Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.
Institute for Quantitative Biosciences (QB3), University of California, Berkeley, CA 94720.
Proc Natl Acad Sci U S A. 2023 Mar 21;120(12):e2221309120. doi: 10.1073/pnas.2221309120. Epub 2023 Mar 14.
DNA compaction is required for the condensation and resolution of chromosomes during mitosis, but the relative contribution of individual chromatin factors to this process is poorly understood. We developed a physiological, cell-free system using high-speed egg extracts and optical tweezers to investigate real-time mitotic chromatin fiber formation and force-induced disassembly on single DNA molecules. Compared to interphase extract, which compacted DNA by ~60%, metaphase extract reduced DNA length by over 90%, reflecting differences in whole-chromosome morphology under these two conditions. Depletion of the core histone chaperone ASF1, which inhibits nucleosome assembly, decreased the final degree of metaphase fiber compaction by 29%, while depletion of linker histone H1 had a greater effect, reducing total compaction by 40%. Compared to controls, both depletions reduced the rate of compaction, led to more short periods of decompaction, and increased the speed of force-induced fiber disassembly. In contrast, depletion of condensin from metaphase extract strongly inhibited fiber assembly, resulting in transient compaction events that were rapidly reversed under high force. Altogether, these findings support a speculative model in which condensin plays the predominant role in mitotic DNA compaction, while core and linker histones act to reduce slippage during loop extrusion and modulate the degree of DNA compaction.
DNA 压缩对于有丝分裂过程中染色体的凝聚和解聚是必需的，但单个染色质因子对此过程的相对贡献仍知之甚少。我们使用高速卵提取物和光学镊子开发了一种生理的无细胞系统，以研究单个 DNA 分子上实时有丝分裂染色质纤维的形成和力诱导的解聚。与将 DNA 压缩约 60%的间期间提取物相比，中期提取物将 DNA 长度缩短了 90%以上，这反映了这两种条件下整个染色体形态的差异。核心组蛋白伴侣 ASF1 的耗竭抑制核小体组装，使中期纤维压缩的最终程度降低了 29%，而连接组蛋白 H1 的耗竭则产生了更大的影响，总压缩减少了 40%。与对照组相比，两种耗竭都降低了压缩速度，导致解压缩的短时间间隔增加，并增加了力诱导纤维解聚的速度。相比之下，从中期提取物中耗尽浓缩物强烈抑制了纤维组装，导致在高力下迅速逆转的短暂压缩事件。总的来说，这些发现支持了一个推测性的模型，即浓缩物在有丝分裂 DNA 压缩中起主要作用，而核心和连接组蛋白在环挤出过程中减少滑动并调节 DNA 压缩程度。
