Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115.
Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115.
Proc Natl Acad Sci U S A. 2018 Feb 20;115(8):1919-1924. doi: 10.1073/pnas.1719907115. Epub 2018 Feb 5.
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 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.
我们最近在小鼠神经干细胞/祖细胞（NSPC）中发现了 27 个频发的 DNA 双链断裂（DSB）簇（RDC）。大多数 RDC 发生在长的、晚期复制的 RDC 基因上，并且只有在轻度抑制 DNA 复制后才能发现。RDC 基因具有有趣的特征，包括编码表面蛋白，这些蛋白组织大脑结构和神经元连接，并且在神经精神疾病和/或癌症中具有遗传相关性。RDC 的鉴定依赖于高通量全基因组转位测序（HTGTS），该方法基于其转位到特定染色体位置的“诱饵”DSB 来绘制频发 DSB。3D 基因组组织中的细胞异质性允许使用 HTGTS 诱饵在三个小鼠染色体上对 14 个不同染色体上的 RDC 进行明确识别。然而，还暗示了其他候选 RDC，这表明有些 RDC 被遗漏了。为了更完全地鉴定 RDC，我们利用了这样一个发现，即如果两个 DSB 位于同一染色体上，则它们的连接发生的频率更高。因此，我们使用 CRISPR/Cas9 在 NSPC 中将特定的 DSB 引入每个小鼠染色体中，这些染色体作为 HTGTS 文库的诱饵。该分析证实了之前鉴定的 27 个 RDC，并鉴定了许多新的 RDC。基于基因在其中的长度、组织、转录水平和复制时间，NSPC RDC 分为三组。虽然大部分不太稳健，但新定义的最大 RDC 组与原始的 27 个 RDC 具有许多有趣的特征。我们的发现还揭示了在没有诱导复制应激的情况下 NSPC 中的 RDC，并支持这样一种观点，即后者处理增强了已经活跃的内源性过程。
