Abstract：
The dynamic regulation of the physical states of chromatin in the cell nucleus is crucial for maintaining cellular homeostasis. Chromatin can exist in solid- or liquid-like forms depending on the surrounding ions, binding proteins, post-translational modifications and many other factors. Several recent studies suggested that chromatin undergoes liquid-liquid phase separation (LLPS) in vitro and also in vivo; yet, controversial conclusions about the nature of chromatin LLPS were also observed from the in vitro studies. These inconsistencies are partially due to deviations in the in vitro buffer conditions that induce the condensation/aggregation of chromatin as well as to differences in chromatin (nucleosome array) constructs used in the studies. In this work, we present a detailed characterization of the effects of K+, Mg2+ and nucleosome fiber length on the physical state and property of reconstituted nucleosome arrays. LLPS was generally observed for shorter nucleosome arrays (15-197-601, reconstituted from 15 repeats of the Widom 601 DNA with 197 bp nucleosome repeat length) at physiological ion concentrations. In contrast, gel- or solid-like condensates were detected for the considerably longer 62-202-601 and lambda DNA (~48.5 kbp) nucleosome arrays under the same conditions. In addition, we demonstrated that the presence of reduced BSA and acetate buffer is not essential for the chromatin LLPS process. Overall, this study provides a comprehensive understanding of several factors regarding chromatin physical states and sheds light on the mechanism and biological relevance of chromatin phase separation in vivo.
摘要：
细胞核中染色质物理状态的动态调节对于维持细胞稳态至关重要。取决于周围的离子，染色质可以以固体或液体形式存在，结合蛋白，翻译后修饰和许多其他因素。最近的几项研究表明，染色质在体外和体内都会经历液-液相分离(LLPS)；然而，从体外研究中也观察到关于染色质LLPS性质的有争议的结论。这些不一致部分是由于诱导染色质缩合/聚集的体外缓冲条件的偏差以及研究中使用的染色质(核小体阵列)构建体的差异。在这项工作中,我们给出了K+效应的详细表征，Mg2+和核小体纤维长度对重组核小体阵列的物理状态和性质的影响。在生理离子浓度下，通常观察到较短的核小体阵列（15-197-601，从具有197bp核小体重复长度的Widom601DNA的15个重复序列重建）。相比之下,在相同条件下，对于更长的62-202-601和λDNA（〜48.5kbp）核小体阵列，检测到凝胶状或固体状缩合物。此外,我们证明了还原型BSA和乙酸盐缓冲液的存在对于染色质LLPS过程不是必需的。总的来说,这项研究提供了有关染色质物理状态的几个因素的全面理解，并揭示了染色质相分离的机制和生物学相关性在体内。
