwig Institute for Cancer Research, Department of Cellular and Molecular Medicine, University of California San Diego School of Medicine, La Jolla, CA, USA

    Peter Ly, Ofer Shoshani, Dong Hyun Kim, Weijie Lan & Don W. Cleveland

  2. Wellcome Sanger Institute, Hinxton, UK

    Simon F. Brunner, Sam Behjati & Peter J. Campbell

  3. Whitehead Institute for Biomedical Research, Cambridge, MA, USA

    Tatyana Pyntikova & David C. Page

  4. University College London Cancer Institute, London, UK

    Adrienne M. Flanagan

  5. Department of Histopathology, Royal National Orthopaedic Hospital NHS Trust, Stanmore, UK

    Adrienne M. Flanagan

  6. Department of Paediatrics, University of Cambridge, Cambridge, UK

    Sam Behjati

  7. Howard Hughes Medical Institute, Whitehead Institute for Biomedical Research, Cambridge, MA, USA

    David C. Page

  8. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

    David C. Page

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Contributions

P.L. and D.W.C. conceived the project, designed the experiments, and wrote the manuscript. P.L., O.S., and D.H.K. conducted the experiments and analyzed the data. S.F.B. and P.J.C. performed and analyzed the sequencing experiments. P.L. and W.L. prepared Xenopus egg extracts. T.P. and D.C.P. provided critical FISH reagents. S.B. and A.M.F. contributed osteosarcoma samples. All authors provided input on the manuscript.

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Correspondence to Peter Ly or Don W. Cleveland.

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Integrated supplementary information

Supplementary Figure 1 CRISPR–Cas9-mediated engineering of a neomycin-resistance gene (NeoR) into the Y-chromosome AZFa locus.

a) Step-by-step outline of genome editing strategies used to generate DLD-1 cells and their corresponding clonal derivatives used in this study. NeoR, neomycin-resistance gene. b) Schematic of CRISPR–Cas9-mediated integration of NeoR into the Y-chromosome AZFa locus. The locations of PCR primers used to identify clones with correct NeoR integration are shown. Forward primers outside the left homology arm (HA) produced a PCR product specific for correct integration. As a control, another set of forward primers within the left HA amplified both correct and non-specific integration events. Coordinates represent reference assembly GRCh38. c) PCR using the primers shown in b or the indicated sequence-tagged site (STS). Four correctly targeted clones were identified, of which the indicated clone was chosen for further modifications, as shown in a. d) Representative colony formation plate scans (n = 3 biological replicates) from 3 independent DLD-1 clones carrying a CENP-AC–H3 rescue gene, which were generated as described in a, following the indicated treatment conditions.

Supplementary Figure 2 Endogenous CENP-A reaccumulates and localizes to the Y-chromosome centromere after doxycycline/auxin washout.

a–c) DLD-1 cells carrying a CENP-AC–H3 rescue gene were treated with or without DOX/IAA followed by washout for the indicated duration. a) Whole-cell lysates were immunoblotted with antibodies against CENP-A or GAPDH as a loading control. b,c) Representative immunofluorescence images using antibodies against CENP-A and GFP on b) interphase cells or c) mitotic cells. Anti-GFP antibody specifically recognizes endogenous, wild-type CENP-A fused to an EYFP-AID tag. Scale bars, 5 µm. d) DLD-1 cells carrying a CENP-AC–H3 rescue gene were treated with DOX/IAA for 3 d followed by 4-d washout and selection in G418 for 10 d. Cells were processed for immunofluorescence with a GFP antibody, followed by DNA FISH using a Y centromere-specific probe. Magnified insets show positive GFP signals detected at the Y centromere. Scale bar, 10 µm.

Supplementary Figure 3 Recognition of micronuclear DNA damage without active DNA double-strand-break repair.

a) Representative immunofluorescence images of DLD-1 cells treated with 1 µM doxorubicin for 1 h or DOX/IAA for 3 d stained with antibodies against the indicated DNA damage components. Scale bar, 5 µm. b) Representative immuno-FISH images of DLD-1 cells treated with 1 µM doxorubicin for 1 h or DOX/IAA for 3 d. Cells were processed for immunofluorescence with γH2AX and 53BP1 antibodies followed by DNA FISH using a Y chromosome paint probe. Scale bar, 5 µm.

Supplementary Figure 4 Most micronuclei disassemble their nuclear envelopes with normal kinetics during mitotic entry.

a) Experimental schematic for nuclear envelope breakdown assay using Xenopus egg extracts, which contain abundant kinases whose activity can disassemble typical nuclear membranes and promote chromosome condensation, the latter visualized by H2B–mRFP. b) Time-lapse imaging of semi-permeabilized RPE-1 cells expressing lamin A–GFP and H2B–mRFP following the addition of interphase or CSF-arrested egg extracts. Scale bar, 5 µm. c) Lamin A–GFP disassembly kinetics following the addition of interphase or CSF-arrested egg extract (mean ± SEM). d-e) Lamin A–GFP disassembly kinetics following CSF-arrested egg extract addition for d) primary nuclei and e) micronuclei. Inset depicts the overlay between primary nuclei and micronuclei with normal disassembly. Green lines represent mean ± SEM, and red lines indicate individual examples of micronuclei with partial or delayed lamin A–GFP disassembly. f) Example image series of a micronucleus that fails to efficiently disassemble its nuclear envelope upon addition of CSF-arrested egg extracts. Scale bar, 2 µm. g) Quantification of the frequency of micronuclei undergoing normal or delayed disassembly in the presence of CSF-arrested egg extracts. Data in c–e and g represent n = number of nuclei or micronuclei as indicated.

Supplementary Figure 5 Schematic for the generation of alternating FISH patterns by chromothripsis or a series of inversions.

(associated with Fig. 2d)

Supplementary Figure 6 Population dynamics during chronic centromere inactivation and selection.

a) Experimental schematic for chronic centromere inactivation. p, passage; p0, starting population; p0’, final passage of cells selected with G418 without Y centromere inactivation. b-c) Measurements of b) cumulative cell doublings and c) population doubling time at each passage. d) Quantification of Y chromosome location from interphase cells hybridized to whole Y chromosome paint probes (n = number of cells examined). e) Quantification of Y chromosome structural status from metaphase spreads hybridized to MSY/YqH FISH probes (n = number of metaphases examined). f) Examples of a rare ring chromosome and uncharacterized derivative chromosome following prolonged CEN-SELECT.

Supplementary Figure 7 Chromosomal rearrangements generated from ionizing-radiation exposure or mitotic spindle assembly checkpoint inactivation.

(associated with Fig. 4a). a-b) Increasing doses of ionizing radiation were used to induce widespread DNA damage, which produced co-localized nuclear foci of γH2AX and 53BP1. DLD-1 cells were treated with the indicated doses of radiation and fixed after recovery for 30 min. Cells were processed for immunofluorescence using antibodies against γH2AX and 53BP1. Quantification of co-localized foci (n = number of cells examined per dose) is shown in a, and representative images are shown in b. Scale bar, 5 µm. c-d) An inhibitor of the Mps1 mitotic kinase (reversine) was used to inactivate the spindle assembly checkpoint during mitosis to drive premature anaphase onset. DLD-1 cells stained with SiR-DNA were treated with the indicated doses of reversine and imaged by time-lapse microscopy. Quantification of mitotic duration is shown in c (n = number of cells examined per dose; NEBD, nuclear envelope breakdown), and the frequency of mitotic events developing chromosome segregation errors is shown in d. At the highest concentration used (800 nM), nearly half of mitoses resulted in errors with micronuclei detected in ~10% of cells. e) Experimental schematic for Fig. 4a. f) Examples of irradiated cells showing structural rearrangements for the indicated chromosomes by metaphase FISH. Scale bar, 5 µm. g) Sample sizes for each condition and set of probes for Fig. 4a. h) The total number of inter- and intra-chromosomal rearrangements observed for the indicated conditions.

Supplementary Figure 8 Molecular sequence features of chromosomal-rearrangement breakpoint junctions.

a) Bar graph depicting the total number of structural variants detected per chromosome normalized to chromosome size and copy-number. Data were pooled from all 20 sequenced clones. Inner pie chart shows the distribution of each Y-Y junction orientation. b) Scatter plot depicting the absolute number of structural variants per chromosome compared to its length. c) Distribution of 71 breakpoints reconstructed from 817 split-end reads with the indicated lengths of microhomology for Y-chromosome specific or non-Y chromosome junctions compared to microhomology lengths from 7,100 randomly simulated junctions (see Methods). Data were compiled from all sequenced clones. d) Distribution of microhomology or insertion lengths with each dot representing an individually reconstructed Y-Y breakpoint junction. e) Schematic of intra-DSB distance measurements (left) and distribution of Y chromosome fragment sizes pooled across all sequenced clones (right). f) Rainfall plot of substitution patterns across the MSY region from four clones. Red dots below the line represent sites of consecutive C>T basepair substitutions with a 1 kb intermutation distance.

Supplementary Figure 9 Cytogenetic verification of rearrangement partners identified by whole-genome sequencing.

Representative metaphase FISH images of the indicated clones hybridized to the corresponding chromosome paint probes. Scale bar, 5 µm.

Supplementary Figure 10 Y chromosome rearrangement and DNA copy-number profiles of additional clones.

Red vertical line indicates position of the integrated NeoR gene. All three inter-chromosomal translocations shown were confirmed by metaphase FISH. X-axes are clipped at 30 Mb to exclude the Yq heterochromatic region.

Supplementary Figure 11 Patients with osteosarcoma presenting complex Y chromosome rearrangements coupled to interchromosomal translocations.

a–c) X-axes are clipped at 30 Mb to exclude the Yq heterochromatic region. Vertical grey lines represent inter-chromosomal rearrangements with a) chromosome 11, b) chromosomes 1, 19, and 22, and c) chromosome 15. d) Distribution of each Y-Y junction orientation (n = number of breakpoint junctions). e) Distribution of microhomology or insertion lengths with each dot representing an individually reconstructed Y-Y breakpoint junction. f) Distribution of intra-DSB distances pooled across all three examples.

Supplementary Figure 12 DNA copy-number analyses of two somatically expressed Y-chromosome genes.

a) DNA copy-number plots for KDM5D and EIF1AY across all clones obtained by sequencing. b) Magnified DNA copy-number profiles at the KDM5D or EIF1AY gene regions, represented by red shading, in the indicated clones. c) Reverse transcription polymerase chain reaction (RT–PCR) analysis of KDM5D, EIF1AY, or GAPDH transcripts from the indicated clones or female HEK-293 cells as a negative control. Bar graphs show the copy-number status of the respective gene.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 and Supplementary Note

Reporting Summary

Supplementary Table 1

Summary of DLD-1 clones analysed by cytogenetics and whole-genome sequencing.

Supplementary Table 2

Breakpoint junctions with microhomology or non-templated DNA insertion sequences.

Supplementary Table 3

List of primer sequences.

Supplementary Video 1

Normal micronuclear envelope disassembly during mitosis.

Supplementary Video 2

Aberrant micronuclear envelope disassembly during mitosis.

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Ly, P., Brunner, S.F., Shoshani, O. et al. Chromosome segregation errors generate a diverse spectrum of simple and complex genomic rearrangements. Nat Genet 51, 705–715 (2019). https://doi.org/10.1038/s41588-019-0360-8

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