Three classes of recurrent DNA break clusters in brain progenitors identified by 3D proximity-based break joining assay Pei-Chi Weia,b,c,1, Cheng-Sheng Leea,b,c,1, Zhou Dua,b,c, Bjoern Schwera,b,c,2, Yuxiang Zhanga,b,c, Jennifer Kaoa,b,c, Jeffrey Zuritaa,b,c, and Frederick W. Alta,b,c,3 aHoward Hughes Medical Institute, Harvard Medical School, Boston, MA 02115; bProgram in Cellular and Molecular Medicine, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115; and cDepartment of Genetics, Harvard Medical School, Boston, MA 02115 Contributed by Frederick W. Alt, January 9, 2018 (sent for review November 17, 2017; reviewed by Fred H. Gage and Irving L. Weissman) We recently discovered 27 recurrent DNA double-strand break Over the past decade, we have developed and refined high- (DSB) clusters (RDCs) in mouse neural stem/progenitor cells (NSPCs). throughput genome-wide translocation sequencing (HTGTS) Most RDCs occurred across long, late-replicating RDC genes and to identify recurrent endogenous DSBs (13–15). Application of were found only after mild inhibition of DNA replication. RDC genes the HTGTS approach recently allowed us to map a set of re- share intriguing characteristics, including encoding surface proteins currently breaking genes in mouse neural stem/progenitor that organize brain architecture and neuronal junctions, and are cells (NSPCs) (16). genetically implicated in neuropsychiatric disorders and/or cancers. HTGTS maps, at nucleotide resolution, genome-wide DSBs RDC identification relies on high-throughput genome-wide trans- based on their ability to translocate to a “bait” DSB introduced location sequencing (HTGTS), which maps recurrent DSBs based on at a specific chromosomal location (13–15). Bait DSBs can be their translocation to “bait” DSBs in specific chromosomal locations. either introduced ectopically by designer nucleases (14) or pro- Cellular heterogeneity in 3D genome organization allowed unequivocal vided by endogenous DSBs, including RAG-initiated V(D)J re- identification of RDCs on 14 different chromosomes using HTGTS combination DSBs (17–19) or clusters of activation-induced baits on three mouse chromosomes. Additional candidate RDCs cytidine deaminase-initiated DSBs in IgH locus switch (S) re- were also implicated, however, suggesting that some RDCs were gions during class switch recombination (CSR) in mature B cells missed. To more completely identify RDCs, we exploited our finding (20). The ability of HTGTS to identify recurrent DSBs across the that joining of two DSBs occurs more frequently if they lie on the genome relies on cellular heterogeneity in 3D genome organization same cis chromosome. Thus, we used CRISPR/Cas9 to introduce spe- (2); however, due to the increased potential for interaction, the cific DSBs into each mouse chromosome in NSPCs that were used as joining frequency between two separate DSBs is greatly enhanced bait for HTGTS libraries. This analysis confirmed all 27 previously if the two lie on the same cis chromosome (2, 14, 17), and is en- identified RDCs and identified many new ones. NSPC RDCs fall into hanced even further if the two lie within the same topological or three groups based on length, organization, transcription level, and loop domain (2, 17, 21). Indeed, B lymphocytes exploit enhanced replication timing of genes within them. While mostly less robust, the largest group of newly defined RDCs share many intriguing Significance characteristics with the original 27. Our findings also revealed RDCs in NSPCs in the absence of induced replication stress, and support Human brain neuron genomes can differ from one another, giving the idea that the latter treatment augments an already active rise to brain mosaicism. We developed a sensitive DNA break endogenous process. joining assay that uses “bait” DNA breaks introduced on different chromosomes to detect endogenous “prey” DNA breaks across nonhomologous end-joining | neural stem cells | replication stress | the mouse brain progenitor cell genome. This approach revealed neurodevelopment | recurrent DNA break clusters 27 recurrently breaking sites, many of which occur in long neural- specific genes associated with mental illnesses and cancer. We lassical nonhomologous end-joining (C-NHEJ) is a major have exploited the finding that bait and prey DSB join more CDNA double-strand break (DSB) repair pathway in somatic frequently when on the same chromosome to increase assay cells that was first implicated based on its requisite role in V(D)J sensitivity. This approach confirms previously identified break- recombination in the developing lymphocytes (1, 2). Subse- ing neural genes and identifies new ones, often with the same quently, we found that inactivation of XRCC4, a core C-NHEJ intriguing characteristics. Our study offer potential insights into factor (3) specifically abrogates both lymphocyte and neuronal brain diversification and disease. development due to unrepaired DSBs in progenitor cells (4). Similar findings have been reported for inactivation of DNA Author contributions: P.-C.W., C.-S.L., B.S., and F.W.A. designed research; P.-C.W., C.-S.L., and J.K. performed research; P.-C.W., C.-S.L., Z.D., and J.Z. contributed new reagents/ ligase 4 (5, 6), with which XRCC4 partners in C-NHEJ end- analytic tools; P.-C.W., C.-S.L., Z.D., Y.Z., and F.W.A. analyzed data; and P.-C.W. and ligation (7). The unrepaired DSBs that cause blocked lym- F.W.A. wrote the paper. phocyte development in C-NHEJ–deficient mice are generated Reviewers: F.H.G., The Salk Institute for Biological Studies; and I.L.W., Stanford University. in antigen receptor genes by the RAG endonuclease, the pro- The authors declare no conflict of interest. tein complex that initiates V(D)J recombination (2). The na- Published under the PNAS license. NEUROSCIENCE ture of the DNA breaks that cause neuronal apoptosis in the Data deposition: Sequencing data have been deposited in the Gene Expression Omnibus absence of XRCC4 or DNA ligase 4 has remained unresolved, (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. GSE106822). however. Nonetheless, these previous studies promoted spec- 1P.-C.W. and C.-S.L. contributed equally to this work. ulation that specific DSBs might play a role in brain develop- 2Present address: Department of Neurological Surgery and Eli and Edythe Broad Center of ment or disease (8, 9). In this regard, more recent studies have Regeneration Medicine and Stem Cell Research, University of California, San Francisco, highlighted the potential of genomic alterations to contribute to CA 94158. brain diversification and disease (10, 11). Somatic “brain only” 3To whom correspondence should be addressed. Email: [email protected]. mutations and genomic variations also have been implicated This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in neurodevelopmental and neuropsychiatric disorders (12). 1073/pnas.1719907115/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1719907115 PNAS | February 20, 2018 | vol. 115 | no. 8 | 1919–1924 Downloaded by guest on September 27, 2021 DSB joining within a topological domain to promote robust joining chromosome (2, 14, 17). Thus, for more complete coverage of of activation-induced cytidine deaminase-initiated S region DSBs the genome, we designed 17 additional sgRNAs to generate separated by 100 s of kb to effect exon shuffling during CSR (20), HTGTS bait DSBs, on each of the remaining 16 mouse auto- and developing lymphocytes exploit joining within chromosomal somes and the X chromosome (Fig. 1A and SI Appendix, Table loops to mediate physiological V(D)J recombination (18, 19). S1). Because the rejoining of two resected DSBs in close prox- To identify recurrent DSB clusters (RDCs) in the NSPC ge- imity to the bait break site is the most frequently detected event − − − − nome, we applied HTGTS to NSPCs from Xrcc4 / p53 / mice, in HTGTS analyses, the sgRNAs were designed to target geno- as this background enhances HTGTS detection of genomic mic sequences that were at least 5 Mb away from known RDC DSBs due to their persistence (2). HTGTS analyses from bait genes to avoid potentially confounding effects of resection events DSBs on three separate chromosomes revealed 27 RDCs found extending into adjacent RDC genes (16). In addition, to ensure by at least two of the three chromosomal baits, along with many that mapping from HTGTS bait DSBs was not influenced by additional “candidate” RDCs found with only a bait from one repetitive sequences, we selected bait genomic locales that did chromosome (16). Notably, all 27 RDCs occurred within genes not contain telomeric or simple repeat sequences. To maximize − − − − (“RDC genes”), and these genes shared an intriguing set of RDC DSB detection efficiency, we used Xrcc4 / p53 / primary characteristics, including encoding surface proteins that organize mouse NSPCs for the current HTGTS experiments, as deficiency brain architecture and neuronal junctions. Moreover, human for XRCC4 facilitates DSB persistence and detection of trans- counterparts of most mouse RDC genes had already been im- locations (2, 16). plicated genetically in neuropsychiatric disorders and cancer We used the same general approach to identify RDCs as de- (16). RDC genes also tend to be very long, moderately tran- scribed previously (16) (SI Appendix,Fig.S1). The 17 Cas9:sgRNA − − − − scribed, and late replicating. In the latter context, most RDCs constructs were introduced individually in Xrcc4 / p53 / NSPCs appeared only after