Investigating the role of human topoisomerases in maintaining chromosome topology and preventing genomic instability

DNA的复制和转录等过程都需要并诱导DNA拓扑结构发生巨大变化。由于这些变化发生在染色质的背景下，它们必须与形成染色体组织本身的过程相协调，例如染色质环的形成和高阶染色体折叠。DNA拓扑结构的变化是由DNA拓扑异构酶控制的，DNA拓扑异构酶是一种有效的酶，通过放松、解开和去除受约束的DNA来调节扭转应力。大量的体内研究已经揭示了拓扑异构酶在调节低等真核生物DNA拓扑结构中的基本作用，然而，在人类细胞中使用抑制剂或长期功能缺失的低分辨率实验提供了有限的关于拓扑异构酶如何在基因组和染色质背景下控制三维基因组中的扭转应力的信息。我们建议将最先进的高通量测序和成像方法与人类细胞的急性功能丧失策略相结合，系统地研究拓扑异构酶在染色体拓扑中的作用，并了解它们的功能如何控制基因表达和防止基因组在3D基因组中的不稳定性。我们的工作将集中在了解拓扑异构酶功能的急性丧失如何：(1)影响三维基因组的超螺旋结构，转录聚合酶的位置和基因表达；(2)非b DNA结构如r环的形成；(3)空间基因组组织和折叠；(4)促进基因组在三维基因组中的不稳定性。我们的最终目标是全面了解各种类型的拓扑异构酶如何在染色质和染色体组织的背景下调节扭转应力，并阐明它们的功能如何与基本的细胞过程（如复制、转录和环挤压）协调，以调节基因表达和防止基因组不稳定。

Processes that traverse DNA, such as replication and transcription, both require and induce dramatic changes in DNA topology. As these changes occur in the context of chromatin, they must be coordinated with processes that shape chromosome organization per se, such as the formation of chromatin loops and higher-order chromosome folding. Changes in DNA topology are controlled by DNA topoisomerases, a family of potent enzymes that regulate torsional stress by relaxing, unknotting and decatenating constrained DNA. Seminal in vivo studies have uncovered fundamental roles of topoisomerases in regulating DNA topology in lower eukaryotes, however, low resolution experiments using inhibitors or long-term loss-of-function knockdowns in human cells have provided limited information on how topoisomerases control torsional stress in the genomic and chromatin context across the 3D genome. We propose to combine state-of-the-art high-throughput sequencing and imaging methodologies with acute loss-of-function strategies in human cells to systematically study the role of topoisomerases on chromosome topology and to understand how their functions control gene expression and prevent genomic instability across the 3D genome. Our efforts will be focusing on understanding how acute loss of topoisomerase function: (1) influences the supercoiling landscape across the 3D genome, the position of transcribing polymerase and gene expression; (2) the formation of non-B DNA structures such as R-loops; (3) the spatial genome organization and folding and, (4) promotes genomic instability across the 3D genome. Our ultimate goal is to comprehensively understand how the various types of topoisomerases regulate torsional stress in the context of chromatin and chromosome organization and to shed light on how their functions coordinate with fundamental cellular processes such as replication, transcription and loop extrusion, to regulate gene expression and prevent genomic instability.