Linker histones stabilize the intrinsic salt-dependent folding of nucleosomal arrays: Mechanistic ramifications for higher-order chromatin folding
Linker histones stabilize the intrinsic salt-dependent folding of nucleosomal arrays: Mechanistic ramifications for higher-order chromatin folding
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DOI：
10.1021/bi981684e
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发表时间：
1998-10-20
期刊：
影响因子：
2.9
通讯作者：
Hansen, JC
中科院分区：
文献类型：
--
作者：
Carruthers, LM;Bednar, J;Hansen, JC
由核心组蛋白八聚体和Lytechinus 5S rDNA的12个208 bp串联重复序列（208-12个核体阵列）重建的确定核体阵列具有在MgCl2中形成不稳定折叠种的能力，其压实程度相当于标准的高阶30 nm直径染色质结构[Schwarz， P. M, and Hansen， J. C. (1994) J. Biol]。化学学报，26(2),389 - 389。为了研究连接体组蛋白在染色质凝聚中的机制功能，纯化的组蛋白1-15用208-12核小体阵列在50 mM NaCl中组装。随后开发了新的纯化程序，产生了208-12染色质模型系统的制备，其中大多数样品包含每个5S rDNA重复一个组蛋白八聚体和每个组蛋白八聚体一个组蛋白1-15分子。纯化的208-12染色质的完整性已在低盐条件下使用分析性超离心，定量琼脂糖凝胶电泳，电子低温显微镜和核酸酶消化进行了广泛的表征。结果表明，与208-12核小体阵列结合的组蛋白1-15以一种与天然鸡红细胞染色质难以区分的方式限制进入和退出的连接体DNA。在NaCl和MgCl2中进行的折叠实验表明，1-15结合显著地稳定了核小体阵列的中间和广泛折叠状态，而不会从根本上改变固有的核小体阵列折叠途径。这些结果首次证明了高阶染色质结构的形成和稳定需要明显不同的大分子决定因素，从而为染色质折叠机制提供了新的见解。
Defined nucleosomal arrays reconstituted from core histone octamers and twelve 208 bp tandem repeats of Lytechinus 5S rDNA (208-12 nucleosomal arrays) possess the ability to form an unstable folded species in MgCl2 whose extent of compaction equals that of canonical higher-order 30 nm diameter chromatin structures [Schwarz, P. M,, and Hansen, J. C. (1994) J. Biol. Chem. 269, 16284-16289]. To address the mechanistic functions of linker histones in chromatin condensation, purified histone 1-15 has been assembled with 208-12 nucleosomal arrays in 50 mM NaCl. Novel purification procedures subsequently were developed that yielded preparations of 208-12 chromatin model systems in which a majority of the sample contained both one histone octamer per 5S rDNA repeat and one molecule of histone 1-15 per histone octamer. The integrity of the purified 208-12 chromatin has been extensively characterized under low-salt conditions using analytical ultracentrifugation, quantitative agarose gel electrophoresis, electron cryomicroscopy, and nuclease digestion. Results indicate that histone 1-15 binding to 208-12 nucleosomal arrays constrains the entering and exiting linker DNA in a way that produces structures that are indistinguishable from native chicken erythrocyte chromatin, Folding experiments performed in NaCl and MgCl2 have shown that 1-15 binding markedly stabilizes both the intermediate and extensively folded states of nucleosomal arrays without fundamentally altering the intrinsic nucleosomal array folding pathway These result provide new insight into the mechanism of chromatin folding by demonstrating for the first time that distinctly different macromolecular determinants are required for formation and stabilization of higher-order chromatin structures.