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

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. Overall design: GRO-seq libraries were performed to assess transcription activity in embryonic stem cells and embronic stem cell-derived neural progenitor cells RDC-genes.

本实验室通过高通量全基因组易位测序（HTGTS）技术，在小鼠神经干细胞/前体细胞（NSPCs）中鉴定出了稳定存在的复发性DNA双链断裂簇（RDC）基因。已鉴定的绝大多数RDC基因的基因组改变与孤独症、精神分裂症等精神疾病相关，其中多个基因在脑癌中存在异常。在小鼠中最具稳定性的RDC基因均为长基因，且具有活跃转录特征，在轻度抑制复制应激后其转录水平会得到增强。我们在RDC基因中观察到的共同转录与复制特征提示，频繁出现的RDC相关DNA双链断裂可能由转录与复制过程的碰撞所引发。不过，RDC形成的潜在分子机制仍未明确。为阐明此类细胞中DNA双链断裂的产生与修复机制，我们建立了一套由胚胎干细胞诱导生成的神经前体细胞体外模型。通过CRISPR/Cas9在小鼠三条染色体上引入HTGTS诱饵DNA双链断裂位点后，我们在胚胎干细胞中仅鉴定出5个RDC基因，在其诱导分化得到的神经前体细胞中则鉴定出27个RDC基因。所有在诱导型神经前体细胞中鉴定出的RDC基因，均属于我们在原代神经及前体细胞中已鉴定出的基因集合。上述结果表明，我们建立的体外分化系统能够有效且稳定地重现我们此前的研究发现，可为机制研究提供有力支撑。整体实验设计：我们构建了全局运行测序（GRO-seq）文库，用于评估胚胎干细胞及其诱导分化得到的神经前体细胞中RDC基因的转录活性。