Osmotic disruption of chromatin induces Topoisomerase 2 activity at sites of transcriptional stress [CC-seq-v2]

Transcription generates superhelical stress in DNA that poses problems for genome stability, but determining when and where such stress arises within chromosomes is challenging. Here, using G1-arrested S. cerevisiae cells, and employing rapid fixation and ultra-sensitive enrichment, we utilise the physiological activity of endogenous topoisomerase 2 (Top2) as a probe of transcription-induced superhelicity. We demonstrate that Top2 activity is surprisingly uncorrelated with transcriptional activity, suggesting that superhelical stress is obscured from Top2 within chromatin in vivo. We test this idea using osmotic perturbation—a treatment that transiently destabilises chromatin in vivo—revealing that Top2 activity redistributes within sub-minute timescales into broad zones patterned by long genes, convergent gene arrays, and transposon elements—and also by acute transcriptional induction. We propose that latent superhelical stress is normally absorbed by the intrinsic topological buffering capacity of chromatin, helping to avoid spurious topoisomerase activity arising within the essential coding regions of the genome. Genome-wide maps of Top2ccs in G1-arrested S. cerevisisae under various conditions, generated by CC-seq-v2.

转录过程会在DNA中产生超螺旋应力，该应力会对基因组稳定性造成威胁，但要确定此类应力在染色体内的产生时机与位置极具挑战。本研究采用G1期阻滞的酿酒酵母（S. cerevisiae）细胞，结合快速固定与超灵敏富集策略，以内源拓扑异构酶2（Top2）的生理活性作为转录诱导超螺旋的检测探针。研究发现Top2活性与转录活性出乎意料地并无关联，这表明体内染色质环境中的超螺旋应力无法被Top2感知。我们通过渗透压扰动——一种可在体内瞬时破坏染色质稳定性的处理方式——验证了这一假说，结果显示Top2活性会在亚分钟级时间尺度内重新分布，形成由长基因、收敛型基因阵列与转座子元件以及急性转录诱导所塑造的宽泛区域。本研究提出，潜在的超螺旋应力通常会被染色质固有的拓扑缓冲能力所吸收，从而避免在基因组的关键编码区域内产生异常的拓扑异构酶活性。本研究通过CC-seq-v2技术，生成了不同条件下G1期阻滞的酿酒酵母（S. cerevisiae）细胞内Top2共价结合复合物（Top2 covalent complexes, Top2ccs）的全基因组图谱。