Differential nucleosome organization in human interphase and metaphase chromosomes [Chemical Mapping]

Mitosis induces profound changes in chromosome structure, impacting nucleosome organization. Here, we employed chemical mapping to achieve single-base-pair resolution of nucleosome positioning in human interphase and metaphase chromosomes, unveiling distinct organizing principles between the two states. During interphase, we observe greater variability in internucleosome spacing, with a higher abundance of shorter linkers compared to metaphase, reflecting the genome's adaptability in accommodating nucleosome arrays for gene expression. Local analyses further uncover differential nucleosome landscapes at key euchromatin landmarks, including promoters, enhancers, and insulators, in each state. Moreover, we analyzed the relationship between DNA cyclizability and dyad positioning during mitosis. Our results indicate that in metaphase, nucleosomes exhibit significantly higher cyclizability around the dyad, whereas during interphase, nucleosomes more frequently position DNA with higher cyclizability in the nucleosome shoulder near regulatory genomic regions. Together, this study offers novel insights into the intricate interplay between DNA mechanics and nucleosome dynamics during mitosis. Overall design: Through PiggyBac transgenic expression of an shRNA directed against a shared DNA sequence present in all human H4 genes and RNAi-resistant H4S47C cDNA, we generated H4S47C-substituted HeLa S3 stable cell lines suitable for efficient chemical cleavage of nucleosomes. Using these cells, we performed chemical nucleosome mapping experiments in both interphase and synchronized mitotic cells, with two replicates for each condition.

有丝分裂会引发染色体结构的剧烈改变，进而影响核小体的组织状态。本研究采用化学图谱法（chemical mapping）实现了人类间期与中期染色体中核小体定位的单碱基分辨率检测，揭示了两种细胞周期状态下截然不同的核小体组织原则。在间期阶段，核小体间距的变异程度更高，且相较于中期染色体，短连接区的丰度更高，这反映了基因组在适配核小体阵列以调控基因表达方面的适应性。局部分析进一步揭示了两种细胞状态下关键常染色质（euchromatin）标志性区域——包括启动子、增强子与绝缘子——的核小体景观差异。此外，本研究还分析了有丝分裂过程中DNA环化能力与核小体二分体（dyad）定位之间的关联。研究结果显示，在中期染色体中，核小体二分体周围区域的DNA环化能力显著更高；而在间期阶段，核小体更常将具有较高环化能力的DNA定位于调控基因组区域附近的核小体肩区（nucleosome shoulder）区域。综上，本研究为理解有丝分裂过程中DNA力学特性与核小体动态变化之间的复杂相互作用提供了全新视角。

实验设计：通过PiggyBac转座子（PiggyBac）介导的转基因表达，靶向所有人类H4基因共有的DNA序列的短发夹RNA（shRNA）以及耐RNA干扰的H4S47C互补DNA（cDNA），我们构建了可高效实现核小体化学切割的H4S47C修饰型HeLa S3稳定细胞系。利用该细胞系，我们分别对间期细胞与同步化的有丝分裂细胞开展了化学核小体图谱实验，每组实验设置两次生物学重复。