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.
Keywords: chromatin fiber length; confocal fluorescence microscopy; ionic conditions.