染色质免疫共沉淀测序技术研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2021-0807

摘要/Abstract

摘要：

染色质免疫共沉淀测序（chromatin immunoprecipitation followed by sequencing,ChIP-seq）是研究目的蛋白与DNA相互作用的重要方法,被广泛应用于转录因子、组蛋白修饰等分布与功能的研究。研究人员通过对细胞分离、染色质片段化以及测序文库构建等关键步骤不断优化,使ChIP-seq适合少量细胞的分析。近年来发展迅速的CUT&RUN（cleavage under targets and release using nuclease）、CUT&Tag（cleavage under targets and tagmentation）技术,利用特异性抗体使酶“靶向”结合目标蛋白,通过MNase酶切或Tn5转座酶切割染色质,简化了实验操作流程。本文介绍了ChIP-seq的原理及其数据分析方法,比较ChIP-seq优化方法和衍生技术。总结了在植物生长发育过程中,转录因子和组蛋白修饰在生物钟调控、激素信号转导、光信号途径、胁迫响应等方面研究与染色质免疫共沉淀测序技术的应用。

关键词: 染色质免疫共沉淀, 转录因子, 组蛋白修饰, 数据分析

Abstract:

Chromatin immunoprecipitation followed by sequencing（ChIP-seq）is an important method to study the interaction between DNA and target proteins,it has been widely used to study transcription factor and the functions and distributions of post-translation histone modification. In recent years,researchers have been optimizing the key steps of this method,including cell separation,chromatin fragmentation,immunoprecipitation and sequencing library construction,allowing the ChIP-seq suitable for the analysis of small amount of cells. In cleavage under targets and release using nucleus（CUT&RUN）and cleavage under targets and tagmentation（CUT&Tag）,the enzyme is “targeted” to the target protein through specific antibodies,and the chromatin near the target protein is digested by MNase or Tn5 transposase,which simplifies the operation process. The paper provided an overview of the principle behind ChIP-seq and its data analysis methods,compared the ChIP-based optimization methods and derived methods,and summarized the studies of transcription factors and histone modification in the regulation of biological clock,hormone signal transduction,signal transduction pathway and stress response in the process of plant growth and development,as well as the application of ChIP-seq technique.

Key words: chromatin immunoprecipitation, transcription factors, histone modification, data analysis

陈桂芳, 杨佳怡, 高运华, 任歌. 染色质免疫共沉淀测序技术研究进展[J]. 生物技术通报, 2022, 38(7): 40-50.

CHEN Gui-fang, YANG Jia-yi, GAO Yun-hua, REN Ge. Research Progress in Chromatin Immunoprecipitation Followed by Sequencing[J]. Biotechnology Bulletin, 2022, 38(7): 40-50.

图/表 5

参考文献 76

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	ULI-NChIP-seq	scChIP-seq	ChIP-exo	MOW ChIP-seq	ChIP-nexus	Nano-ChIP-seq
细胞分离/ 单细胞捕获	流式细胞分选	液滴微流控芯片	-	微流控芯片	-	-
染色质制备	MNase酶切	MNase酶切	甲醛交联超声断裂 lambda核酸外切酶 RecJ_f 核酸外切酶	甲醛交联超声断裂	甲醛交联超声断裂	甲醛交联超声断裂
文库制备	-	-	-	-	使用含有BamH I酶切位点的测序接头,环化连接酶使DNA成环,酶切后进一步建库	使用发夹结构引物; BciV I酶切DNA,使含有突出末端便于连接测序接头
细胞数	10³-10^5[39]	单细胞建库^[42]	10^6[43-44]	10²-10^4[45]	10^6[47]	10^4[49]

	ULI-NChIP-seq	scChIP-seq	ChIP-exo	MOW ChIP-seq	ChIP-nexus	Nano-ChIP-seq
细胞分离/ 单细胞捕获	流式细胞分选	液滴微流控芯片	-	微流控芯片	-	-
染色质制备	MNase酶切	MNase酶切	甲醛交联超声断裂 lambda核酸外切酶 RecJ_f 核酸外切酶	甲醛交联超声断裂	甲醛交联超声断裂	甲醛交联超声断裂
文库制备	-	-	-	-	使用含有BamH I酶切位点的测序接头,环化连接酶使DNA成环,酶切后进一步建库	使用发夹结构引物; BciV I酶切DNA,使含有突出末端便于连接测序接头
细胞数	10³-10^5[39]	单细胞建库^[42]	10^6[43-44]	10²-10^4[45]	10^6[47]	10^4[49]

	ChIP-seq	CUT&RUN	CUT&Tag
细胞处理	甲醛交联/裂解细胞	改变细胞膜通透性	改变细胞膜通透性
染色质制备	超声断裂/MNase酶切	染色质与特异性抗体、pA-MN反应;Ca²⁺激活MNase酶切处理染色质	染色质与特异性抗体、pA-Tn5反应;Mg²⁺激活Tn5,进行切割与接头连接
细胞数	10⁶-10⁷	10²-10^3[54] 可实现单细胞建库^[55]	10²-10³ 可实现单细胞建库^[59]

	ChIP-seq	CUT&RUN	CUT&Tag
细胞处理	甲醛交联/裂解细胞	改变细胞膜通透性	改变细胞膜通透性
染色质制备	超声断裂/MNase酶切	染色质与特异性抗体、pA-MN反应;Ca²⁺激活MNase酶切处理染色质	染色质与特异性抗体、pA-Tn5反应;Mg²⁺激活Tn5,进行切割与接头连接
细胞数	10⁶-10⁷	10²-10^3[54] 可实现单细胞建库^[55]	10²-10³ 可实现单细胞建库^[59]