Linker DNA conformational variability has been proposed to direct nucleosome array folding into more or less compact chromatin fibers but direct experimental evidence for such models are lacking. Here, we tested this hypothesis by designing nucleosome arrays with A-tracts at specific locations in the nucleosome linkers to induce inward (AT-IN) and outward (AT-OUT) bending of the linker DNA. Using electron microscopy and analytical centrifugation techniques, we observed spontaneous folding of AT-IN nucleosome arrays into highly compact structures, comparable to those induced by linker histone H1. In contrast, AT-OUT nucleosome arrays formed less compact structures with decreased nucleosome interactions similar to wild-type nucleosome arrays. Adding linker histone H1 further increased compaction of the A-tract arrays while maintaining structural differences between them. Furthermore, restriction nuclease digestion revealed a strongly reduced accessibility of nucleosome linkers in the compact AT-IN arrays. Electron microscopy analysis and 3D computational Monte Carlo simulations are consistent with a profound zigzag linker DNA configuration and closer nucleosome proximity in the AT-IN arrays due to inward linker DNA bending. We propose that the evolutionary preferred positioning of A-tracts in DNA linkers may control chromatin higher-order folding and thus influence cellular processes such as gene expression, transcription and DNA repair.

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通过核小体接头DNA的构象变化调节染色质折叠

已经提出了接头DNA构象变异性来指导核小体阵列折叠成或多或少的致密染色质纤维，但是缺乏这种模型的直接实验证据。在这里，我们通过设计在核小体接头中特定位置带有A序列的核小体阵列，以诱导接头DNA的向内（AT-IN）和向外（AT-OUT）弯曲来验证这一假设。使用电子显微镜和分析离心技术，我们观察到AT-IN核小体阵列自发折叠成高度紧凑的结构，可与接头组蛋白H1诱导的结构相比。相反，AT-OUT核小体阵列与野生型核小体阵列相似，形成的紧凑结构较少，核小体相互作用减少。添加接头组蛋白H1进一步增加了A序列阵列的紧实度，同时保持了它们之间的结构差异。此外，限制性核酸酶消化揭示了紧密AT-IN阵列中核小体接头的可及性大大降低。电子显微镜分析和3D计算蒙特卡洛模拟与深刻的之字形接头DNA构型和AT-IN阵列中由于向内接头DNA弯曲而更接近核小体邻近性相一致。我们提出，DNA接头中A链的进化优先定位可能会控制染色质的高阶折叠，从而影响细胞的过程，例如基因表达，转录和DNA修复。限制性核酸酶消化揭示了紧密AT-IN阵列中核小体接头的可及性大大降低。电子显微镜分析和3D计算蒙特卡洛模拟与深刻的之字形接头DNA构型和AT-IN阵列中由于向内接头DNA弯曲而更接近核小体邻近性相一致。我们提出，DNA接头中A链的进化优先定位可能会控制染色质的高阶折叠，从而影响细胞的过程，例如基因表达，转录和DNA修复。限制性核酸酶消化揭示了紧密AT-IN阵列中核小体接头的可及性大大降低。电子显微镜分析和3D计算蒙特卡洛模拟与深刻的之字形接头DNA构型和AT-IN阵列中由于向内接头DNA弯曲而更接近核小体邻近性相一致。我们提出，DNA接头中A链的进化优先定位可能会控制染色质高阶折叠，从而影响细胞过程，例如基因表达，转录和DNA修复。