Genome-wide translocation sequencing reveals mechanisms of chromosome breaks and rearrangements in B cells

Abstract

Whereas chromosomal translocations are common pathogenetic events in cancer, mechanisms that promote them are poorly understood. To elucidate translocation mechanisms in mammalian cells, we developed high-throughput, genome-wide translocation sequencing (HTGTS). We employed HTGTS to identify tens of thousands of independent translocation junctions involving fixed I-SceI meganuclease-generated DNA double-strand breaks (DSBs) within the c-myc oncogene or IgH locus of B lymphocytes induced for activation-induced cytidine deaminase (AID)-dependent IgH class switching. DSBs translocated widely across the genome but were preferentially targeted to transcribed chromosomal regions. Additionally, numerous AID-dependent and AID-independent hot spots were targeted, with the latter comprising mainly cryptic I-SceI targets. Comparison of translocation junctions with genome-wide nuclear run-ons revealed a marked association between transcription start sites and translocation targeting. The majority of translocation junctions were formed via end-joining with short microhomologies. Our findings have implications for diverse fields, including gene therapy and cancer genomics.

Figure 1. High Throughput Genomic Translocation Sequencing

(A and B) Circos plots of genome-wide translocation landscape of representative c-myc (A) or IgH (B) HTGTS libraries. Chromosome ideograms comprise the circumference. Individual translocations are represented as arcs originating from specific I-SceI breaks and terminating at partner site. (C) Top: a cassette containing either 25 or one I-SceI target(s) was inserted into intron 1 of c-myc (see Fig. S1A–C). Bottom: a cassette composed of a 0.5 kb spacer flanked by I-SceI target replaced the IgH Sγ1 region. Relative orientation of I-SceI sites is indicated by red arrows. Position of primers for generation and sequencing HTGTS libraries is shown. (D) An expression cassette for I-SceI fused to a glucocorticoid receptor (I-SceI-GR) was targeted into Rosa26 (see Fig. S1D–G). The red fluorescent protein Tomato (tdT) is co-expressed via an IRES. (E) Schematic representation of HTGTS methods; left: circularization-PCR, right: adapter-PCR. See text for details. (F) Background for HTGTS approaches, calculated as percent of artifactual human:mouse hybrid junctions when human DNA was mixed 1:1 with mouse DNA from indicated samples.

Figure 2. Genome-wide distribution and orientation of translocations from c-myc DSBs

Genome-wide map of translocations originating from the c-myc^{25x I-SceI} cassette (chr15) in αCD40/IL4-activated and I-SceI-infected B cells. Single junctions are represented by dots located at corresponding chromosomal position. The dot scale is 2 Mb. Clusters of translocations are indicated with color codes, as shown in legend. (+) and (−) orientation junctions (see Fig. S3) are plotted on righ and left side of each ideogram, respectively. Hotspots (see Fig. 4A), are listed in blue on top, with notation on the left side of chromosomes to indicate position. Data are from HTGTS libraries from 7 different mice. Centromere (Cen) and telomere (Tel) positions are indicated. See also Fig. S2.

Figure 3. Distribution of IgH and c-myc breakpoint-proximal junctions

(A) Distribution of junctions around chr15 breaksite in the pooled c-myc^25xI-SceI HTGTS library. Top: 10 kb around breaksite (represented as a split). Middle: 250 kb around breaksite (represented by red bar); Bottom: 2.5 Mb around breaksite. (+) and (−)-oriented junctions are plotted on top and bottom of chromosome diagrams, respectively. (B,C) Distribution of translocation junctions at IgH in the pooled ΔSγ1^2xI-SceI (B) or c-myc^25xI-SceI (C) HTGTS libraries. Translocations in WT (top) and AID^−/− (bottom) B cells are shown. Positions of S regions within the 250 kb IgH C_H region are indicated. Color codes are as in Fig. 2. Dot size, position of centromere (red oval) and telomere (green rectangle), and orientation of the sequencing primer are indicated. See also Fig. S4.

Figure 4. Identification of specific and general translocation hotspots

(A) Graph representing translocation numbers in frequently hit genes and non-annotated chromosomal regions. Only hotspots with more than 5 hits are shown and are ordered based on frequency of translocations in the pooled c-myc^25xI-SceI/WT HTGTS library (top bars). Respective frequencies of translocations in the pooled c-myc^25xI-SceI/AID^−/− HTGTS library are displayed underneath (bottom bars). Green bars represent frequent hits involving cryptic I-SceI sites. Blue and yellow portions of top bars represent translocations found in c-myc^1xI-SceI and c-myc^25xI-SceI/ROSA^I-SceI-GR libraries, respectively. Genes translocated in human and mouse lymphoma or leukemia are in red. The dashed line represents the cutoff for significance over random occurrence for each of the two groups (see Table S3). (B and C) Genome-wide distribution of translocations relative to TSSs. Junctions from c-myc^25xI-SceI/WT (B) or c-myc^25xI-SceI/AID^−/− (C) libraries (excluding 2 Mb around chr15 breaksite and IgH S regions) are assigned a distance to the nearest TSS and separated into “active” and “inactive” promoters as determined by GRO-seq. Translocation junctions are binned at 100 bp intervals. n represents the number of junctions within 20 kb (upper panels) or 2 kb (lower panels) of TSS. Asterisks indicate cryptic genomic I-SceI sites. See also Fig. S5.

Figure 5. Translocations Preferentially Translocate Near TSSs

WT and AID^−/− c-myc^25xI-SceI HTGTS libraries were analyzed. In each panel, translocation junctions are in the first and second rows (WT and AID^−/− as indicated). The third and fourth rows represent sense and antisense nascent RNA signals from GRO-seq. The IgH μ, γ1, ε genes are shown in (C), the next most-frequently hit hotspots in (A) and three selected oncogene hotspots in (B). The transcriptional start site (arrow) is at the bottom of each panel. The size of each genomic region and number of junctions in each are shown.

Figure 6. Translocations cluster to transcribed regions

Translocation density maps from pooled c-myc^25xI-SceI/WT and c-myc^25xI-SceI/AID^−/− HTGTS libraries are aligned with combined sense and antisense nascent RNA signals for chr 15, 11, and 17 using the UCSC genome browser. Chromosome gene densities are displayed below GRO-seq traces. Chromosomal orientation from left to right is centromere (C) to Telomere (T). See also Figs. S6, S7.

Figure 7. Identification of cryptic I-SceI sites in the mouse genome by HTGTS

(A) Cryptic I-SceI site translocation targets. The canonical I-SceI recognition sequence is on top; nucleotides divergent from the consensus are in red. Chromosomal position and gene location of each cryptic site are indicated. “Hits” represent total number of unique junctions in a 4 kb region centered around each site in the pool of all HTGTS libraries (see also Table S6). In vitro cutting efficiency, evaluated as in Suppl. Exp. Procedures, is indicated. NA, intergenic or not annotated; nd, not determined. (B) In vitro cutting of PCR products encompassing indicated cryptic I-SceI sites. C+, positive control: PCR fragment containing a canonical I-SceI site. U, uncut; I, I-SceI-digested. (C) PCR to detect translocations between c-myc^25xI-SceI and cryptic I-SceI sites in Scd2, Dmrt1 and Mmp24 genes. (Top) Position of primers used for PCR amplification. (Middle) Average frequency of translocations ±SEM. (Bottom) Number of translocations/10⁵ cells from three independent c-myc^25xI-SceI WT mice. (D) Transcription in genes containing I-SceI sites determined by GRO-seq. Translocation junctions are in the first (AID^−/−) and second (WT) rows; sense and antisense nascent RNA signals are in the third and fourth rows. (E). Distance of cryptic I-SceI hotspots from the nearest TSS in pooled HTGTS libraries from WT and AID^−/− c-myc^25xI-SceI B cells.