Osmotic disruption of chromatin induces Topoisomerase 2 activity at sites of transcriptional stress [Mnase-Seq]

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.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构成威胁，但要确定这类应力在染色体中的产生时机与位点极具挑战。本研究使用G1期阻滞的酿酒酵母（Saccharomyces cerevisiae, S. cerevisiae）细胞，结合快速固定与超灵敏富集技术，以内源拓扑异构酶2（Topoisomerase 2, Top2）的生理活性作为转录诱导超螺旋的检测探针。我们发现，Top2的活性与转录活性意外地不存在相关性，这提示体内染色质环境中的Top2无法感知超螺旋应力。为验证这一假说，我们采用渗透压扰动处理——一种可在体内短暂破坏染色质稳定性的实验手段——结果显示，Top2活性会在亚分钟级的时间尺度上发生重分布，形成由长基因、会聚型基因阵列以及转座子元件所塑造的宽幅区域，同时也会受急性转录诱导的调控。我们提出，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产生异常的拓扑异构酶活性。