Investigations Into DNA Double Strand Breaks in Neural Development and Function

Abstract

Genomic integrity is dependent on the repair of DNA double-strand breaks (DSBs) by classical non-homologous end-joining (C-NHEJ). C-NHEJ is essential for suppressing aberrant chromosomal rearrangements, and is also critical for immune and nervous system development. Germline deletion of C-NHEJ ligation factors XRCC4 and Lig4 in mice leads to extensive apoptosis of early postmitotic neurons and embryonic lethality, likely due to the activation of p53-dependent checkpoints by unrepaired DSBs in neural cells. Notably, the genomic locations and etiology of these DSBs were unknown for many years. We now apply high-throughput genome-wide translocation sequencing (HTGTS) to map DSBs in neural stem/progenitor cells at nucleotide resolution. We identify 27 recurrent DSB clusters (RDCs), which are detected at enhanced frequency upon aphidicolin treatment, and map to genes that are long, transcribed, and late-replicating, implicating replication stress as a mechanistic basis for their fragility. Notably, human orthologs of 9 RDCs overlapped with copy number variations identified from single-cell sequencing of human neurons, suggesting that RDCs may contribute to somatic mosaicism in neuronal genomes. In this regard, the majority of RDC-genes encode proteins that serve critical neural functions, and nearly all are implicated in various neuropsychiatric disorders, while many are associated with cancer. We discuss our findings with regard to the potential roles of RDCs in neural diversity and disease. We also describe neural blastocyst complementation (NBC), a novel approach that expedites the generation of neurobiological mouse models, and will thus facilitate investigations into neurological phenotypes of RDC-gene mutations and DSB mapping in vivo. Genetically modified mouse models are conventionally generated by microinjection of donor embryonic stem cells (ESCs) into host blastocysts, resulting in chimeric mice that require breeding for germline transmission before analysis. NBC uses a diphtheria toxin-mediated strategy to ablate dorsal telencephalic progenitors in host embryos, which then develop into mice without forebrain structures. Injection of donor ESCs allows donor-derived progenitors to reconstitute the vacant forebrain niche in the absence of host competition, resulting in first-generation chimeras that can be directly analyzed for forebrain phenotypes. We present experiments to validate NBC, and discuss its utility in the study of neural development and function.