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Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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생물학

核小体阵列从重组核心组蛋白和核小体定位的DNA组装

Published: September 10, 2013
doi:
10.3791/50354
, , , , ,
1Biochemistry and Molecular Biology,Colorado State University

Summary

的方法，提出了用于从重组核心组蛋白模型核小体阵列的重构和串联重复的核小体定位的DNA。我们还描述沉降速度实验在分析型超速离心机，和原子力显微镜（AFM）是如何被用来监测核小体阵列饱和的重构后的程度。

Abstract

被沿DNA分子重复间隔核心组蛋白八聚体被称为核小体阵列。核小体阵列中的两种方法之一获得：纯化来自体内来源的，或重组的来自重组核心组蛋白的体外和串联重复的核小体定位的DNA。后一种方法具有允许的更组分均匀的组装和精确定位的核小体阵列的优点。沉降速度实验中约大分子通过分析在它们通过离心力作用下溶液迁移率的大小和形状的分析型超速离心机产率的信息。该技术中，随着原子力显微镜，可以用于质量控制，确保多数的DNA模板被重构后饱和的核小体。在这里，我们描述了需要重建的长度毫克量和成分上D中的协议efined核小体阵列适用于染色质的结构和功能的生物化学和生物物理研究。

Introduction

真核基因组中不存在作为裸DNA，而是被压缩和被结合的蛋白质的组织。 DNA和蛋白质的这些复合物被称为染色质。染色质的基本重复单位是核小体。核小体是由组蛋白八聚体和146个碱基对的DNA周围的组蛋白八聚体包裹约1.6倍1的。组蛋白八聚体是由每个核心组蛋白H2A，H2B，H3和H4的两个副本。被沿DNA分子重复间隔核心组蛋白八聚体被称为核小体阵列。核小体阵列的扩展结构已经被称为10纳米纤维或“珠子串”结构，是目前在低盐条件下，2 体外 。在10纳米纤维能够通过阵列内部的压实和/或跨阵列齐聚2冷凝成高阶结构。这些高次结构的可诱导的盐的存在下，或者可以通过染色质的建筑蛋白的核小体阵列3,4的结合的影响。染色质压实水平呈负相关转录体内 5,6率。最近的研究突出在如分化，肿瘤发展和其他7,8流程的基因组的结构组织的重要性。用核小体阵列来研究染色质结构和功能已变得很普遍。这里，我们描述了从重组核心组蛋白和核小体定位核小体的DNA阵列的组件的方法。

使用重组DNA与核小体定位序列的串联重复允许对包含规则间隔的核小体阵列的重构。两个比较流行的定位序列是5S rRNA基因序列和“601”序列9,10。 601序列是由SELEX实验和铁道部导出Ë强烈持仓核比5S序列11。因此，601阵列的接头DNA长度更均匀。通过凝胶过滤得到的4,12串联重复的核小体定位的DNA。重组组蛋白是从大肠杆菌纯化变性条件下13 大肠杆菌 。使用重组组蛋白允许人们小心地控制在核小体阵列的组蛋白组合物。例如，核心组蛋白轴承特异性突变14或翻译后修饰15,16可以取代野生型核心组蛋白。

沉降速度实验监控解决方案大分子沉淀在施加离心力17的速度。这产生大约样品中大分子的大小和形状的信息。沉降速度实验，因而为研究染色质纤维溶液状态的变化适当的工具结构由于染色质浓缩18。重要的是，它是第一个必要使用沉降速度实验如在核小体阵列重建质量控制步骤。如果DNA的核小体和不结合在适当的摩尔比，该阵列可以是欠或过饱和的核心组蛋白。因此，从沉降速度实验所获得的信息用于确保该DNA被正确地饱和的核小体。使用替代方法来估算的DNA与核小体的饱和度是很重要的，特别是如果处理的是以前未知的DNA模板。因此，我们还描述了使用原子力显微镜（AFM）的核小体阵列的分析方法。 AFM是一个功能强大的技术，允许的许多参数，如饱和度的水平，组蛋白变体的存在的影响或MgCl 2的19,20的作用效果可视化。原子力显微镜也一直APPLI编辑使用时间推移成像21，研究核小体动力学。 体外核小体组装12聚体阵列是特别适合于原子力显微镜的研究，因为他们属于在正确的尺寸范围原子力显微镜成像22。在本研究中，我们使用了核小体阵列的原子力显微镜作为一种质量控制以及肯定从AUC的数据（“眼见为实”）的一种手段。除了简单的可视化，原子力显微镜允许样本作为一个附加度量值的高度轮廓测量。

Protocol

1。重组核心组蛋白八聚体进入组装理由：在核小体阵列重建的第一步是准备从冻干重组核心组蛋白的原生核心组蛋白八聚体。组蛋白相结合，在等摩尔量和透析的样品出来变性缓冲到缓冲折叠组装成的组蛋白八聚体。 纯化和冻干的重组核心组蛋白（H2A，H2B，H3，H4）的描述13。 溶解约5毫克每冻干核心组蛋白在3ml去折叠缓冲液（6M盐酸胍，20毫摩尔Tris pH?…

Representative Results

为了说明该协议，我们重组从重组非洲爪蟾核心组蛋白和DNA组成的12串联601定位序列（601 207×12）的207 bp的重复序列核小体阵列。我们首先组装从冻干的核心组蛋白八聚体原生，然后用S200柱（ 图1A）纯化，八聚体，通过FPLC。更大的络合物较早洗脱从S200列。组蛋白一般洗脱顺序为：非特异性组蛋白聚集体，组蛋白八聚体，四聚体H3/H4和H2A/H2B二聚体（ 图1A）。…

Discussion

核小体模型阵列是染色质的结构和功能的体外研究中一个非常有用的工具。例如，它们已被广泛用于研究的染色质纤维凝结的机理在溶液30-34，并且使得有可能获得一个tetranucleosome 35的X-射线结构。最近它们已被证明在解密的特定核心组蛋白变体，突变体和翻译后修饰14-16,36结构效应是有用的。在这里，我们描述了用于从核小体定位的DNA和重组核心组蛋白模型核小体?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作是由美国国立卫生研究院资助GM45916和GM66834到JCH从Rett综合症国际基金会的奖学金，以AK这项工作的支持还得到美国国立卫生研究院授予GM088409和霍华德休斯医学研究所吉隆坡的贡献

Materials

Name of Reagent/Material Company Catalog Number Comments
(3-Aminopropyl)triethoxysilane Sigma-Aldrich A3648-100ML
6-8 kDa MWCO Dialysis Tubing Fisher 21-152-5
HiLoad Superdex 200 16/60 Column GE 17-1069-01
Vivaspin 50 kDa MWCO Centrifugal Concentrator Sartorius VS2031
12-14 kDa MWCO Dialysis Tubing Fisher 08-667A
Illustra Sephacryl S-1000 Superfine GE 17-0476-01
XL-A/I Analytical Ultracentrifuge Beckman-Coulter
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Tags

Keywords: Nucleosomal ArrayRecombinant Core HistonesNucleosome Positioning DNASedimentation VelocityAnalytical UltracentrifugeAtomic Force MicroscopyChromatin StructureChromatin Function
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Rogge, R. A., Kalashnikova, A. A., Muthurajan, U. M., Porter-Goff, M. E., Luger, K., Hansen, J. C. Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA. J. Vis. Exp. (79), e50354, doi:10.3791/50354 (2013).
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