Abstract
High-throughput, genome-wide translocation sequencing (HTGTS) studies of activated B cells have revealed that DNA double-strand breaks (DSBs) capable of translocating to defined bait DSBs are enriched around the transcription start sites (TSSs) of active genes. We used the HTGTS approach to investigate whether a similar phenomenon occurs in primary neural stem/progenitor cells (NSPCs). We report that breakpoint junctions indeed are enriched around TSSs that were determined to be active by global run-on sequencing analyses of NSPCs. Comparative analyses of transcription profiles in NSPCs and B cells revealed that the great majority of TSS-proximal junctions occurred in genes commonly expressed in both cell types, possibly because this common set has higher transcription levels on average than genes transcribed in only one or the other cell type. In the latter context, among all actively transcribed genes containing translocation junctions in NSPCs, those with junctions located within 2 kb of the TSS show a significantly higher transcription rate on average than genes with junctions in the gene body located at distances greater than 2 kb from the TSS. Finally, analysis of repair junction signatures of TSS-associated translocations in wild-type versus classical nonhomologous end-joining (C-NHEJ)-deficient NSPCs reveals that both C-NHEJ and alternative end-joining pathways can generate translocations by joining TSS-proximal DSBs to DSBs on other chromosomes. Our studies show that the generation of transcription-associated DSBs is conserved across divergent cell types.
Highlights
High-throughput, genome-wide translocation sequencing (HTGTS) studies of activated B cells have revealed that DNA double-strand breaks (DSBs) capable of translocating to defined bait DSBs are enriched around the transcription start sites (TSSs) of active genes
To perform HTGTS for DSB identification, we used a Cas9:sgRNAbased approach to introduce HTGTS bait DSBs into primary neural stem/progenitor cells (NSPCs) isolated from Xrcc4−/−p53−/− mice, as previously described [4]
To investigate the repair junction structures of translocations between bait DSBs and prey DSBs located within 2 kb of TSSs, we examined HTGTS data from Chr12-single-guide RNAs (sgRNAs)-1–based experiments or Chr15-MycsgRNA–based experiments in WT or Xrcc4−/−p53−/− NSPCs (Fig. 4)
Summary
High-throughput, genome-wide translocation sequencing (HTGTS) studies of activated B cells have revealed that DNA double-strand breaks (DSBs) capable of translocating to defined bait DSBs are enriched around the transcription start sites (TSSs) of active genes. Our studies of neural stem/ progenitor cells (NSPCs) further revealed that recurrent DSB clusters (RDCs) occur within the bodies of a set of at least 27 long genes that mostly are involved in neural functions [4]. Because of its dual role in C-NHEJ and DSB checkpoint responses, ATM deficiency can lead to long-term DSB persistence, leading, for example, to V(D)J recombination breaks in early B-cell development that persist developmentally into mature B cells [23,24,25]. These ATM roles likely contribute to immunodeficiency and lymphoid cancers associated with ATM deficiency, whether such ATM functions are involved with the neurodegenerative phenotype associated with ATM deficiency in humans remains unknown [21, 22]
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