Three classes of recurrent DNA break clusters in brain progenitors identified by 3D proximity-based break joining assay

Abstract

We recently discovered 27 recurrent DNA double-strand break (DSB) clusters (RDCs) in mouse neural stem/progenitor cells (NSPCs). Most RDCs occurred across long, late-replicating RDC genes and were found only after mild inhibition of DNA replication. RDC genes share intriguing characteristics, including encoding surface proteins that organize brain architecture and neuronal junctions, and are genetically implicated in neuropsychiatric disorders and/or cancers. RDC identification relies on high-throughput genome-wide translocation sequencing (HTGTS), which maps recurrent DSBs based on their translocation to "bait" DSBs in specific chromosomal locations. Cellular heterogeneity in 3D genome organization allowed unequivocal identification of RDCs on 14 different chromosomes using HTGTS baits on three mouse chromosomes. Additional candidate RDCs were also implicated, however, suggesting that some RDCs were missed. To more completely identify RDCs, we exploited our finding that joining of two DSBs occurs more frequently if they lie on the same cis chromosome. Thus, we used CRISPR/Cas9 to introduce specific DSBs into each mouse chromosome in NSPCs that were used as bait for HTGTS libraries. This analysis confirmed all 27 previously identified RDCs and identified many new ones. NSPC RDCs fall into three groups based on length, organization, transcription level, and replication timing of genes within them. While mostly less robust, the largest group of newly defined RDCs share many intriguing characteristics with the original 27. Our findings also revealed RDCs in NSPCs in the absence of induced replication stress, and support the idea that the latter treatment augments an already active endogenous process.

Keywords: neural stem cells; neurodevelopment; nonhomologous end-joining; recurrent DNA break clusters; replication stress.

Fig. 1.

Identification of NSPC RDCs by a proximal DSB joining approach. (A) Map illustrating 19 murine autosomes and X chromosome (gray hollow bars), the 20 HTGTS bait DSB locations (arrowheads), and the 113 RDC locations. Cen, centromere; Tel, telomere. Horizontal lollipop symbols mark the locations of RDCs in the murine NSPC genome. (B) Graph showing a total of 113 RDCs either identified previously by three HTGTS bait DSBs located at chromosomes 12, 15, and 16 (green dots) or newly identified by at least two of the 20 HTGTS bait DSBs (black dots) as indicated in A. The y-axis indicates the number of different HTGTS baits significantly joined to each DSBs in each RDC; the x-axis, the number of RDCs. The genes within the top six most frequently identified RDCs are listed in the orange box, and RDCs identified by more than seven chromosomal baits are listed in the blue box. The numbers of RDCs in the orange and blue boxes are indicated. The robustness scores for the RDCs are provided in Dataset S1 and SI Appendix, Fig. S3B.

Fig. 2.

Joining frequency of genome-wide HTGTS bait DSBs to strong and weak RDC DSBs. (A, Upper) An HTGTS bait DSB (orange box) induced by Cas9:sgRNA (Chr-12-sgRNA-1, black arrowhead) joining to the prey DSBs (blue box) in the Npas3 RDC ∼40 Mb downstream of the bait DSB on chromosome 12. Cen, centromere; Tel, telomere. The green arrowhead indicates the HTGTS primer; dashed line/arrows indicate joining possibilities between the bait DSB and RDC DSBs. (A, Lower) The HTGTS prey junctions (black bars) distributed across the Npas3 gene and its surrounding genomic area on chromosome 12 in the APH-treated (+) or control (−) Xrcc4^−/−p53^−/− NSPCs. Yellow rectangles indicate overall RDC locations; RefGene (blue track) indicates the gene location. A total of 17,701 randomly selected HTGTS prey junctions from APH-treated or control experiments were plotted. (B, Upper) The joining between transchromosomal Cas9:sgRNA-induced HTGTS bait DSBs to the Npas3 RDC DSBs on chromosome 12. (B, Lower) The HTGTS prey junctions distributed across the Npas3 gene and its surrounding area. Each panel represents an independent experiment using a bait on the indicated chromosome. The location of RDCs identified by each chromosomal bait are indicated with yellow boxes and generally represent a subset of the longest RDCs identified. (C, Upper) The joining between HTGTS bait DSB induced by Cas9:sgRNA (Chr-12-sgRNA-1) and the Dgkb RDC DSBs ∼15 Mb downstream of the bait DSB. (C, Lower) Graph showing HTGTS prey junctions distributed across and surrounding the Dgkb gene. (D, Upper) Graph illustrating the joining between a transchromosomal Cas9:sgRNA-induced HTGTS bait DSB and the Dgkb RDC DSBs at chromosome 12. (D, Lower) HTGTS prey junctions distributed in and around the Dgkb gene. Panels are organized as described for B. (Scale bars: 1 Mb.) *Panels generated using previously published HTGTS datasets (16). Dataset S1 presents the MACS-based adjusted P values of RDCs.

Fig. 3.

Proximal intrachromosomal HTGTS bait DSBs facilitates spontaneous RDC identification. (A, Upper) The joining between a HTGTS bait DSB to Ctnna2 RDC DSBs located ∼5 Mb downstream of the bait DSB at chromosome 6. The figure is organized as described in Fig. 2A. (A, Lower) The HTGTS junction distribution across the Ctnna2 gene and its surrounding area in Xrcc4^−/−p53^−/− NSPCs. RDC areas are shaded in yellow. Ctnna2 RDC HTGTS junctions are significantly enriched in both the DMSO-treated control (P = 4.5 × 10⁻²) and APH-treated experiments (P = 7.0 × 10⁻⁷⁵). Transcription activity of the Ctnna2 gene and its surrounding genomic DNA by GRO-seq is shown in the centromeric-to-telomeric direction (blue) and the telomeric-to-centromeric direction (red). The scale indicates normalized GRO-seq counts [reads per kilobase per million (RPKM)]. (B) Joining of HTGTS bait DSBs to Maml2/Mtmr2 RDC DSBs located ∼12 Mb upstream on chromosome 9. The figure is organized as described in the panel. Maml2/Mtmr2 RDCs are significant in the control (P = 4.5 × 10⁻²) and APH-treated experiments (P = 2.17 × 10⁻²³).

Fig. 4.

Characteristics and functional classification of murine NSPC RDCs and their relevance to diseases. (A) Venn diagram of the indicated classes among the 113 RDCs, including 27 previously identified RDC genes (blue); 76 group 1 RDCs (pink), including 25 previously identified RDC genes; 34 group 2 RDCs (gray), including two previously identified RDC genes; and three group 3 RDCs (yellow). Additional group 1, 2, and 3 examples are shown in Figs. 2 and 3 and SI Appendix, Fig. S2. For viewing additional RDC junction distributions, all datasets are available in the GEO database (accession no. GSE106822). (B and C) Length (B) and transcription rate determined by GRO-seq (RPKM) (C) of the 27 RDC genes (blue), genes in newly identified group 1 RDCs (pink), genes >80 kb in newly identified group 2 RDCs (gray), and all genes in group 3 RDCs (orange). The number of genes analyzed in each group is indicated. Whiskers indicate minimum and maximum values; the top and bottom edges of the boxplots correspond to the 25th and 75th percentiles, respectively; and the horizontal line indicates medium values. *P < 0.05; ****P < 0.0001 (Mann–Whitney U test); n.s., P ≥ 0.05. (D–F) Timing of replication of the newly identified group 1 RDC genes (D), group 2 RDCs (E), and group 3 RDCs (F). Average and SEM are shown. Details are provided in Materials and Methods and SI Appendix, Materials and Methods. Green, early; blue, late. The corresponding locations of each indicated RDC are provided in Dataset S1. (G and H) Venn diagram of the indicated gene function (G) and link to diseases (H) among the 51 newly identified group 1 RDCs. Details are provided in SI Appendix, Table S5.