Symmetric Neural Progenitor Divisions Require Chromatin-Mediated Homology-Directed DNA Repair
Keil, Jason
2021
Abstract
The cerebral cortex is generated by a highly coordinated series of cellular proliferations, beginning with symmetric neural progenitor cell (NPC) divisions to expand the pool of progenitors, and subsequent asymmetric NPC divisions that give rise to molecularly-distinct subtypes of excitatory post-mitotic neurons. Recent analyses have convergently implicated chromatin-regulating enzymes in a broad range of disordered cortex development. Regulation of chromatin coordinates spatiotemporal gene expression during neurodevelopment, but is also necessary for other processes including DNA damage repair crucial to proliferating NPCs. INO80, which encodes the catalytic subunit of the INO80 chromatin remodeling complex, is a candidate gene for human microcephaly. However, Ino80 function in the developing brain has not been empirically studied. Here, leveraging a conditional allele of Ino80, I created Ino80 cKO models using multiple Cre-driver lines to dissect the spatial and temporal specificity with which Ino80 functions in cortex development. I uncovered molecularly dissociable roles for Ino80 in chromatin-mediated transcriptional regulation and genome maintenance in corticogenesis. Conditional deletion of Ino80 led to apoptosis of early NPCs and postnatal microcephaly, consistent with the microcephaly reported in individuals who carry recessive INO80 mutations. Cortical NPCs deficient in INO80 demonstrated impaired DNA double strand break (DSB) repair, which led to p53 activation, massive apoptosis, and microcephaly. Using a novel in vivo DSB repair assay, I found that Ino80 deletion selectively impaired homology-directed repair (HDR) of DSBs, implicating chromatin-mediated DSB repair as essential to proper cortex development. Consistent with impaired DSB repair and downstream p53-dependent apoptosis as the cause of microcephaly, co-deletion of Trp53 and Ino80 led to a remarkable reversal of all Ino80 phenotypes. Notably, the role of INO80 in DNA repair was mechanistically distinct from previously described function in mediating gene transcription with the transcription factor YY1. Therefore, INO80 plays molecularly dissociable roles in transcriptome and genome maintenance during cortical development. Unexpectedly, sensitivity to loss of INO80-mediated HDR differed starkly based on the mode of NPC division. Ino80 deletion caused extensive DNA damage and apoptosis in symmetric NPC-NPC divisions, but not in asymmetric NPC-neuron divisions. These observations were confirmed using conditional deletion of the HR gene Brca2, demonstrating that the sensitivity of symmetric divisions to impaired HR was generalizable to multiple members of the HR pathway and not specific to chromatin-mediated HR. Together, these experiments revealed a critical role for Ino80 in chromatin-mediated HR DNA repair in the embryonic cortex. This role is molecularly distinct from previously identified gene regulatory functions, the disruption of which did not contribute to NPC apoptosis or the microcephaly phenotype. Furthermore, symmetric NPC divisions were exquisitely sensitive to impaired HDR, whereas asymmetric divisions were largely unaffected. This dichotomy suggested that distinct modes of division may employ distinct strategies for genome fidelity. Insights from this work have important implications for the genetics of microcephaly, understanding of chromatin-mediated DSB repair, and the acquisition of brain somatic mutations during expansive NPC-NPC and neurogenic NPC-neuron divisions.Deep Blue DOI
Subjects
Neurodevelopment DNA damage repair Chromatin regulation Neurogenesis
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