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 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.

Induction of Recurrent Break Cluster Genes in Neural Progenitor Cells Differentiated from Embryonic Stem Cells In Culture

“…To extend these studies, we exploited our finding that joining of two DSBs occurs more frequently if they lie on the same cis chromosome (12,13). Thus, we introduced DSBs into each of the 20 mouse chromosomes as baits for HTGTS libraries from control or APH-treated NSPCs (14). This analysis confirmed previously identified RDCs and identified many more.…”

“…Based on identification frequency with different baits and RDC-DSB density, we ranked relative RDC-gene robustness. On this basis, 19 originally identified plus 11 newly identified RDC-genes were highly robust (14). Of note, four of these highly robust RDC-genes were identifiable in untreated controls, but became more robust with APH-treatment, consistent with ectopic replication stress augmenting an ongoing endogenous process (11,14).…”

“…On this basis, 19 originally identified plus 11 newly identified RDC-genes were highly robust (14). Of note, four of these highly robust RDC-genes were identifiable in untreated controls, but became more robust with APH-treatment, consistent with ectopic replication stress augmenting an ongoing endogenous process (11,14). The great majority of highly robust RDC-genes are very long (>0.5Mb), variably transcribed, encode proteins that regulate synaptic function/cell adhesion, and have been associated with neuropsychiatric and development disorders and/or cancer (11,14).…”

A journey with common fragile sites: From S phase to telophase

“…Strikingly, single‐cell sequencing of human frontal cortex neurons has revealed that up to 41% of them had at least one very large (in the order of the Mb) de novo copy number variation (CNV), most of them being deletions . CNVs have also been detected at high frequency in mouse neural stem/progenitor cells, many of them in large genes . The inherent susceptibility to genomic instability of genes coding physiologically important proteins is difficult to understand from an evolutionary point of view.…”

“…94 CNVs have also been detected at high frequency in mouse neural stem/progenitor cells, many of them in large genes. 13,95 The inherent susceptibility to genomic instability of genes coding physiologically important proteins is difficult to understand from an evolutionary point of view. However, pathologic somatic mutations have been described in healthy individuals at subclinical frequencies, indicating that brain function may cope with mutated clone, depending on their size.…”