PMC

(A and B) The myeloid cells with premature sister chromatid separation are frequently seen in PB smears (A) and BM (B) of Asxl1^+/− and Asxl1^−/− mice with MDS. Red arrows indicate the abnormal nuclear bridging. Scale bars, 5 μm (A) and 10 μm (B). (C and D) Representative cells with premature sister chromatid separation in cultured WT, Asxl1^+/−, and Asxl1^−/− LK cells. Red arrows indicate the premature sister chromatid separation. The frequency of cells with premature sister chromatid separation is shown in (C). Y axis shows the percentage of cells with premature sister chromatid separation within all binucleated cells. Data are represented as means ± SEM from three independent experiments. ***P < 0.001 and **P < 0.01. Scale bars, 5 μm. (E) The frequency of cells with premature sister chromatid separation in the HeLa^GFP-H2B cells with hASXL1 KD and hASXL1 KD plus mAsxl1 rescues. KD of ASXL1 leads to increased frequency of cells with premature sister chromatid separation in HeLa^GFP-H2B cells. Reintroducing full-length mAsxl1 rescued the premature sister chromatid separation in HeLa cells with ASXL1 KD. Data are represented as means ± SEM from three independent experiments. ***P < 0.001 and **P < 0.01. (F and G) SMC1A or RAD21 KD leads to premature sister chromatid separation in HeLa^GFP-H2B cells. Representative photomicrographs show the cells with premature sister chromatid separation, as indicated by red arrowheads (G). The frequency of cells with premature sister chromatid separation is shown in (F). Y axis shows the percentage of cells with premature sister chromatid separation within all binucleated cells. Data are represented as means ± SEM from three independent experiments. ***P < 0.001 and **P < 0.01. Scale bars, 5 μm. (H) Western blotting shows the expression of full-length ASXL1 and ASXL1 amino acids 401 to 587 in HeLa^GFP-H2B cells transfected with vector only, full-length ASXL1, or ASXL1 amino acids 401 to 587. β-Actin serves as loading control. (I and J) ASXL1 amino acids 401 to 587 induce chromatin bridging in HeLa^GFP-H2B cells. Quantification of the frequency of cells with premature sister chromatid separation in HeLa^GFP-H2B cells transfected with pcDNA3.1⁺, full-length ASXL1, or ASXL1 amino acids 401 to 587 (J). Data are represented as means ± SEM from three independent experiments. **P < 0.01 for ASXL1 amino acids 401 to 587 fragment versus pcDNA3.1⁺ or full-length ASXL1. Red arrows indicate the premature sister chromatid separation. Scale bars, 5 μm.

A, Examples of chromatid breaks 3 h after 0.5 Gy γ-irradiation. The nascent chromatid breaks (as indicated by arrows) were identified by the chromatid gap larger than the width of the chromatid and the lack of telomere signals. The short arrows indicate the subtle terminal chromatid breaks. Telomeres were detected by red signals and DNA was stained blue by DAPI. B and C, Frequencies of chromatid breaks before or after γ-irradiation. Error bars represent standard deviations. For all time points analyzed, the frequencies of chromatid breaks in LMP1-expressing cells were significantly higher (P<0.05) than empty vector-infected cells.

In the absence of BRCA1 or BRCA2 function, chromatid breaks accumulate, resulting in aberrant chromatid exchanges or other processes involving illegitimate end-joining. If two chromatid breaks are joined to produce a chromosome structure containing two centromeres, a dicentric quadri-radial chromosome is formed, which leads to cell death at mitosis. If an exchange is made with a chromatid fragment without a centromere, processing and cell division can produce a viable cell with a translocation. All of the hallmarks of BRCA-deficient cancers can be explained by the production of chromatid breaks and illegitimate end-joining. Without exchange events between different chromosomes, interstitial deletions, terminal deletions and insertions of chromosome fragments can originate from the chromatid break. In the absence of homologous recombination, the resulting phenotypes can be seen either by spectral karyotyping or by array-comparative genomic hybridization (aCGH), which detects large losses and gains across the genome.

A: Frequencies of chromatid breaks measured at the end of APH (+) or DMSO (−) treatment. P≤0.05 for all frequencies after APH treament compared with DMSO-treated cells. B: Examples of chromatid breaks (indicated by arrows). The upper panel shows non-pericentromeric chromatid breaks in fragile sites. The lower panel shows pericentromeric chromatid breaks.