Graduate School of Environmental Studies, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan.
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551 Singapore.
Nucleic Acids Res. 2018 Jan 25;46(2):635-649. doi: 10.1093/nar/gkx1135.
To gain insight into the conformational properties and compaction of megabase-long chromatin molecules, we reconstituted chromatin from T4 phage DNA (165 kb) and recombinant human histone octamers (HO). The unimolecular compaction, induced by divalent Mg2+ or tetravalent spermine4+ cations, studied by single-molecule fluorescence microscopy (FM) and dynamic light scattering (DLS) techniques, resulted in the formation of 250-400 nm chromatin condensates. The compaction on this scale of DNA size is comparable to that of chromatin topologically associated domains (TAD) in vivo. Variation of HO loading revealed a number of unique features related to the efficiency of chromatin compaction by multivalent cations, the mechanism of compaction, and the character of partly compact chromatin structures. The observations may be relevant for how DNA accessibility in chromatin is maintained. Compaction of saturated chromatin, in turn, is accompanied by an intra-chain segregation at the level of single chromatin molecules, suggesting an intriguing scenario of selective activation/deactivation of DNA as a result of chromatin fiber heterogeneity due to the nucleosome positioning. We suggest that this chromatin, reconstituted on megabase-long DNA because of its large size, is a useful model of eukaryotic chromatin.
为了深入了解兆碱基长染色质分子的构象性质和压缩情况，我们使用 T4 噬菌体 DNA（165kb）和重组人组蛋白八聚体（HO）重新构建了染色质。通过单分子荧光显微镜（FM）和动态光散射（DLS）技术研究，二价 Mg2+或四价 spermine4+阳离子诱导的单分子压缩导致 250-400nm 的染色质凝聚物的形成。这种规模的 DNA 大小的压缩与体内染色质拓扑相关域（TAD）的压缩相当。HO 加载的变化揭示了与多价阳离子对染色质压缩的效率、压缩机制以及部分压缩染色质结构的特征相关的一些独特特征。这些观察结果可能与染色质中 DNA 可及性的维持方式有关。饱和染色质的压缩反过来伴随着单链染色质分子水平上的链内分离，这表明由于核小体定位导致染色质纤维异质性，DNA 的选择性激活/失活会出现有趣的情况。我们建议，由于其较大的尺寸，在兆碱基长 DNA 上重建的这种染色质是真核染色质的有用模型。
