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Selective Y centromere inactivation triggers chromosome shattering in micronuclei and repair by non-homologous end joining


Chromosome missegregation into a micronucleus can cause complex and localized genomic rearrangements1,2 known as chromothripsis3, but the underlying mechanisms remain unresolved. Here we developed an inducible Y centromere-selective inactivation strategy by exploiting a CENP-A/histone H3 chimaera to directly examine the fate of missegregated chromosomes in otherwise diploid human cells. Using this approach, we identified a temporal cascade of events that are initiated following centromere inactivation involving chromosome missegregation, fragmentation, and re-ligation that span three consecutive cell cycles. Following centromere inactivation, a micronucleus harbouring the Y chromosome is formed in the first cell cycle. Chromosome shattering, producing up to 53 dispersed fragments from a single chromosome, is triggered by premature micronuclear condensation prior to or during mitotic entry of the second cycle. Lastly, canonical non-homologous end joining (NHEJ), but not homology-dependent repair, is shown to facilitate re-ligation of chromosomal fragments in the third cycle. Thus, initial errors in cell division can provoke further genomic instability through fragmentation of micronuclear DNAs coupled to NHEJ-mediated reassembly in the subsequent interphase.

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Figure 1: An inducible CENP-A replacement strategy enables functional and selective inactivation of the Y chromosome centromere in human cells.
Figure 2: Y centromere inactivation triggers Y chromosome missegregation and accumulation into micronuclei.
Figure 3: Chromosomes in micronuclei are susceptible to extensive and catastrophic shattering in mitosis.
Figure 4: Shattered chromosomal fragments are re-ligated by canonical non-homologous end joining.
Figure 5: Repair by non-homologous end joining does not occur efficiently within micronuclei.


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We thank K. Jepsen and the UC San Diego IGM Genomics Center (MCC P30 CA023100) for DNA library preparation and sequencing, A. Shiau, S. Dowdy, E. Hatch, M. Hetzer, X. Wu and T. Pyntikova (Whitehead Institute, USA) for providing reagents, D. Jenkins, I. Goyal and Y. Sun for technical assistance, and the UC San Diego School of Medicine Microscopy Core (NINDS P30 NS047101) for shared use of equipment. This work was funded by a grant from the NIH (GM029513) to D.W.C., who receives salary support from the Ludwig Institute for Cancer Research. D.C.P. is supported by the Howard Hughes Medical Institute and NIH (HG007852). P.L. was supported by a Cancer Cell Biology Training Grant from the NCI (5T32CA067754-18) and a Postdoctoral Fellowship from the Hope Funds for Cancer Research (HFCR-14-06-06).

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Authors and Affiliations



P.L. and D.W.C. conceived the project, designed the experiments, and wrote the manuscript. P.L. conducted the experiments and analysed the data. D.F. constructed the parental AID-tagged CENP-A cell line and provided key experimental input. P.L. and D.H.K. performed purification of micronuclei. O.S. assisted with FISH experiments. L.S.T., H.S. and D.C.P. analysed the sequencing data. All authors contributed comments on the final manuscript.

Corresponding author

Correspondence to Don W. Cleveland.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Construction of human DLD-1 cells with auxin-degradable CENP-AAID and a doxycycline-inducible CENP-AC−H3 rescue that is capable of maintaining centromere identity and function.

(a) Amino acid sequence of wild-type CENP-A (WT) and the carboxy-terminal tail chimera (CH-3) swapped with the corresponding tail of histone H3. Schematic not drawn to scale. CATD; centromere targeting domain. (b) Schematic for the construction of DLD-1 cell lines used in all experiments. (c) Unfixed DLD-1 CENP-AEYFP−AID cells stably expressing H2B-mRFP were imaged 2d after IAA addition. Scale bar, 5 μm. (d) DLD-1 cells as in (b) were treated with combinations of dox and IAA for 24 h and whole-cell extracts were analyzed by immunoblotting for CENP-A. The predicted molecular weight of CENP-A fused to an EYFP-AID tag is 66 kDa. (e) Representative immunofluorescent images of engineered DLD-1 cells treated with combinations of dox and IAA for 24 h. Both CENP-AWT and CENP-AC−H3 rescues correctly localized to centromeres. Enlarged images of CENP-A staining following dox/IAA addition is shown below. ACA; anti-centromere antibodies. Scale bar, 5 μm. (f) Dox-inducible CENP-A is expressed at low basal levels without supplemented doxycycline, allowing for the simultaneous addition of dox and IAA without epigenetic loss of centromere identity. (g) CENP-AC−H3-rescued cells are capable of sustaining long-term clonal growth and viability using a 2-week colony formation assay. Data were normalized to untreated cells and represent the mean ± SEM of n = 3 independent experiments each performed in biological triplicate. Asterisks indicate significance by two-tailed Student’s t-test compared to untreated cells. P = 0.0019, NS = not significant. (h) Quantification of DLD-1 CENP-AC−H3 cell growth rate with or without dox/IAA over a 9d period performed in biological triplicate. Line represents linear regression analysis. (i) Estimated doubling time calculated from h. (j) 5d CENP-AC−H3-rescued cells were subjected to propidium iodine staining followed by flow cytometry analysis for DNA content with and without 6 h treatment with 100 ng ml−1 nocodazole. Source data for g and h have been provided in Supplementary Table 1.

Supplementary Figure 2 Induced Y centromere inactivation provokes Y chromosome missegregation into micronuclei.

(a) DLD-1 cells were rescued with CENP-AWT or CENP-AC−H3 for 5d and the percentage of micronucleated cells were quantified by DAPI staining. Data represent the mean ± SEM of n = 3 independent experiments (1,453–1,945 cells per condition). P-values indicate significance by two-tailed Student’s t-test compared to untreated cells. (b) DLD-1 cells were rescued with CENP-AWT and CENP-AC−H3 for 5d and micronuclei were quantified for the percentage harboring centromere Y or centromere 4 signal(s). Data represent the mean ± SEM of n = 3 independent experiments (380–754 micronuclei) or the mean of 2 independent experiments (CENP-AWT, dox/IAA; 290 micronuclei). P-values indicate significance by two-tailed Student’s t-test compared as denoted. (c) Comparison between the frequency of cells with the specified chromosome in micronuclei when treated as indicated by extrapolating the percentage of micronucleated cells and the percentage of micronuclei containing either chromosome Y or 4. (d) Quantification of the number of Y centromere foci observed in spontaneously-derived or induced micronuclei following 5d CENP-AC−H3 rescue. Data represent the mean ± SEM of n = 65 (WT, -dox/IAA), 76 (WT, + dox/IAA), 67 (C-H3, − dox/IAA) or 102 (C-H3, + dox/IAA) micronuclei. (e) Comparison between the number of Y centromere foci per micronucleus and micronuclear diameter from 5d CENP-AC−H3-rescued cells. Data were compiled from n = 150 micronuclei pooled from 3 independent experiments, and means are indicated by the line. R2-value represents correlation of size and foci number by linear regression analysis. (f) Summary of cellular characteristics comparing untreated (CENP-AEYFP−AID) cells with dox/IAA-treated (CENP-AC−H3) cells. Source data for a and b have been provided in Supplementary Table 1.

Supplementary Figure 3 Induced Y chromosome micronuclei share common features of spontaneously derived micronuclei including micronuclear envelope disruption and the acquisition of DNA damage.

(a) DLD-1 CENP-AC−H3 cells stably expressing 2xRFP-NLS treated with or without 5d dox/IAA (experimentally versus spontaneously derived micronuclei, respectively) were fixed and DAPI-stained. Representative images from dox/IAA-treated cells and quantifications for micronuclear RFP compartmentalization are shown on the left. Data on the right panel represent the mean of 2 independent experiments (166–294 total micronuclei). Scale bar, 5 μm. (b) DLD-1 CENP-AC−H3 cells treated with 5d dox/IAA were immunostained for the DNA damage marker γH2AX and nuclear envelopes with Lamin B1. Representative images (scale bar, 5 μm) of micronuclei without and with varying degrees of detectable DNA damage signals are shown. (c) γH2AX fluorescent signal intensities from b were measured from 200 micronuclei (pooled from 3 independent experiments) and individually plotted. a.u., arbitrary units. Source data for a and c have been provided in Supplementary Table 1.

Supplementary Figure 4 Characterization of chromosome fragmentation events and induction of premature chromosome condensation using calyculin A.

(a) Representative example of interphase cells hybridized to Y chromosome paint (green) and Y centromere (red) FISH probes following 3d CENP-AC−H3 rescue. Scale bar, 10 μm. (b) Additional example of Y chromosome fragmentation event derived from 3d CENP-AC−H3-rescued cells. Scale bar, 10 μm. (c) Centromere and fragment counts from Fig. 3f, g were cross-plotted per mitotic shattering event. Red line indicates linear regression analysis. (d) Representative image of DAPI-stained, metaphase-like spreads induced by 1 h treatment with calyculin A, showing examples for G1-, S-, and G2-phase spreads. G1-phase chromosomes appear as single chromatids, S-phase appears as highly pulverized and abnormal nuclei (and excluded from quantitative analyses), and G2-phase appears as normal mitotic chromosomes with two distinguishable sister chromatids. Scale bar, 25 μm. (e) DLD-1 cells treated with 1 μM of the CDK4/6 inhibitor PD-0332991 (also known as Palbociclib) or 10 μM of the CDK1 inhibitor RO-3306 for 24 h were subjected to propidium iodine staining followed by flow cytometry analysis for DNA content. (f) Experimental schematic for panels shown in Fig. 4b, d, e, f, g, h.

Supplementary Figure 5 Paired-end sequencing information for each source of DNA.

(a) Base pair sizes (mean ± SD, n = 1,000 reads) of sequencing fragments for each sample. (b) Percentage of sequencing reads in which both ends of a pair mapped to the reference genome following removal of duplicate and mitochondrial reads. (c) The concentration of discordant sequencing reads for each chromosome follows a second order reaction and rises as the square of the concentration of total sequencing reads (see Methods). Each dot represents a single chromosome from three independent genomic or micronuclear DNA samples, and the green dot indicates the Y chromosome. The curved line shows a predictive model of discordant pairs that is described under the Methods section. Source data for a and b have been provided in Supplementary Table 1.

Supplementary Figure 6 Unprocessed film scans of all immunoblots with corresponding protein size markers.

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Ly, P., Teitz, L., Kim, D. et al. Selective Y centromere inactivation triggers chromosome shattering in micronuclei and repair by non-homologous end joining. Nat Cell Biol 19, 68–75 (2017).

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