In vitro reconstitution of chromatin domains [ChIP-Seq]

A key step towards defining the structure-function relationship of the genome is to identify the molecular mechanisms that drive higher-order genome folding. To this end, we reconstituted five S. cerevisiae chromosomes in vitro and developed a high-resolution MNase-based chromosome conformation capture assay to measure their 3D organization. We show that the formation of regularly spaced and phased nucleosome arrays is sufficient to drive higher-order genome folding into domains that resemble in vivo genome organization and thereby demonstrate that neither loop extrusion nor transcription are required for domain formation. The domain boundaries correspond to nucleosome-free regions and insulation strength scales with their width. Integrated molecular dynamics simulations show that domain compaction is dependent on nucleosome linker length, with longer linkers forming more compact structures. Together, our work demonstrates that fundamental properties of chromatin fibers are important determinants of higher-order genome folding and provides a proof-of-principle for bottom-up 3D genome studies.阐明基因组结构与功能的关联，核心步骤之一在于揭示介导高阶基因组折叠的分子机制。为此，我们在体外重构了五株酿酒酵母（Saccharomyces cerevisiae, S. cerevisiae）染色体，并开发了基于微球菌核酸酶（Micrococcal Nuclease, MNase）的高分辨率染色体构象捕获测定技术，以表征其三维空间组织特征。本研究证实，规则间距且相位有序的核小体（nucleosome）阵列的形成，足以介导高阶基因组折叠为类体内基因组组织的结构域，由此证明结构域的形成无需依赖环挤出（loop extrusion）或转录过程。结构域边界与无核小体区域一一对应，且结构域的绝缘强度随其宽度呈正相关。集成分子动力学模拟结果显示，结构域的压缩程度依赖于核小体连接臂长度，更长的连接臂可形成更为紧凑的染色质结构。综上，本研究证实染色质纤维的基本属性是高阶基因组折叠的重要决定因素，并为自下而上的三维基因组研究提供了原理验证。