53BP1 can limit sister-chromatid rupture and rearrangements driven by a distinct ultrafine DNA bridging-breakage process

Abstract

Chromosome missegregation acts as one of the driving forces for chromosome instability and cancer development. Here, we find that in human cancer cells, HeLa and U2OS, depletion of 53BP1 (p53-binding protein 1) exacerbates chromosome non-disjunction resulting from a new type of sister-chromatid intertwinement, which is distinct from FANCD2-associated ultrafine DNA bridges (UFBs) induced by replication stress. Importantly, the sister DNA intertwinements trigger gross chromosomal rearrangements through a distinct process, named sister-chromatid rupture and bridging. In contrast to conventional anaphase bridge-breakage models, we demonstrate that chromatid axes of the intertwined sister-chromatids rupture prior to the breakage of the DNA bridges. Consequently, the ruptured sister arms remain tethered and cause signature chromosome rearrangements, including whole-arm (Robertsonian-like) translocation/deletion and isochromosome formation. Therefore, our study reveals a hitherto unreported chromatid damage phenomenon mediated by sister DNA intertwinements that may help to explain the development of complex karyotypes in tumour cells.

Fig. 1

53BP1 depletion leads to increased chromosome non-disjunction in human cancer cells. Quantitation of anaphase bridge and lagging chromatin in 53BP1Δ and 53BP1^hypo cells a U2OS, b HeLa and c RPE1. Numbers of cell counted: U2OS = 529, B4 = 327, B18 = 344, D29 = 372; HeLa = 540, B2 = 351, D4 = 426, D10 = 317; RPE1 = 435, B7 = 449, D6 = 382, D24 = 458 from 3–4 separate preparations. d IR sensitivity assay on HeLa and B2 (53BP1^hypo) cells (N =  three independent experiments). Statistical significance was determined by two-way ANOVA. e The formation of IR-induced DNA damage foci in HeLa and B2 (53BP1^hypo) cells. Thirty minutes post 2 Gy IR, the cells were immunostained with anti-53BP1 and anti-γH2AX antibodies. Enlarged regions demonstrating the recruitment of 53BP1 (red) at the IR-induced DNA breaks, marked by γH2AX (green). f Representative image of telomere fusions on metaphase chromosomes of B2 (53BP1^hypo) cells overexpressing TRF2^ΔBΔM. An example of fusions on single (arrowheads) and both (arrows) sister telomeres indicated (left). Middle: percentage of chromosome fusion events in HeLa, B2 (53BP1^hypo), D4 and D10 (53BP1Δ) cells, >75 metaphases of each cell line were analysed from three independent experiments. Right: histogram showing telomere fusion events in HeLa, B2 (53BP1^hypo), D4 and D10 (53BP1Δ). Total number of chromosomes analysed in HeLa = 3890, B2 = 3792, D4 = 4720 and D10 = 4790 from >60 metaphase spreads. Statistical significance was determined by T-test (* p < 0.0, ** p < 0.01, *** p < 0.001, ns nonsignificant). Error bars represent s.d. of three independent experiments. Scale bars, 5 μm

Fig. 2

Chromosome non-disjunction in 53BP1-depleted cancer cells is mediated by ultrafine DNA bridges. a Representative images of DAPI-stained HeLa D4 (53BP1Δ), HeLa B2 (53BP1^hypo) and U2OS B18 (53BP1Δ) cells showing anaphase bridges, bridge-like structures and lagging chromatin pairs (arrowheads). Insets show enlarged view of the numbered cells exhibiting bridge-like (arrows; 1 & 3), bridge structures (arrow; 2). b Increased formation of multi-lagging chromosomes in HeLa B2 (53BP1^hypo) cells as compared to HeLa. Quantification of single or multiple lagging chromatin in HeLa and B2 (53BP1^hypo) cells. more than 100 cells with lagging chromatin were counted from three separate preparations. c Deconvolved high-resolution images showing the two separating chromatin arms (red) connected by PICH-UFBs (green) in HeLa B2 (53BP1^hypo, Left) and in U2OS B18 (53BP1Δ, Right). Insets shows enlarged views of the selected region. d Deconvolved image showing hRIF1 (red) localises at a PICH-coated UFB (green), intertwining a pair of lagging chromatin in HeLa B2 (53BP1^hypo). Inset shows enlarged view of selected region. e Percentage of mid-anaphase cells with PICH-UFBs in U2OS B18 (53BP1Δ), HeLa D4 (53BP1Δ) and HeLa B2 (53BP1^hypo). Numbers of anaphase counted: U2OS = 91, B18 = 90; HeLa = 123, D4 = 105; HeLa = 138, B2 = 139 from three separate preparations. f Average number of PICH-UFB per mid-anaphase cell in U2OS B18 (53BP1Δ), HeLa D4 (53BP1Δ) and HeLa B2 (53BP1^hypo). Error bars represent s.d. of three independent experiments. Statistical significance was determined by T-test (* p < 0.05, ** p < 0.01, *** p < 0.001). Scale bars, 5 μm

Fig. 3

DNA entanglements between sister chromatids are the major cause of chromosome non-disjunction in 53BP1^hypo cells. a Diagram depicts differential sister-chromatid labelling (green; EdU) to distinguish between anaphase bridges caused by (i) inter-chromosomal fusion, shown as non-symmetric or by (ii) DNA intertwining between sister chromatids, shown as symmetric. b Experimental setup of differentially labelling sister chromatids. c Quantitation of anaphase DAPI bridges with symmetrical and non-symmetrical staining patterns in HeLa B2 (53BP1^hypo) cells. Over 65 anaphase cells with chromatin bridges were counted from two independent experiments. Total numbers of DNA bridge (n = 74) were counted. d Representative image of a HeLa B2 (53BP1^hypo) cell showing a symmetrical (but opposite) staining pattern along a DAPI anaphase bridge (Top). Yellow arrow in the inset shows EdU (green) staining was present on only one-half of the bridge. Area chart displaying the relative intensity along the differentially labelled sister chromatids (Bottom). e Representative example of a chromatin bridge, linked by a PICH-UFB (red), displaying a staining pattern resembling sister-chromatid exchange (SCE) (Top). Yellow arrows in the inset indicates the sister-chromatid exchange positions. Area chart displaying the relative intensity of differentially labelled sister chromatids with sister-chromatid exchange regions (Bottom). f A pair of lagging chromosomes showing differential staining (green arrow) were inter-linked by a PICH-UFB (red arrow) in HeLa B2 (53BP1^hypo) cells. g Quantitation of dicentric and radial chromosomes in HeLa B2 (53BP1^hypo) and HeLa D4 (53BP1Δ) metaphase cells. Top: an example of a dicentric chromosome in B2 (53BP1^hypo) cells (arrows indicate centromeres). Over 6000 chromosomes in 88 metaphase spreads from three independent experiments were examined. Chromosomes were hybridised with telomere and centromere PNA FISH probes. DNA was stained with DAPI. Scale bars, 5 μm

Fig. 4

The formation of sister DNA entanglements in 53BP1-depleted HeLa cells is dependent on RAD51. a Quantitation of 53BP1-depleted HeLa (left) and U2OS (right) anaphase cells forming FANCD2-negative UFBs. Numbers of anaphase counted: HeLa = 135, B2 = 112, D4 = 105; U2OS = 91, B18 = 90 from three independent experiments. b Maximum z-projection high-resolution image showing multiple short FANCD2-negative PICH-coated UFBs (arrows), linking the separating chromatin and lagging chromosomes in B2 (53BP1^hypo) cells. Inset shows that PICH stained UFBs (green) are not associated with FANCD2 foci (red). c Maximum z-projection high-resolution image showing the association of FANCD2 foci (red) on PICH-UFBs (green) in aphidicolin-treated HeLa B2 (53BP1^hypo) cells. Inset shows enlarged view of PICH-coated UFBs are positive of FANCD2 foci at their termini. d Representative images showing γH2AX present on chromatin bridges and lagging chromatin pairs in HeLa 53BP1^hypo cells. Left: maximum z-projection image showing γH2AX (red) at the junction (arrows) of the differentially labelled sister-chromatid bridges (EdU; green). Right: a pair of lagging sister chromatin intertwined by a PICH-UFB (red) and positive of γH2AX (blue) at their termini. Bottom Right: panels showing single-plane images of the intertwining lagging sister chromatin. Blue arrows indicate γH2AX present at the tips of the chromatin. e HeLa B2 (53BP1^hypo) cell showing the presence of γH2AX signals (red) at the termini of chromatin that were tethered by PICH-coated UFBs (green). f HeLa B2 (53BP1^hypo) cells were transfected with control or Rad51 siRNA oligos, followed by IF analysis using anti-Rad51. Nuclei are outlined (grey). g RAD51 knockdown caused the formation of FANCD2-assoicated (red) PICH-UFBs (green) in HeLa B2 (53BP1^hypo) cells. h Quantitation of HeLa B2 (53BP1^hypo) anaphase cells with FANCD2-positive UFBs following RAD51 knockdown. i Quantitation of HeLa B2 (53BP1^hypo) anaphase cells with FANCD2-negative UFBs following RAD51 knockdown. Numbers of anaphase cells scored: B2 + control siRNA = 350, B2 + RAD51 siRNA = 220 from three independent experiments. j A model showing the potential roles of 53BP1 and RAD51 in the formation of FANCD2-negative sister DNA bridges in the 53BP1-depleted cells. Error bars represent s.d of three independent experiments. Statistical significance was determined by T-test (* p < 0.05, ** p < 0.01, *** p < 0.001). Scale bars, 5 μm

Fig. 5

Sister-chromatid rupture is associated with HR-mediated DNA intertwining. a Diagram depicting the formation of telomere-positive DNA bridges, resulting from DNA entanglements between sister chromatids. b A single-plane high-resolution image showing the presence of telomeres (red) at the termini of chromatin tethered by an UFB (PICH; green) in a HeLa B2 (53BP1^hypo) anaphase cell. c Maximum z-projection image of an UFB-tethered chromatin bridge missing telomeric regions (asterisks) at their terminal ends in HeLa B2 (53BP1^hypo) (left) and in U20S B18 (53BP1Δ) cells (right). Insets show enlarged images of the DNA/chromatin bridges. d Quantitation of DAPI bridges with and without telomeres in HeLa, HeLa 53BP1^hypo, U2OS 53BP1Δ and in HeLa 53BP1^hypo cells overexpressing TRF2^ΔBΔM. Note: Majority of DNA bridges are negative for telomere signals in 53BP1-depleted cells, except after TRF2^ΔBΔM overexpression. Total numbers of DAPI bridge analysed were B2 = 85, B2 + TRF2^ΔBΔM = 60, D4 = 45 and B18 = 55 from three independent experiments. Statistical significance was determined by T-test (***, p < 0.001). e A representative image showing telomeres were detected on chromatin bridges induced by telomere end-joining. Inset indicates the presence of telomere signals (green) at chromatin bridges (arrows). f Representative images of HeLa B2 (53BP1^hypo) cells showing UFBs tethering a pair of lagging chromatin at their termini, at where telomere (red) signals are absent (asterisks), but remained connected by PICH-UFBs. Insets showing consecutive single z-plane images of the lagging chromatin. g Quantitation of telomeres present at one or both ends of lagging chromatin pairs. Note that all lagging chromatin pairs lack one telomere end in HeLa B2 (53BP1^hypo) and D4 (53BP1Δ) cells. In contrast, HeLa 53BP1^hypo cells stably overexpressing GFP-tagged 53BP1 (B2G53BP1) cells contain lagging chromatin having telomere signals at both, or single ends. Total numbers of lagging chromatin pair analysed were B2 = 35, D4 = 41 and B2G53BP1 = 44, from three independent experiments. h Representative images of B2G53BP1 cells showing intact lagging chromosome with telomere signals (red) at both termini. Insets showing enlarged view of the lagging chromatin with telomere signals present at both their termini. Error bars represent s.d of three independent experiments. Scale bars, 5 μm

Fig. 6

Gross chromosomal hyper-rearrangements mediated by sister-chromatid bridging in 53BP1-depleted cancer cells. a Formation of new chromosome 16 and 7 derivatives in HeLa 53BP1^hypo and 53BP1Δ clones. Left panels: whole chromosome 16 painting revealing 16p arm deletion and arm/centromeric translocations in HeLa b9 (53BP1^hypo) cells. Middle panels: 16q deletions and arm/centromeric translocations in HeLa B2 (53BP1^hypo) cells; Isochromosome 16q formation in HeLa b15 (53BP1^hypo) cells. Right panels: whole chromosome 7 painting revealing centromeric translocation in HeLa D4 (53BP1Δ) cells and arm deletion and centromeric translocation in HeLa B2 (53BP1^hypo) cells. b Ideogram of human chromosome 16, marking the positions of FISH probes used in Fig. 6c–f. c 16q distal arm deletion, centromeric translocation and 16p isochromosome formation was identified in HeLa B2 (53BP1^hypo) populations with the indicated percentages. d 16p whole-arm deletion, 16q whole-arm translocation and 16q distal-arm translocation were detected in HeLa b9 (53BP1^hypo) populations with the indicated percentages. e 16q isochromosome formation was detected in HeLa b15 (53BP1^hypo) populations with the indicated percentages. f Normal chromosome 16 showing both p-arm and q-arm is maintained in all HeLa cells

Fig. 7

Sister-chromatid rupture-bridging is strongly linked to distinct chromosomal rearrangements. a Positions of FISH probes at WWOX gene locus on chromosome 16. b Representative FISH images showing DNA thread structures linking the promoter region (left) and at CFS-FRA16D site (right) of WWOX sister alleles on HeLa B2 (53BP1^hypo) metaphase chromosomes. c DNA thread structures (91O9 probe; red) were also detected in HeLa b9 (53BP1^hypo) cells. Arrows indicate DNA threads linking the well-separated chromatid arms. Probe 352J17 was used as a control. d Relative distance between sister signals of FISH probes, 352J17 and 91O9, in HeLa and 53BP1^hypo cells. FISH signals showing a line or connected dot is considered as zero distance—DNA thread formation. Eighteen metaphase spreads were counted. Note: HeLa B2 (53BP1^hypo) cells retain only two intact WWOX alleles. e Examples of centromere-tethered lagging sister chromatin in 53BP1-depleted HeLa and U2OS cells. A pair of lagging sister chromatin as differentially labelled by EdU (green), intertwined by a PICH-UFB (red) at their centromeres (CENB, blue) in HeLa B2 (53BP1^hypo) anaphase cell (Top). Pairs of broken lagging chromatin tethered at centromeres (red) by PICH-UFBs (green) in HeLa D4 (53BP1Δ) (Middle) and U2OS B18 (53BP1Δ) cells (Bottom). f Frequencies of lagging-chromatin pairs with UFBs linking at centromeres in HeLa 53BP1^hypo and 53BP1Δ cells. Numbers of lagging chromatin pairs analysed, B2 = 49 and D4 = 44 from three independent experiments. g Immuno-FISH analysis revealed loss of whole chromatid arms on lagging chromatid pairs tethered by UFBs at centromeres. A representative image of HeLa 53BP1^hypo cell showing a PICH-UFB (green) intertwines the sister centromeres (blue) of a pair of lagging chromatin, at where the telomeres (red) are missing (asterisks). h Frequencies of sister-chromatid rupture-bridging phenomenon in unperturbed HeLa (4/40; 10%), U2OS (3/43; 7%) and Saos-2 (2/25; 8%) cells. Representative images showing ruptured chromatin tethered by PICH-UFBs (green), sometimes at centromeres (blue). Asterisks mark the ruptured positions at where the rest of chromatids is lost, as determined by telomere FISH (red). Scale bars, 5 μm. Error bars represent s.d. of three independent experiments. Statistical significance was determined by T-test (***, p < 0.001; ns, nonsignificant)

Fig. 8

Models of gross chromosomal rearrangements driven by conventional anaphase bridge-breakage and sister-chromatid rupture-bridging pathways. a Conventional anaphase bridge-breakage (also known as breakage-fusion-bridge cycle) model driven gross chromosomal rearrangements (GCRs). Aberrant chromatids or chromosomes, such as dicentric/radial chromosomes and sister-arm fusion lead to chromatin bridge formation in anaphase. The breakage of anaphase bridges during or after cytokinesis results in chromosome damage, which subsequently can lead to deletions, translocations and/or the re-formation of dicentric chromosomes and sister-arm fusion. Cells enter another anaphase bridge-breakage cycle in the next mitosis that accumulate further chromosome alterations. b Sister-chromatid rupture-bridging model driven GCRs (in the current study). Illegitimate formation/accumulation of ultrafine sister DNA intertwinements lead to a symmetrical rupture of sister-chromatid axes (asterisks). The resulting sister arms remain tethered by UFB structures resulting in anaphase bridges or lagging chromatin pairs formation (when the rupture occurs at centromeres). Further breakage may occur on the UFB-tethered anaphase bridges in late mitosis (e.g. during abscission in cytokinesis), which lead to arm deletions or translocations. On the other hand, the centromere-tethered lagging chromatin pairs, which lose the entire opposite arms, may escape abscission and co-segregate into one of the daughter cells. Hence, this provides an ideal precursor for isochromosome formation, or causes whole-arm translocations in the next cell cycle. Our current model, therefore, provides an alternative explanation on the formation of whole-arm rearrangements that may arise in cancer karyotypes