High Multiplex Translocation Sequencing Reveals the Versatile Role of Condensin II in Chromosomal Translocations

Chromosomal translocations play pivotal roles in various physiological and pathological processes, such as immunoglobulin production and tumor progression; however, the infrequency of chromosomal translocation events has impeded the exploration of the underlying mechanisms. To tackle this challenge, we devised a strategy to report and enrich cells with translocations in vitro, in conjunction with a novel method termed High Multiplex Translocation Sequencing (HMTS), to capture genome-wide translocations from multiple bait regions simultaneously. Analysis of HMTS data unveiled a preference for translocations to occur at Topologically Associating Domain (TAD) boundaries, and experimental disruption of the TAD boundary indeed led to a reduction in translocation frequency, exemplified by translocations involving ERG. Knockdown of Cohesin or condensin II was observed distinct roles in translocations. Cohesin deficiency promoted long-distance translocations, while condensin II deficiency promoted short distance translocation, inside TAD, and decreased intra-chromatin long-distance translocation, particularly at TAD boundaries. For inter-chromatin, although condensin II deficiency also decreased the translocation at TAD boundaries, and highly transcription regions while paradoxically slightly increased inter-chromatin translocation ratio, suggesting that condensin II physiologically mediated inter-chromatin interaction at TAD boundary regions but simultaneously restricted interaction from other regions, such as centromere. Our new translocation sequencing method revealed the versatile role of condensin II in controlling intra-chromatin short-distance, long-distance, and inter-chromosome translocations. Overall design: We sought to investigate whether disruption of TAD boundaries would affect the translocation frequency of genes located near TAD boundaries. To address this question, we deleted a fragment containing two CTCF-peaks near the TAD boundary of ERG gene. These two CTCF motifs are oriented in a convergent manner with neighboring CTCF motifs. We confirmed the deletion of the CTCF peak region by PCR analysis and Sanger sequencing. Subsequenty, we used in situ Hi-c to confirm whether the TAD boudnary where disrupted following the deletion of the pair of CTCF peaks.

染色体易位（Chromosomal translocations）在多种生理及病理过程中发挥关键作用，例如免疫球蛋白生成与肿瘤进展；然而染色体易位事件发生频率极低，阻碍了对其潜在机制的探索。为应对这一挑战，我们开发了一种可在体外标记并富集易位细胞的策略，结合一种名为高多重易位测序（High Multiplex Translocation Sequencing, HMTS）的新型方法，可同时捕获多个诱饵区域的全基因组易位。对HMTS数据的分析揭示，易位更倾向于发生在拓扑关联结构域（Topologically Associating Domain, TAD）边界处，而对TAD边界的实验性破坏确实导致了易位频率降低，以涉及ERG基因的易位为例。研究发现黏连蛋白（Cohesin）与凝缩蛋白II（condensin II）的敲低对易位产生了截然不同的作用：黏连蛋白缺失促进了长距离易位，而凝缩蛋白II缺失则促进了TAD内部的短距离易位，并降低了染色质内的长距离易位，尤其是在TAD边界区域。对于染色质间易位，尽管凝缩蛋白II缺失同样降低了TAD边界及高转录区域的易位频率，但反常地略微升高了染色质间易位比例，这表明凝缩蛋白II在生理上介导了TAD边界区域的染色质间相互作用，同时限制了来自着丝粒等其他区域的相互作用。我们的新型易位测序方法揭示了凝缩蛋白II在调控染色质内短距离、长距离易位以及染色质间易位中的多方面作用。整体实验设计：我们旨在探究TAD边界的破坏是否会影响TAD边界附近基因的易位频率。为解答这一问题，我们敲除了ERG基因TAD边界附近包含两个CCCTC结合因子（CTCF）峰的片段，这两个CTCF基序与邻近的CTCF基序以收敛方向排列。我们通过聚合酶链式反应（PCR）分析与桑格测序（Sanger sequencing）确认了该CTCF峰区域的敲除。随后，我们使用原位Hi-C技术确认了该对CTCF峰敲除后，对应的TAD边界是否被破坏。