使用哺乳动物细胞中的微球菌核酸酶进行染色质免疫沉淀测定
Summary
染色质免疫沉淀(ChIP)是了解基因调控的分子机制的有力工具。然而,该方法在通过机械剪切获得可重复染色质碎片方面遇到了困难。在这里,我们为使用酶消化的ChIP测定提供了改进的协议。
Abstract
为了表达生物体内的细胞表型,活细胞相应地执行基因表达,转录程序在基因表达中起着核心作用。细胞转录机及其染色质修饰蛋白协调以调节转录。为了在分子水平上分析转录调控,可以使用几种实验方法,如电泳移动移位、瞬态报告器和染色质免疫沉淀(ChIP)测定。本文详细介绍了经过修饰的ChIP测定,因为它具有直接显示组蛋白修饰和细胞中蛋白质和DNA之间的相互作用的优点。成功的 ChIP 测定的关键步骤之一是染色质剪切。虽然声波常用于剪切染色质,但很难识别可重现的条件。我们采用微球核酸酶(MNase)的酶消化,而不是通过声波剪切染色质,从而获得更可重复的结果。在本文中,我们使用MNase提供一个简单的ChIP测定协议。
Introduction
哺乳动物细胞的基因表达是严格而动态的调节,转录是关键步骤之一。基因转录主要受转录因子和组蛋白的调节。转录因子是一种蛋白质,它与特定的DNA序列结合并控制基因转录。这些因素要么促进或抑制RNA聚合酶II(PolII)的招募,它启动从基因组DNA作为模板1的mRNA合成。组蛋白修饰,如乙酰化和甲基化组蛋白尾残留物正和消极影响基因转录通过改变染色质结构2。由于基因表达的变化会影响细胞上下文,因此有必要研究转录的分子机制。
迄今为止,已有几种研究基因转录调控的方法可用。电泳移动移位测定(EMSA),也称为凝胶移位测定,用于分析蛋白质-DNA相互作用3。从感兴趣的细胞中孵育出一种放射性同位素(例如,32 P)标记的DNA探针,并在聚丙烯酰胺凝胶上电泳。其自动放射图显示,DNA-蛋白质复合物的迁移速度比凝胶中的探针慢。在存在针对蛋白质的抗体的情况下,DNA-蛋白质-抗体复合物在凝胶中迁移的速度比DNA-蛋白质复合物要慢。这个超移带揭示了DNA和蛋白质之间的特定结合。然而,EMSA只确定细胞无细胞系统中的特定DNA-蛋白质相互作用,因此,该相互作用是否控制活细胞的转录仍不得而知。瞬态报告器测定,通常称为荧光酶报告器测定,是为解决细胞中的基因表达调节而开发的。通常 , 感兴趣的基因的上游基因组区域入到报告质粒中 , 瞬时转染成细胞 , 并测量报告者的活动。各种缺失突变体允许识别负责基因调控的区域。尽管报告员检测是识别转录因子和控制转录的结合DNA序列的有用工具,但这种方法有一个主要缺点,即报告质粒不含染色质结构,不能反映”真实”转录机械。此外,组蛋白修改的更改不能由系统确定。
染色质免疫沉淀(ChIP)方法的发展是基于杰克逊和查克利的报告,”全细胞”固定与甲醛保留染色质结构4,5。自那时以来,许多相关的技术已经开发和改进6。在ChIP测定中,细胞用甲醛固定,以交叉连接DNA和蛋白质。染色质是支离破碎的,然后用感兴趣的抗体进行免疫沉淀。免疫复合物被洗净,DNA被纯化。PCR扩增与引基因靶向基因组的特定区域揭示感兴趣的蛋白质在基因组的占用。
虽然ChIP是识别蛋白质(如转录因子)和修饰组蛋白与DNA相互作用的有力工具,但该方法在实践中涉及一些困难,如染色质破碎步骤。声波已广泛应用于剪切染色质;但是,识别可重现的条件很麻烦。微球核酸酶(MNase)治疗是染色质剪切的替代方法。MNase是一种内源性酶,可消化双链、单链、圆形和线性DNA和RNA。确定最佳染色质分片的条件(包括染色质和酶的量、温度和孵育时间)相对容易。我们修改和简化了现有的协议,并建立了一个简单易重用的方法。本文提供了在哺乳动物细胞中使用MNase的ChIP测定方案。
Protocol
Representative Results
Discussion
虽然声波通常用于获得零碎的染色质,但识别可重现的条件既耗时又繁琐。在此协议中,我们使用MNase消化,因为酶消化应该更容易识别可重复的条件。在MNase消化后,需要一个短暂的声波步骤(参见步骤2.2)来破坏细胞膜并释放消化的染色质。因此,我们协议中的声波功率应该尽可能低。我们对所有使用的细胞使用相同的声波条件(参见图1和表1)来获得膜的完全分解。
…Divulgations
The authors have nothing to disclose.
Acknowledgements
这项研究得到了基因科技对希望之城的版税的支持。这项工作没有得到国家卫生研究院的全部或部分支持。
Materials
| 0.5 M EDTA (pH 8.0) | Thermo Scientific | AM9010 | |
| 2 M KCl | Thermo Scientific | AM9010 | |
| 2X iQ SYBR Green supermix | Bio-Rad | 1706862 | |
| 5 M NaCl | Thermo Scientific | AM9010 | |
| 50 bp DNA ladder | New England Biolabs | N3236S | |
| Agarose | Research Product International | A20090 | |
| Branched octylphenoxy poly(ethyleneoxy)ethanol | Millipore Sigma | I8896 | IGEPAL CA-630 |
| ChIP-grade protein G magnetic beads | Cell signaling technology | 9006S | |
| Chromatin Immunoprecipitation (ChIP) Dilution Buffer | Millipore Sigma | 20-153 | Buffer composition: 0.01% SDS, 1.1% Triton X- 100, 1.2mM EDTA, 16.7mM Tris-HCl, pH 8.1, 167mM NaCl. |
| Gel Loading Dye Purple (6X) | New England Biolabs | B7024S | |
| Glycine | Bio-Rad | 161-0724 | Electropheresis grade |
| Glycogen | Millipore Sigma | G1767 | 19-22 mg/mL |
| Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100x) | Thermo Scientific | 78445 | |
| High Salt Immune Complex Wash Buffer | Millipore Sigma | 20-155 | Buffer composition: 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 500mM NaCl. |
| Histone H3K4me3 antibody (pAb) | Active Motif | 39915 | |
| LiCl Immune Complex Wash Buffer | Millipore Sigma | 20-156 | Buffer composition: 0.25M LiCl, 1% IGEPAL CA630, 1% deoxycholic acid (sodium salt), 1mM EDTA, 10mM Tris, pH 8.1. |
| Low Salt Immune Complex Wash Buffer | Millipore Sigma | 20-154 | Buffer composition: 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 150mM NaCl. |
| Magna GrIP Rack (8 well) | Millipore Sigma | 20-400 | Any kind of magnetic separation stands that are compatible with a 1.5 mL tube is fine. |
| Micrococcal nuclease | New England Biolabs | M0247S | comes with 10 x buffer (500 mM Tris-HCl, 50 mM CaCl2, pH 7.9 @ 25 °C) and 100 x BSA (10 mg/ml) |
| NaHCO3 | JT Baker | 3506-01 | |
| Normal rabbit IgG | Millipore Sigma | 12-370 | |
| PIPES | Millipore Sigma | P6757 | |
| Proteinase K | Millipore Sigma | 3115887001 | |
| Real-time PCR system | Bio-Rad | CFX96, C1000 | |
| RNA pol II CTD phospho Ser5 antibody | Active Motif | 39749 | |
| SDS | Boehringer Mannheim | 100155 | Electropheresis grade |
| sodium acetate | Millipore Sigma | S5636 | |
| Sonicator equipped with a microtip probe | QSONICA | Q700 | Any kind of sonicators that are compatible with a 1.5 mL tube is fine. |
| UltraPure Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) | Thermo Scientific | 15593031 | pH 8.05 |
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