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Chromosome missegregation during anaphase triggers p53 cell cycle arrest through histone H3.3 Ser31 phosphorylation

Abstract

Maloriented chromosomes can evade the spindle assembly checkpoint and generate aneuploidy, a common feature of tumorigenesis1,2,3,4. But chromosome missegregation in non-transformed cells triggers a p53-dependent fail-safe mechanism that blocks proliferation of normal cells that inadvertently become aneuploid5,6. How this fail-safe is triggered is not known7,8. Here we identify a conserved feedback mechanism that monitors missegregating chromosomes during anaphase through the differential phosphorylation of histone H3.3 at Ser319. We do this by inducing transient chromosome missegregation in diploid cells10. During anaphase, H3.3 Ser31 is phosphorylated along the arms of lagging or misaligned chromosomes. Within minutes, Ser31 phosphorylation (Ser31P) spreads to all of the chromatids of both daughter cells, which persists into G1. Masking H3.3 Ser31P by antibody microinjection prevents nuclear p53 accumulation in the aneuploid daughters. Previous work demonstrated that prolonged prometaphase and DNA damage during abnormal mitosis can activate p5311,12,13,14,15. We show that p53 activation in response to chromosome missegregation can occur without prolonged mitosis or DNA damage. Our study provides insight into how aneuploidy caused by chromosome missegregation is normally monitored and suppressed.

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Figure 1: Histone H3.3 Ser31 becomes phosphorylated along arms of misaligned chromosomes.
Figure 2: Histone H3.3 Ser31 becomes phosphorylated along arms of missegregated chromosomes in anaphase.
Figure 3: Histone H3.3 Ser31 phosphorylation spreads from individual lagging chromosomes to both nuclei following anaphase.
Figure 4: Chromosome missegregation in the absence of DNA damage.
Figure 5: Microinjection of anti-histone H3.3 Ser31P blocks p53 stabilization in cells that missegregate a chromosome.

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Acknowledgements

S.F. was supported by the Hormel Institute SURE programme. This work was supported by the Hormel Foundation, Austin ‘Paint the Town Pink’, US Department of Defense (CDMRP) grant CA130436 to E.H.H. and National Institutes of Health grant R01 CA166011 to Z.D.

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Authors and Affiliations

Authors

Contributions

E.H.H. identified the H3.3 Ser31 differential phosphorylation; E.H.H., K.T.V. and Z.D. provided the cell lines, reagents, and research resources; E.H.H., C.A.D., K.B.K., S.F. and A.L. carried out the experiments; E.H.H., C.A.D. and K.T.V. analysed the data, E.H.H., K.T.V. and Z.D. wrote the paper.

Corresponding authors

Correspondence to Edward H. Hinchcliffe or Zigang Dong.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Histone H3.3 Ser31 is phosphorylated during mitosis.

(a) Cell cycle regulation of H3.3 Ser31 phosphorylation measured with a monospecific anti-phospho Ser31 antibody (AbCam ab62207). Phosphorylation levels (fluorescence intensity) abruptly appear in prophase, persist through metaphase chromosome alignment, and decrease after anaphase onset, returning to near basal levels in the subsequent interphase. Data represent 1 out of 3 independent experiments. Bar = 5 μm. (b) Ser31 phosphorylation detection on mitotic chromosomes by antibodies is Calyculin A-dependent. BSC-1 cells treated with S-trityl-L-cysteine (kinesin-5 inhibitor) in the presence or absence of PP1/PP2A phosphatase inhibitor Calyculin A. In the absence of phosphatase inhibitor, the anti-Ser31P antibody detects phosphorylated H3.3 Ser31 at pericentromeric heterochromatin (visualized as fluorescent dots). When the same cells are co-treated with the phosphatase inhibitor, the anti-Ser31P labels the entire chromosomes (lower panels). Data represent 1 out of 3 independent experiments. Bar = 5 μm. (c) Immunoblot of isolated BSC-1 cell extracts. The anti-H3.3 Ser31P detects a single major band coincident with the band detected by an anti-H3.3 antibody. Data representative of 2 separate experiments. MW markers are in kDa. (d) H3.3 Ser31 phosphorylation patterns are sensitive to Calyculin A treatment. Upper panels: a field of cells treated with Calyculin A, with one cell just before onset of mitosis (pair of nascent spindle poles—arrows). The nucleus is decorated with anti-Ser31P, while surrounding interphase cells are not. Middle panels: cell in prometaphase/metaphase, treated with Calyculin A. The anti-Ser31P labels the whole chromosomes. Bottom panels: daughter cells treated with Calyculin A following cytokinesis, with a midbody. Both the nuclei are highly positive for Ser31P. Compare to (a) above. Bar = 5 μm in top panels, 10 μm in bottom panels. (e) Characterization of second anti-H3.3 Ser31P antibody (AbCam ab92628). Immunofluorescence comparison of anti-Ser31P labelling of BSC-1 cells in interphase, metaphase and telophase. The interphase nuclei lack H3.3 Ser31P, the metaphase cell has high labelling (arrow) while the adjacent telophase cell has decreased fluorescence intensity. Bar = 5 μm. (f) Metaphase cell with one misaligned chromosome. Bar = 10 μm. (g) Post-anaphase cell with lagging chromosomes in both astral regions (arrows). Bar = 10 μm. (h) Post-mitotic daughter cells with missegregated chromosomes that have formed a micronucleus (arrow). The micronucleus and both daughter nuclei are H3.3 Ser31P positive. Data represent 1 out of 2 independent experiments. Bar = 10 μm. (i) Immunoblot analysis of anti-H3.3 Ser31P. Equal loading of mitotic-shake off cell extracts (treated with Calyculin A) and unsynchronized cell extracts (w/o Calyculin A) were separated by SDS-PAGE. The blot was cut in half and incubated with either anti-H3.3 (total protein) or anti-H3.3 Ser31P antibodies. The ± refers to the presence or absence of Calyculin A during lysis. There is a single band in the mitotic extract detected by the anti-Ser31P antibody, coincident with the H3.3 band. The faint Ser31P band in the in unsynchronized would represent mitotic cells present in the culture. Representative of 3 separate experiments. Unprocessed gel scans of blots in panels 1c and 1i can be found in Supplementary Fig. 6.

Supplementary Figure 2 Chromosome missegregation following transient cell chilling and re-warming.

(a) Chromosome missegregation caused by chromosomes lagging in the astral region of the spindle. Frames from a time-lapse sequence of a BSC-1 H2B-GFP expressing cell following chilling/re-warming, with a single misaligned chromosome (arrow). This misaligned pair of sister chromatids undergoes disjunction at T = 39 min. After nuclear envelope reformation (T = 71 min), the missegregated chromosome forms a micronucleus. (b) Chromosome missegregation caused by chromosomes lagging in the spindle midzone. Frames from a time-lapse sequence of a BSC-1 H2B-GFP expressing cell following chilling/re-warming, which aligns all chromosomes before anaphase onset (T = 49 min). A chromosome pair lags in the central spindle (arrow). Both chromatids migrate into the right cell. After nuclear envelope reformation (T = 69 min), the missegregated chromosomes form a micronucleus. (c) Lagging chromosomes in anaphase induce nuclear H3.3 Ser31 in both daughter nuclei, even if there is no micronucleus present. Upper panels: frames from a time-lapse sequence of a post-chill/re-warmed BSC-1 H2B-GFP expressing cell. During anaphase a chromosome lags in the furrow region (arrow), but becomes engulfed in the reforming nuclear envelope at T = 40 min (25 min post-anaphase onset). Lower panels: same cell after fixation and antibody labelling. Both daughter nuclei are strongly labelled with anti-Ser31P, compared to surrounding interphase nuclei. We have identified two cells from independent experiments that show this phenotype. Bar = 5 μm.

Supplementary Figure 3 Chromosome missegregation following transient cell chilling and re-warming results in prolonged cell cycle arrest.

(a) Frames from a time-lapse sequence of a BSC-1 H2B-GFP expressing cell following chilling/re-warming, with a single misaligned chromosome (arrow). After nuclear envelope reformation, the missegregated chromosome forms a micronucleus (pseudo-colored magenta), and each daughter nucleus is pseudo-colored yellow. Both daughter cells remain in interphase for the duration of the observations (56 h, 30 min after anaphase onset). Cells in the field of view continue to undergo repeated rounds of mitosis (dividing cells pseudo-colored red), indicating that the chilling/re-warming does not affect cell cycle progression. Data represent 1 out of 3 cells independently monitored in separate experiments. T = h:min. Bar = 10 μm.

Supplementary Figure 4 H3.3 Ser31 phosphorylation on the arms of misaligned and missegregated chromosomes in RPE-1 and Cos7 cells.

(a) Immunofluorescence of a late anaphase RPE (hTERT-immortalized human retinal pigmented epithelial) cell induced by S-trityl-L-cysteine (kinesin-5 inhibitor) treatment and washout, to missegregate chromosomes (arrows). The lagging chromosomes have high H3.3 Ser31 phosphorylation levels along their arms. Data represent 1 out of 5 cells independently monitored. Bar = 5 μm. (bf) Anti-H3.3 Ser31P labelling pattern in control (untreated) mitotic RPE-1 cells. Data representative of 2 separate experiments. Phosphorylation appears as pericentromeric dots in prophase, persist through metaphase chromosome alignment (d), and decrease after anaphase onset (e, f). (g) H3.3 Ser31P decorates pericentromeric heterochromatin in Cos7 cells treated with S-trityl-L-cysteine (kinesin-5 inhibitor). Bar = 5 μm. (hj) Misaligned chromosomes in a Cos7 cell have high levels of Ser31P along their arms (arrows). Bar = 5 μm in c, = 10 μm in d. (j) Anaphase cell with a chromosome lagging in the spindle midzone (arrow). The chromosome has high levels of Ser31P along its arms. Data representative of 3 independent experiments. Bar = 10 μm.

Supplementary Figure 5 Inhibitors to Chk-1 kinase to not lower H3.3 Ser31 phosphorylation levels along missegregated chromosomes.

(a) BSC-1 cell induced to missegregate chromosomes by treatment with S-trityl-L-cysteine (kinesin-5 inhibitor) and washout in the presence of 5 μM MK-8776 (Chk-1 inhibitor). Chromosome lagging in furrow region has high Ser31 phosphorylation along the arms (arrow). (b) BSC-1 cell induced to missegregate chromosomes by treatment with S-trityl-L-cysteine (kinesin-5 inhibitor) and washout in the presence of 5 μM SB 218078 (Chk-1 inhibitor). Chromosome lagging in furrow region has high Ser31 phosphorylation along the arms (arrows). Bar = 5 μm. (c) Quantitation of Ser31 phosphorylation (fluorescence levels) of chromosomes lagging in anaphase in cells treated with S-trityl-L-cysteine, then washed out in the presence of MK-8776, DMSO alone, or SB 218078. Center bar = median intensity, whiskers (error bars) = range of intensities, the maroon box = 50th percentile, and the gold box = 75th percentile. P values (two-tailed t-test): 0.97 and 0.14. N values represent the number of individual mitotic cells analysed for each condition in 4 separate experiments.

Supplementary Figure 6 Unprocessed gel scansOriginal scans for immunoblots in Supplementary Fig. 1.

(a) Gel scan for Supplementary Fig. 1c. (b) Gel scan for Supplementary Fig. 1i.

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Hinchcliffe, E., Day, C., Karanjeet, K. et al. Chromosome missegregation during anaphase triggers p53 cell cycle arrest through histone H3.3 Ser31 phosphorylation. Nat Cell Biol 18, 668–675 (2016). https://doi.org/10.1038/ncb3348

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