Cell cycle control of higher-order chromatin assembly around naked DNA in vitro.
Cell cycle control of higher-order chromatin assembly around naked DNA in vitro.
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DOI：
10.1083/jcb.115.6.1479
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发表时间：
1991-12
期刊：
影响因子：
--
通讯作者：
Mitchison TJ
中科院分区：
文献类型：
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作者：
Hirano T;Mitchison TJ
我们已经开发了一种体外系统，在该系统中，高阶染色质结构以细胞周期依赖的方式组装在裸DNA周围。从非洲爪蟾卵中制备了特异于间期和有丝分裂状态的无膜可溶性提取物。当高分子量DNA与间期提取物一起孵育时，组装出蓬松的染色质样结构。相反，有丝分裂提取物产生高度浓缩的染色体样结构。免疫荧光研究表明，识别一类有丝分裂特异性磷蛋白的单克隆抗体MPM-2染色浓缩的有丝分裂染色质的“核心”或“轴”，但不染色间期染色质。通过加入有丝分裂提取物，间期染色质结构同步转化为凝聚态。染色质的浓缩状态与MPM- 2染色结构的外观和结构重排相关。这些结果表明，MPM-2识别的有丝分裂特异性磷蛋白可能直接参与染色体支架样结构的组装和染色质凝聚。虽然这两种提取物以相同的速率促进核小体组装，但拓扑异构酶II（topo II）活性在有丝分裂提取物中比间期提取物高4至5倍。在有丝分裂组装混合物中加入拓扑酶II抑制剂VM-26会干扰MPM-2染色结构的组织，并影响染色质凝聚的最后阶段。这种体外系统应该是有用的，用于确定顺式和反式作用元件负责高阶染色质组装及其在细胞周期中的结构变化。
We have developed an in vitro system in which higher-order chromatin structures are assembled around naked DNAs in a cell cycle-dependent manner. Membrane-free soluble extracts specific to interphase and mitotic states were prepared from Xenopus eggs. When high molecular weight DNA is incubated with interphase extracts, fluffy chromatin-like structures are assembled. In contrast, mitotic extracts produce highly condensed chromosome-like structures. Immunofluorescence studies show that a monoclonal antibody MPM-2, which recognizes a class of mitosis- specific phosphoproteins, stains the "core" or "axis" of condensed mitotic chromatin but not interphase chromatin. By adding mitotic extracts, interphase chromatin structures are synchronously converted into the condensed state. The increasingly condensed state of chromatin correlates with the appearance and structural rearrangements of the MPM- 2-stained structures. These results suggest that mitosis-specific phosphoproteins recognized by MPM-2 may be directly involved in the assembly of the chromosome scaffold-like structures and chromatin condensation. Although both extracts promote nucleosome assembly at the same rate, topoisomerase II (topo II) activity is four to five times higher in mitotic extracts compared with interphase extracts. The addition of a topo II inhibitor VM-26 into mitotic assembly mixtures disturbs the organization of the MPM-2-stained structures and affects the final stage of chromatin condensation. This in vitro system should be useful for identifying cis- and trans-acting elements responsible for higher-order chromatin assembly and its structural changes in the cell cycle.