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Induction of Recurrent Break Cluster Genes in Neural Progenitor Cells Differentiated from Embryonic Stem Cells In Culture [GRO-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: GSE142312 | 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 [HTGTS-Seq]

Project description: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.

2020-01-13 | GSE142289 | GEO

Three classes of recurrent DNA break clusters in brain progenitors identified by 3D proximity-based break joining assay

Project description:We recently discovered 27 recurrent DNA double-strand break (DSB) clusters (RDCs) in mouse neural stem/progenitor cells (NSPCs). Most RDCs occurred across long, late-replicating RDC genes and were found only after mild inhibition of DNA replication. RDC genes share intriguing characteristics, including encoding surface proteins that organize brain architecture and neuronal junctions, and are genetically implicated in neuropsychiatric disorders and/or cancers. RDC identification relies on high-throughput genome-wide translocation sequencing (HTGTS), which maps recurrent DSBs based on their translocation to "bait" DSBs in specific chromosomal locations. Cellular heterogeneity in 3D genome organization allowed unequivocal identification of RDCs on 14 different chromosomes using HTGTS baits on three mouse chromosomes. Additional candidate RDCs were also implicated, however, suggesting that some RDCs were missed. To more completely identify RDCs, we exploited our finding that joining of two DSBs occurs more frequently if they lie on the same cis chromosome. Thus, we used CRISPR/Cas9 to introduce specific DSBs into each mouse chromosome in NSPCs that were used as bait for HTGTS libraries. This analysis confirmed all 27 previously identified RDCs and identified many new ones. NSPC RDCs fall into three groups based on length, organization, transcription level, and replication timing of genes within them. While mostly less robust, the largest group of newly defined RDCs share many intriguing characteristics with the original 27. Our findings also revealed RDCs in NSPCs in the absence of induced replication stress, and support the idea that the latter treatment augments an already active endogenous process.

2018-01-19 | GSE106822 | GEO

Long Neural Genes Harbor Recurrent DNA Break Clusters in Neural Stem/Progenitor Cells

Project description:Repair of DNA double-strand breaks (DSBs) by non-homologous end-joining is critical for neural development, and brain cells frequently contain somatic genomic variations that might involve DSB intermediates. We now use an unbiased, high-throughput approach to identify genomic regions harboring recurrent DSBs in primary neural stem/progenitor cells (NSPCs). We identify 27 recurrent DSB clusters (RDCs) and, remarkably, all occur within gene bodies. Most of these NSPC RDCs were detected only upon mild, aphidicolin-induced replication stress, providing a nucleotide-resolution view of replication-associated genomic fragile sites. The vast majority of RDCs occur in long, transcribed, and late-replicating genes. Moreover, almost 90% of identified RDC-containing genes are involved in synapse function and/or neural cell adhesion, with a substantial fraction also implicated in tumor suppression and/or mental disorders. Our characterization of NSPC RDCs reveals a basis of gene fragility and suggests potential impacts of DNA breaks on neurodevelopment and neural functions. We performed high-throughput, genome-wide, translocation sequencing (HTGTS) and GRO-seq in primary mouse neural stem/progenitor cells of the indicated genotypes.

2016-02-15 | E-GEOD-74356 | biostudies-arrayexpress

Linear interaction between transcription and replication shapes DNA break dynamics

Project description:Recurrent DNA break clusters (RDCs) are replication and transcription collision hotspots. Through high-resolution replication sequencing and a capture-ligation assay in mouse neural progenitor cells experiencing replication stress, we unraveled the replication fork architecture dictating RDC location and orientation. Most RDC occurs at the replication forks traversing timing transition regions (TTRs), where sparse replication origins connect unidirectional forks. Leftward-moving forks generate telomere-connected DNA double-strand breaks (DSB) while rightward-moving forks lead to centromere-connected DSBs. Strand-specific mapping for DNA-bounded RNA revealed transient DNA:RNA hybrids present at a higher density in RDC than in other actively transcribed long genes. In addition, mapping nascent RNA and RNA polymerase activity revealed that head-to-head interactions between replication and transcription machinery slow down DNA replication, resulting in 60% DSB contribution to the head-on as compared to 60% for co-directional . Our findings revealed TTR as a novel fragile class and highlighted how the linear interaction between transcription and replication impacts genome stability.

2024-03-18 | GSE254765 | GEO

Long Neural Genes Harbor Recurrent DNA Break Clusters in Neural Stem/Progenitor Cells

2016-02-15 | GSE74356 | GEO

High-throughput sequencing of genome-wide translocations from stimulated wild-type or ATM-deficient B cells

Project description:High-throughput genome-wide translocation sequencing (HTGTS) is a robust approach to identify genome-wide translocation junctions. We performed HTGTS to study the fate of introduced c-myc DSBs in mouse splenic B cells activated for activation cytidine deaminase (AID)-dependent class switch recombination (CSR). We found frequent translocations of c-myc DSBs to AID-initiated DSBs in IgH switch regions in wild-type (WT) and ATM-deficient B cells. However, c-myc also translocated frequently to newly generated DSBs within a 35-megabase region downstream of IgH in ATM-deficient, but not WT, CSR-activated B cells. Moreover, we found such DSBs and translocations in activated B cells that did not express AID or undergo CSR. These findings indicate that ATM deficiency leads to formation of chromosome 12 dicentrics via RAG-initiated IgH DSBs in progenitor B cells and that these dicentrics can be propagated developmentally into mature B cells where they generate new DSBs downstream of IgH via breakage-fusion-bridge cycles. Preparation of libraries from WT or ATM-deficient activated by a-CD40/IL4 or RP105.

2014-06-18 | E-GEOD-58599 | biostudies-arrayexpress

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

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.

2024-03-18 | GSE233842 | GEO

High-throughput sequencing of genome-wide translocations from stimulated wild-type or ATM-deficient B cells

2014-06-18 | GSE58599 | GEO

Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases

Project description:We describe a robust linear amplification-mediated high-throughput genome-wide translocation sequencing (HTGTS) method that identifies endogenous or ectopic "prey" DNA double-stranded breaks (DSBs) across the human genome based on their translocation to "bait" DSBs generated by engineered nucleases. HTGTS with different Cas9:gRNA or TALEN-nuclease on-target baits revealed off-target hotspots for given nucleases that ranged from few or none to dozens or more, and greatly extended known off-target numbers for certain previously characterized engineered nucleases by more than 10-fold. Beyond various types of nuclease off-target collateral damage, we also identified collateral damage in the form of translocations between bona fide nuclease targets on homologous chromosomes. Based on frequent non-specific DSBs making any given human chromosome an HTGTS hotspot region for bait DSBs within it, we found that HTGTS also reveals wide-spread, low-level DSB-generating activities of engineered nucleases. Finally, HTGTS confirmed that the Cas9D10A paired nickase approach suppresses off-targets genome-wide and suggested other strategies to enhance desired nuclease activities.

2014-11-29 | GSE57283 | GEO

Increased neural progenitor proliferation in a hiPSC model of autism induces replication stress-associated genome instability [GRO-seq]

Project description:The association between macrocephaly and autism spectrum disorder (ASD) suggests that the mechanisms underlying excessive neural growth could contribute to ASD pathogenesis. Consistently, neural progenitor cells (NPCs) derived from induced pluripotent stem cells (iPSCs) of ASD individuals with early developmental brain enlargement are inherently more proliferative than control NPCs. Here, we show that hiPSC-derived NPCs from ASD individuals with macrocephaly display an altered DNA replication program and increased DNA damage. When compared to the control NPCs, high throughput genome-wide translocation sequencing (HTGTS) demonstrates that ASD-derived NPCs harbored elevated DNA double-strand breaks in replication stress-susceptible genes, some of which are associated with ASD pathogenesis. Our results provide a mechanism linking hyperproliferation of NPCs with the pathogenesis of ASD by disrupting long neural genes involved in cell-cell adhesion and migration.

2020-01-30 | GSE140486 | GEO

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