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Induction of Recurrent Break Cluster Genes in Neural Progenitor Cells Differentiated from Embryonic Stem Cells In Culture [HTGTS-Seq]

ABSTRACT: Our laboratory identified robust recurrent DNA double-strand break (DSB) cluster (“RDC”) genes in mouse neural stem/progenitor cells (NSPCs) by applying the high-throughput, genome-wide, translocation sequencing (HTGTS) method. Genomic alterations of most of the identified RDC-genes have been associated with psychiatric disorders such as autism and schizophrenia and several are altered in brain cancer. The most robust mouse RDCs are all in genes that tend to be very long, actively transcribed and were enhanced after mild inhibition of replication stress. The common transcription and replication characteristics we observe in RDC-genes suggest that frequent RDC DSBs might be generated by collision between transcription and replication processes. However, the underlying mechanism of RDC formation is still unknown. To elucidate mechanisms that generate and resolve DSBs in these cells, we established an in vitro system of induced neural progenitor cells derived from embryonic stem cells. HTGTS bait-DSBs introduced by CRISPR/Cas9 on three mouse chromosomes identified only 5 RDC-genes in embryonic stem cells and 27 RDC-genes in the neural progenitor cells differentiated from them. All RDC-genes identified in induced neural progenitor cells belonged to the group of genes identified in primary neural and progenitor cells. These results indicate that our in vitro differentiation system is both effective and robust in terms of recapitulating our previous findings and facilitating mechanistic studies.

ORGANISM(S): Mus musculus

PROVIDER: GSE142289 | GEO | 2020/01/13

REPOSITORIES: GEO

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Induction of Recurrent Break Cluster Genes in Neural Progenitor Cells Differentiated from Embryonic Stem Cells In Culture [GRO-seq]

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Project description: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.

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