Mapping catalytically engaged TOP2B in neurons reveals the principles of topoisomerase action within the genome [RNA-Seq]. Mapping catalytically engaged TOP2B in neurons reveals the principles of topoisomerase action within the genome [RNA-Seq]

Topoisomerase IIb (TOP2B) is essential for neural development and function, yet its precise molecular roles remain poorly understood. Here we trapped catalytically engaged TOP2B in DNA cleavage complexes (TOP2Bccs) and mapped their positions genome-wide in cultured mouse cortical neurons. We report that chromosome compartments set the threshold of TOP2B activity within the genome, while specific nucleosome configurations stimulate TOP2B activity within strongly transcribed regions and enhancers. Highly expressed genes that are devoid of usually associated chromatin marks, such as H3K36me3, are deficient in TOP2B activity, indicating that TOP2B may be inefficient at sensing transcription-generated torsional stress directly. Active promoters and the transcription start sites (TSS) with high RNA polymerase II (RNAPII) occupancy show elevated TOP2B ChIP-seq signals but are depleted in TOP2Bccs, indicating that TOP2B is held inactive at active promoters. Surprisingly, TOP2B inhibition with etoposide increased nascent transcription at highly expressed genes and enhancers. While ETP treatment reduced nascent transcription within long genes, these effects were independent of transcript length and instead correlated with the presence of intragenic enhancers. Together these results provide insights into the epigenetic regulation of topoisomerase activity and reveal new roles for TOP2B in neuronal transcriptional regulation. Overall design: RNA-seq to in mouse cortical neruons

拓扑异构酶IIβ（Topoisomerase IIβ, TOP2B）对神经发育与功能至关重要，但其确切的分子功能仍有待深入阐明。本研究将处于催化活性状态的TOP2B捕获于DNA切割复合物（DNA cleavage complexes, TOP2Bccs）中，并在培养的小鼠皮层神经元内完成了其全基因组定位分析。研究发现，染色体区室划定了基因组内TOP2B活性的阈值，而特定的核小体构型则可在高转录区域与增强子内促进TOP2B的活性。缺乏常见相关染色质修饰（如H3K36me3）的高表达基因，其TOP2B活性存在缺陷，这提示TOP2B或许无法高效直接感知转录过程产生的扭转张力。RNA聚合酶II（RNA polymerase II, RNAPII）占据量较高的活性启动子与转录起始位点（Transcription start sites, TSS），其TOP2B染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing, ChIP-seq）信号显著升高，但TOP2Bccs的信号却出现耗竭，表明TOP2B在活性启动子处处于失活状态。令人意外的是，使用依托泊苷（Etoposide, ETP）抑制TOP2B后，高表达基因与增强子区域的新生转录水平反而升高。尽管依托泊苷处理会降低长基因内部的新生转录水平，但该效应与转录本长度并无关联，反而与基因内增强子的存在呈显著相关。综上，本研究结果为拓扑异构酶活性的表观遗传调控机制提供了新见解，并揭示了TOP2B在神经元转录调控中的全新功能。实验整体设计：小鼠皮层神经元的RNA测序（RNA-seq）分析。