The hierarchical packaging of chromatin fibers plays a critical role in gene regulation. The 30-nm chromatin fibers, a central-level structure bridging nucleosomal arrays to higher-order organizations, function as the first level of transcriptional dormant chromatin. The dynamics of 30-nm chromatin fiber play a crucial role in biological processes related to DNA. Here, we report a 3.6-angstrom resolution cryogenic electron microscopy structure of H5-bound dodecanucleosome, i.e., the chromatin fiber reconstituted in the presence of linker histone H5, which shows a two-start left-handed double helical structure twisted by tetranucleosomal units. An atomic structural model of the H5-bound chromatin fiber, including an intact chromatosome, is built, which provides structural details of the full-length linker histone H5, including its N-terminal domain and an HMG-motif-like C-terminal domain. The chromatosome structure shows that H5 binds the nucleosome off-dyad through a three-contact mode in the chromatin fiber. More importantly, the H5-chromatin structure provides a fine molecular basis for the intra-tetranucleosomal and inter-tetranucleosomal interactions. In addition, we systematically validated the physiological functions and structural characteristics of the tetranucleosomal unit through a series of genetic and genomic studies in Saccharomyces cerevisiae and in vitro biophysical experiments. Furthermore, our structure reveals that multiple structural asymmetries of histone tails confer a polarity to the chromatin fiber. These findings provide structural and mechanistic insights into how a nucleosomal array folds into a higher-order chromatin fiber with a polarity in vitro and in vivo.

染色质纤维的分层包装在基因调控中起着关键作用。30 nm 染色质纤维是一种将核小体阵列桥接到更高级组织的中央级结构，是转录休眠染色质的第一级。30 nm 染色质纤维的动力学在与 DNA 相关的生物过程中起着至关重要的作用。在这里，我们报道了 H5 结合的十二核小体的 3.6 埃分辨率低温电子显微镜结构，即在接头组蛋白 H5 存在下重构的染色质纤维，它显示出由四核小体单元扭曲的双开始左旋双螺旋结构。构建了 H5 结合的染色质纤维的原子结构模型，包括完整的染色质，该模型提供了全长连接组蛋白 H5 的结构细节，包括其 N 端结构域和 HMG 基序样 C 端结构域。染色体结构显示 H5 通过染色质纤维中的三接触模式结合核小体二元组。更重要的是，H5 染色质结构为四核小体内和四核小体间相互作用提供了良好的分子基础。此外，我们通过酿酒酵母中的一系列遗传和基因组研究和体外生物物理实验，系统验证了四核小体单元的生理功能和结构特征。此外，我们的结构揭示了组蛋白尾部的多个结构不对称性赋予染色质纤维极性。这些发现为核小体阵列如何在体外和体内折叠成具有极性的高阶染色质纤维提供了结构和机制见解。