Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226, United States of America.
Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226, United States of America; Medical Scientist Training Program, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226, United States of America.
Biophys Chem. 2022 Apr;283:106767. doi: 10.1016/j.bpc.2022.106767. Epub 2022 Feb 2.
Chromatin organization and its dynamic regulation are crucial in governing the temporal and spatial accessibility of DNA for proper gene expression. Disordered chains of nucleosomes comprise the basis of eukaryotic chromatin, forming higher-level organization across a range of length scales. Models of chromatin organization involving phase separation driven by chromatin-associating proteins have been proposed. More recently, evidence has emerged that nucleosome arrays can phase separate in the absence of other protein factors, yet questions remain regarding the molecular basis of chromatin phase separation that governs this dynamic nuclear organization. Here, we break chromatin down into its most basic subunit, the nucleosome core particle, and investigate phase separation using turbidity assays in conjunction with differential interference contrast microscopy. We show that, at physiologically-relevant concentrations, this fundamental subunit of chromatin undergoes phase separation. Individually removing the H3 and H4 tails abrogates phase separation under the same conditions. Taking a reductionist approach to investigate H3 and H4 tail peptide interactions in-trans with DNA and nucleosome core particles supports the direct involvement of these tails in chromatin phase separation. These results provide insight into fundamental mechanisms underlying phase separation of chromatin, which starts at the level of the nucleosome core particle, and support that long-range inter-nucleosomal interactions are sufficient to drive phase separation at nuclear concentrations. Additionally, our data have implications for understanding crosstalk between histone tails and provide a lens through which to interpret the effect of histone post-translational modifications and sequence variants. STATEMENT OF SIGNIFICANCE: Emerging models propose that chromatin organization is based in phase separation, however, mechanisms that drive this dynamic nuclear organization are only beginning to be understood. Previous focus has been on phase separation driven by chromatin-associating proteins, but this has recently shifted to recognize a direct role of chromatin in phase separation. Here, we take a fundamental approach in understanding chromatin phase separation and present new findings that the basic subunit of chromatin, the nucleosome core particle, undergoes phase separation under physiological concentrations of nucleosome and monovalent salt. Furthermore, the histone H3 and H4 tails are involved in phase separation in a manner independent of histone-associating proteins. These data suggest that H3 and H4 tail epigenetic factors may modulate chromatin phase separation.
染色质的组织及其动态调控对于控制 DNA 的时空可及性以实现适当的基因表达至关重要。真核染色质的基础是无序的核小体链，它们在一系列长度尺度上形成更高层次的组织。涉及由染色质相关蛋白驱动的相分离的染色质组织模型已经被提出。最近，有证据表明，核小体阵列可以在没有其他蛋白因子的情况下发生相分离，但关于控制这种动态核组织的染色质相分离的分子基础仍存在问题。在这里，我们将染色质分解为其最基本的亚单位，核小体核心颗粒，并使用浊度测定法结合微分干涉对比显微镜研究相分离。我们表明，在生理相关浓度下，这种染色质的基本亚单位会发生相分离。在相同条件下，单独去除 H3 和 H4 尾巴会阻止相分离。采用还原方法研究 H3 和 H4 尾巴肽与 DNA 和核小体核心颗粒的瞬态相互作用，支持这些尾巴直接参与染色质相分离。这些结果为染色质相分离的基本机制提供了深入的了解，该机制始于核小体核心颗粒的水平，并支持核浓度下长程核小体间相互作用足以驱动相分离。此外，我们的数据对理解组蛋白尾巴之间的串扰具有启示意义，并提供了一个解释组蛋白翻译后修饰和序列变体影响的视角。
