Summary

与ChEC-seq的蛋白质-DNA相互作用的全基因组映射<em>酿酒酵母(Saccharomyces cerevisiae)</em

Published: June 03, 2017
doi:

Summary

我们描述了染色质内源性切割与高通量测序(ChEC-seq),染色质免疫沉淀(ChIP) – 正交法,用于在全基因组范围内用微球菌核酸酶(MNase)融合蛋白测定蛋白质结合位点。

Abstract

蛋白质 – DNA相互作用的全基因组映射对于了解基因调控,染色质重塑和其他染色质定位过程至关重要。甲醛交联随后进行染色质免疫沉淀和高通量测序(X-ChIP-seq)已被用于获得许多对基因组生物学的宝贵见解。然而,X-ChIP-seq具有与交联和超声处理有关的显着的局限性。原生ChIP通过省略交联来避免这些缺点,但通常导致染色质结合蛋白的恢复不良。此外,所有基于ChIP的方法均受抗体品质的考虑。用于测定蛋白质-DNA相互作用的酶学方法,其涉及将目的蛋白质融合到DNA修饰酶中,也用于测定蛋白质-DNA相互作用。我们最近将一种这样的方法,染色质内源性切割(ChEC)与高通量测序合并为ChEC-seq。 ChEC-seq依赖于染色质相互融合将微生物核酸酶(MNase)感兴趣的蛋白质在活细胞中的钙存在下产生靶向的DNA切割。 ChEC-seq不是基于免疫沉淀,因此避免了交联,超声,染色质溶解和抗体质量的潜在问题,同时以最小的背景信号提供高分辨率图谱。我们设想ChEC-seq将成为ChIP的强大对手,提供独立的手段来验证ChIP-seq结果并发现对基因组调控的新见解。

Introduction

映射转录因子(TF),染色质重组体和其他染色质相关调控因子的结合位点是理解所有基于染色质的过程的关键。虽然染色质免疫沉淀和高通量测序(ChIP-seq)方法已被用于获取对基因组生物学的许多重要见解,但它们具有显着的局限性。我们最近引入了一种替代方法,称为染色质内源性切割和高通量测序(ChEC-seq) 1 ,以克服这些缺点。

最常使用初始甲醛交联步骤(X-ChIP-seq)进行ChIP-seq以保护蛋白质-DNA相互作用。然而,一些最近的研究表明,X-ChIP-seq捕获瞬时或非特异性蛋白质 – DNA相互作用2,3,4,5 sup>, 6,7,8 ,导致假阳性结合位点。此外,通常用于在X-ChIP-seq实验中分离染色质的超声处理,优先剪切开放染色质区域,导致片段从这些区域偏向恢复9,10。超声处理还产生片段长度的不均匀混合物,最终限制了结合位点分辨率,尽管加入外切核酸酶消化步骤可以极大地提高分辨率11,12 。来自亲和纯化的天然分离染色质(ORGANIC) 13的基因组的占据区域的本地ChIP方法不使用交联和片段染色质与微球菌核酸酶(MNase),减轻与甲醛交联和超声处理相关的潜在偏差。然而,许多染色质结合蛋白在天然染色质提取所需的相对温和条件下的溶解性差,可能导致动态范围和/或假阴性减少14

虽然ChIP-seq的各种迭代最常用于蛋白质-DNA相互作用的全基因组映射,但是还已经实现了基于目的蛋白质融合到各种DNA修饰酶的几种测绘技术。一种这样的方法是DNA腺嘌呤甲基转移酶鉴定(DamID) 15 ,其中感兴趣的染色质结合蛋白遗传融合到Dam,并且该融合在细胞或动物中表达,导致接近蛋白质结合位点的GATC序列的甲基化。 DamID的优点在于它不依赖于免疫沉淀,因此避免交联,抗体或染色质溶解。它也在体内进行 。然而,DamID的分辨率限于千碱基量表,Dam融合蛋白的甲基化活性是组成型的。基于酶融合的第二种方法是Calling Card-seq 16 ,其使用感兴趣因子融合转座酶,引导位点特异性整合转座子。像DamID一样,Calling Card-seq不是基于免疫沉淀,因此具有类似的优点,增加分辨率的附加益处。然而,呼叫卡-seq可能受到转座酶的序列偏差的限制,并且还依赖于接近转座子插入位点的限制性位点的存在。

在Laemmli实验室开发的第三种酶融合方法是染色质内源性切割(ChEC) 17 。在ChEC中,染色质相关蛋白和MNase之间的融合物在细胞中表达,并且通过钙添加来活化MNase,DNA被切割到接近标记的结合位点因子( 图1 )。结合Southern印迹,ChEC已被用于表征17,18号酵母中多个单个位点的染色质结构和蛋白结合,并结合低分辨率微阵列分析来探测核孔组分与酵母的相互作用基因组19 。 ChEC提供类似DamID和Calling Card-seq的优点,当通过引物扩展19进行分析时,其分辨率几乎为单碱基对。 ChEC也是可控的:MNase的强力DNA切割取决于加入毫摩尔钙,确保MNase在活酵母细胞中观察到的低游离钙浓度下是无活性的。

以前,我们假设将ChEC与高通量测序(ChEC-seq)相结合将提供TF结合位点的高分辨率图谱。事实上,ChEC-seq生成跨基因组1的出芽酵母总调控因子(GRFs)Abf1,Rap1和Reb1的高分辨率 。我们还成功地将ChEC-seq应用于模块化Mediator复合体,这是一种保守的,重要的全局转录共激活因子21 ,扩大了ChEC-seq对不直接接触DNA的巨大体细胞复合物的适用性,可能难以通过ChIP-基于方法。 ChEC-seq是独立验证ChIP-seq结果和产生对染色质定位过程调节的新见解的有力方法。在这里,我们提出了一种在芽殖酵母中实施该方法的分步骤方案。

Protocol

酵母菌的生成 产生带有MNase标记的感兴趣因子的酵母菌株。 PCR使用指定的反应混合物( 表2 )和循环条件( 表3 )从所需载体( 表1 )扩增MNase标记盒。混合5μLPCR反应和1μL6X DNA加载染料。在0.8V的琼脂糖凝胶上以120V的速度运行每个PCR等分试样40分钟。预期产品大小为〜2.3 kb。 使用标准乙酸锂转化22将放大的?…

Representative Results

在成功的ChEC实验的情况下,通过琼脂糖凝胶电泳分析DNA将显示DNA片段化随时间的钙依赖性增加,如通过涂抹和最终完全消化基因组DNA所指示的。在一些情况下,在扩展消化后观察到类似于传统MNase消化所观察到的条带。 Reb1的ChEC分析就是这种情况,Reb1是结合核小体耗尽区域(NDR)的一般调控因子( 图2 )。我们发现,在GRF的情况下,延长的消化导致…

Discussion

我们已经表明,ChEC可以将不同类型的酵母蛋白质映射到染色质上,并且预计它将广泛适用于酵母中TF和其他染色质结合因子的不同家族。 ChEC-seq的优点在于它不需要交联,染色质溶解或抗体。因此,ChEC避免了X-ChIP-seq中可能存在的伪像,例如超级ChIPable伪影3,4和原生ChIP,例如由于不完全的蛋白质溶解引起的假阴性14 。与所有酶融合方法一样,ChEC-seq的主要缺点是…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢Moustafa Saleh和Jay Tourigny对手稿的批判性阅读,Steven Hahn和Steven Henikoff在开发ChEC-seq期间的指导和支持及其在Mediator综合体中的应用。 SG由NIH授权R01GM053451和R01GM075114支持,GEZ由印第安纳大学创业基金支持。

Materials

dNTPs NEB N0447
Q5 high-fidelity DNA polymerase NEB M0491L Other high-fidelity DNA polymerases, such as Phusion, may be used for cassette amplification.
TrackIt 1 Kb Plus DNA ladder ThermoFisher Scientific 10488085
Taq DNA polymerase NEB M0273L
cOmplete Mini EDTA-free protease inhibitor cocktail Sigma-Aldrich 11836170001 It is important that an EDTA-free protease inhibitor mix is used, so as not to inhibit Mnase cleavage by chelation of Ca2+.
PMSF ACROS Organics AC215740010
Digitonin, High Purity EMD Millipore 300410-250MG Make a 2% stock by dissolving 20 mg digitonin in 1 mL DMSO with vigorous vortexing.
Proteinase K, 20 mg/mL Invitrogen 25530049
RNase A, 10 mg/mL ThermoFisher Scientific EN0531
Ampure XP beads Beckman Coulter A63880 Ampure-like beads can be generated using a published protocol (ref 24).
MagneSphere magnetic rack Promega Z5342

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Cite This Article
Grünberg, S., Zentner, G. E. Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae. J. Vis. Exp. (124), e55836, doi:10.3791/55836 (2017).

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