Transcription and DNA Replication Shapes DNA Break Dynamics in Long Neuronal Genes

Transcription and replication conflict (TRC) are one of the main driving forces for genome instability. Yet, TRC rarely been discussed without the context of DNA:RNA entanglement, rending the role of transcription in other TRC unclear . In neural stem and progenitor cells, genes encode protein regulating neuron adhesion are hotspots for recurrent DNA break clusters (RDC). While RDC-containing genes are all actively transcribed, most RDC lack DNA:RNA entanglement. We demonstrated that, through controlled gain and loss of function genetic approaches, transcription activity is essential while not sufficient to induce RDC formation. In combination of a deep neural network and single-nucleotide resolution DNA break mapping approaches, we found RDC break densities mirror the replication fork dynamics. We demonstrated that, for the first time that, head-on TRC results in higher DNA break density than its co-direction counterparts. In summary, our results revealed that transcription has a higher-level regulatory role that has to be coordinated with DNA replication. Two parental lines of mouse ES cell (NXP010, NXP047) were used for CRISPR-Cas genome editing to create daughter lines that lack one allele of Ctnna2 (47-5_Ctnna2_allele-B), Ctnna2 promoter-enhanced deleted ES cell clones (38-3_Ctnna2_allele-B_promoter-A and 18-4_Ctnna2_allele-B_promoter-A), or ES cell lines that lacks one allele of Nrxn1 (22_Nrxn1_allele-B), and ), and Nrxn1 promoter-enhanced deleted ES cell clones (22-5_Nrxn1_allele-B_promoter-A and 22-37_Nrxn1_allele-B_promoter-A). The parental ES cells and the derived ES cell clones were in vitro differentiated to neural progenitor cells (ESC-NPC). To map replication stress-mediated DNA breaks, pX330-spCas9 and sgRNA expression plasmids were transfected to ESC-NPCs, allowing specific DSB to be created as "Bait" for LAM-HTGTS at five unique genomic area (Chr12_13Mb, Chr17_41Mb, Chr5_101Mb, Chr6_70Mb, Chr8_61Mb). Cells were then treated with aphidicolin (02: 0.2 uM. 03: 0.2 uM. 04: 0.4 uM. 05: 0.5 uM. 06: 0.6 uM) or veichle control DMSO for indicated time period. Genomic DNA were extracted four days after treatment and subjected to LAM-HTGTS. Nascent RNA from ESC-NPCs with or without specific genomic alteration were analyzed by GRO-seq, and the R-loop formation was determined by DRIP-seq.