APOBEC3-dependent kataegis and TREX1-driven chromothripsis during telomere crisis

Abstract

Chromothripsis and kataegis are frequently observed in cancer and may arise from telomere crisis, a period of genome instability during tumorigenesis when depletion of the telomere reserve generates unstable dicentric chromosomes^1-5. Here we examine the mechanism underlying chromothripsis and kataegis by using an in vitro telomere crisis model. We show that the cytoplasmic exonuclease TREX1, which promotes the resolution of dicentric chromosomes⁴, plays a prominent role in chromothriptic fragmentation. In the absence of TREX1, the genome alterations induced by telomere crisis primarily involve breakage-fusion-bridge cycles and simple genome rearrangements rather than chromothripsis. Furthermore, we show that the kataegis observed at chromothriptic breakpoints is the consequence of cytosine deamination by APOBEC3B. These data reveal that chromothripsis and kataegis arise from a combination of nucleolytic processing by TREX1 and cytosine editing by APOBEC3B.

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Figure 1.. The effect of TREX1 on telomere crisis induced rearrangements

a, Example of comparison of 1x and 30x WGS analysis of part of chromosome 1 in a clone derived from T2p1 cells induced to undergo telomere crisis. Top: QDNAseq analysis of 1x target coverage genomic sequencing data. Regions of CN loss (pink highlight) and CN gain (blue highlight) are indicated. CN profiles are log₂-transformed. Bottom: DNA CN profile (estimated copy number over genomic windows) and rearrangement joins obtained from Battenberg analysis of 30x target coverage genomic sequencing data. Colored arcs represent joins with the type of rearrangement and orientation as identified on the right. Rearrangement joins are further classified into discrete events as indicated by triangles, squares, and plus symbols (see methods). Interchromosomal rearrangements junctions linking to chromosomes 3 and 18 are indicated. b, Comparison of detection of DNA CN changes per chromosome in 1x and 30x WGS of 391 chromosomes from post-crisis T2p1 clones. Size and color of the dots highlight the approximate number of chromosomes with the indicated CN changes detected in 1x and 30x WGS. No events, simple events, and complex events are defined on the right. c, Examples of DNA copy number profiles (1x (QDNAseq)) of a T2p1 and a TREX1 KO post-crisis clone. d, Analysis pipeline and summary of the number of post-crisis T2p1 and TREX1 KO clones isolated from n = 5 independent telomere crisis experiments, the frequency of simple and complex CN changes detected (1x) and the number of clones selected for 30x WGS. e, Stacked bar plot of chromosomes from T2p1 and TREX1 KO post-crisis clones. Data derived from 1x (WGS of 117 subclones (2,691 chromosomes) and 301 subclones (6,923 chromosomes) from T2p1 and TREX1 KO clones, respectively. P values derived from Chi-squared test for trend in proportions (ns: not significant).

Figure 2.. TREX1 promotes chromothripsis

a, Examples of chromothripsis (see also Fig. 1a and Fig. 3h,I below), chromothripsis-like, Breakage-Fusion-Bridge, and Local Jump patterns in post-crisis clones derived from T2p1 and TREX1 KO cells. DNA CN profiles and rearrangement joins were obtained from 30x target coverage WGS. Annotation as in Fig. 1a. Variant allele frequency tracks are shown below the chromosome ideograms. b, Summary of number of clones that displayed the types of rearrangements shown in (a) as determined by 30x WGS of 14 T2p1 and TREX1 KO post-crisis clones with complex events observed in 1x WGS. c, Summary of the number of chromosomes in post-crisis T2p1 and TREX1 KO clones examined in (b) that display the indicated rearrangements. d, Plot of the number of CN changes associated with the complex events indicated in post-crisis T2p1 and TREX1 KO clones described in (b). P values derived from ANOVA (ns: not significant). Statistical testing of copy number differences in chromothripsis events between T2p1 and TREX1 KOs was not possible because the data set only includes one chromothripsis event in the TREX1 KOs.

Figure 3.. APOBEC3B induces kataegis during telomere crisis.

a, Still photographs from live-cell imaging of T2p1 cells expressing mTurquoise2-RPA70 at the indicated time points after doxycycline treatment showing ssDNA joining the primary nucleus. b, As in (a) but showing an example of ssDNA remaining outside the nucleus. c, As in (a) but showing both H2B-mCherry and mTurquoise2-RPA70 and representing an example of ssDNA joining a daughter nucleus during mitosis (times before and after mitosis are given). Images are representative of n = 3 independent experiments. d, Normalized APOBEC3B mRNA levels in the indicated cell lines based on qRT-PCR and represented relative to U937 cells. Mean and s.d. of n = 3 independent experiments are shown. P values derived from Student’s t test. (ns: not significant). e, Immunoblotting for endogenous APOBEC3B before or after 48 hours of dox treatment in the indicated cell lines. f, Cytidine deaminase activity assay in the indicated cell lines. Expected DNA fragment sizes are indicated. Gel is representative of n = 3 independent experiments. g, Timing of DNA bridge resolution after anaphase in T2p1 and APOBEC3B KO cells expressing H2B-mCherry and mTurquoise2-RPA70 and induced with dox. Data were obtained from two independent experiments (T2p1 = 53, 55; APOBEC3B KO = 65, 67 DNA bridges). h and i, Examples of DNA CN profile and rearrangement joins of T2p1 (h) and APOBEC3B KO (i) post-telomere crisis clones with chromothripsis obtained from 30x WGS. Annotation as in Fig. 1a. Red arrows: kataegis clusters. j, Plot of distance of cytosine mutation clusters (regardless of strand-coordination) indicating the number of mutations and the distance to the nearest rearrangement breakpoint in post-crisis wild T2p1 clones (grey), TREX1 KO clones (green), and APOBEC3B KO clones (red). k, APOBEC signature motif enrichment in C- or G-coordinated clusters of the indicated sizes and intermutational distances < 10 kb (as described in the Methods ref. 32) in post-crisis T2p1 and APOBEC3B KO clones. Above each bar: number of clusters and (number of tCw motifs). l, Statistical evaluation of nucleotide prevalence around cytosines mutated in C- or G-coordinated clusters based on data as in (k). All scale bars = 5 μm.

Figure 4.. TREX1 and APOBEC3B Determine Genome Instability During Telomere Crisis

a, Pie-charts summarizing detected events in the indicated cell lines. Data obtained from 30x WGS. n: Total number of chromosomes with either of the four types of events. b, Example of DNA copy number profiles of T2p1 and APOBEC3B KO post-telomere crisis clones from 1x WGS. c, Stacked bar plot of chromosomes from T2p1 and APOBEC3B KO post-crisis clones. Data derived from 1x (WGS of 117 subclones (2,691 chromosomes) and 375 subclones (8,675 chromosomes) from T2p1 and APOBEC3B KO clones, respectively. P value derived from Chi-squared test for trend in proportions (ns: not significant). d, Schematic displaying the inferred TREX1- and APOBEC3B-dependent events leading to chromothripsis and kataegis during telomere crisis.