A method for the in vitro reconstitution of a defined "30 nm" chromatin fibre containing stoichiometric amounts of the linker histone
A method for the in vitro reconstitution of a defined "30 nm" chromatin fibre containing stoichiometric amounts of the linker histone
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DOI：
10.1016/j.jmb.2004.10.075
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发表时间：
2005-02-04
影响因子：
5.6
通讯作者：
Rhodes, D
中科院分区：
文献类型：
--
作者：
Huynh, VAT;Robinson, PJJ;Rhodes, D
要了解高阶染色质结构在调控细胞过程（如转录）中的作用，需要了解“30 nm”染色质纤维的结构及其折叠和展开途径。我们报道了一个体外染色质重建系统。其使用Widom 601 DNA序列的200 bp重复的12和19个拷贝的阵列。由于该DNA序列以比混合序列DNA高得多的亲和力结合组蛋白八聚体，因此我们在重构中使用竞争DNA来控制组蛋白八聚体和接头组蛋白两者到601 DNA阵列上的加载。使用这种方法，我们已经获得了核小体阵列，具有一个组蛋白八聚体和一个H5结合每200 bp重复，因此具有天然染色质的化学计量组成。为了获得高度紧凑的30运行染色质纤维，我们已经研究了许多折叠缓冲液条件，包括不同的NaCl或MgCl 2，浓度。沉降速度分析表明，重组核小体阵列具有与天然染色质相同的折叠特性，并在高NaCl浓度或1 nM MgCl 2中形成高度紧凑的结构。折叠阵列的负染色和电子冷冻显微镜显示具有3-4 nm均匀直径的均匀折叠颗粒群。所提供的数据提供了很好的证据表明，我们开发的重构方法首次产生了适合详细结构研究的折叠30 nm纤维的定义种群。(C)2004年，爱思唯尔有限公司保留所有权利。
An understanding of the role of higher-order chromatin structure in the regulation of cellular processes such as transcription will require knowledge of the structure of the "30 nm" chromatin fibre and its folding and unfolding pathways. We report an in vitro chromatin reconstitution system. which uses arrays of 12 and 19 copies of a 200 bp repeat of the Widom 601 DNA sequence. Since this DNA sequence binds the histone octamer with much higher affinity than mixed sequence DNA, we have used competitor DNA in the reconstitutions to control the loading of both the histone octamer and linker histone onto the 601 DNA arrays. Using, this method we have obtained nucleosome arrays that have one histone octamer and one H5 bound per 200 bp repeat, and hence have the stoichiometric composition of native chromatin. To obtain highly compact 30 run chromatin fibres, we have investigated a number of folding buffer conditions including varying NaCl or MgCl2, concentrations. Sedimentation velocity analysis shows that the reconstituted nucleosome arrays have the same folding, properties as native chromatin and form highly compact structures in high NaCl concentrations or 1 nM MgCl2,. Negative stain and electron cyro-microscopy of the folded arrays show a homogeneous population of folded particles with a uniform diameter of 3-4 nm. The data presented provide good evidence that the reconstitution method We have developed produces, for the first time, a defined population of folded 30 nm fibres suitable for detailed structural investigation. (C) 2004, Elsevier Ltd. All rights reserved.