自转录活性调节区测序技术在增强子发现研究中的应用

doi:10.16288/j.yczz.24-149

遗传 ›› 2024, Vol. 46 ›› Issue (8): 589-602.doi: 10.16288/j.yczz.24-149

自转录活性调节区测序技术在增强子发现研究中的应用

王纪龙¹(), 李青²^,³(), 战廷正¹^,³()

1.广西医科大学寄生虫学教研室，南宁530021
2.广西医科大学细胞生物学教研室，南宁530021
3.广西高校区域性疾病基础研究重点实验室(广西医科大学)，南宁530021

收稿日期:2024-05-24 修回日期:2024-07-06 出版日期:2024-08-20 发布日期:2024-07-10
通讯作者: 李青，博士，副教授，研究方向：细胞生物学。E-mail: qing_napier@126.com;
战廷正，博士，副教授，研究方向：寄生虫感染与免疫。E-mail: ztznn@163.com
作者简介:王纪龙，硕士研究生，专业方向：病原生物学。E-mail: wjlshuaifeng@163.com
基金资助:
国家自然科学基金项目(82260407);广西自然科学基金项目(2022JJA140599);卫健委寄生虫病原与媒介生物学重点实验室开放课题(NHCKFKT2023-05)

Principle and application of self-transcribing active regulatory region sequencing in enhancer discovery research

Jilong Wang¹(), Qing Li²^,³(), Tingzheng Zhan¹^,³()

1. Department of Parasitology, Guangxi Medical University, Nanning 530021, China
2. Department of Cell Biology and Genetics, Guangxi Medical University, Nanning 530021, China
3. Key Laboratory of Basic Research on Regional Diseases (Guangxi Medical University), Education Department of Guangxi Zhuang Autonomous Region, Nanning 530021, China

Received:2024-05-24 Revised:2024-07-06 Published:2024-08-20 Online:2024-07-10
Supported by:
National Natural Science Foundation of China(82260407);Guangxi Natural Science Foundation(2022JJA140599);Open Project of the Key Laboratory of Parasite and Vector Biology, National Health Commission of China(NHCKFKT2023-05)

摘要/Abstract

摘要：

自转录活性调节区测序(self-transcribing active regulatory region sequencing，STARR-seq)是一种可发现并同时验证全基因组增强子活性的高通量测序方法。其原理为：将待验证序列插入质粒载体并电转入细胞中，该序列在作为增强子提高靶基因转录的同时，其本身也作为靶基因被增强转录。通过对转录组进行测序，并对比未插入片段的测序结果，可获得增强子在基因组位置及活性的信息。在传统增强子研究方法中，通过对染色质开放区域和转录活性区域进行测序以预测增强子，但只能逐一验证预测结果，无法高通量验证增强子活性。STARR-seq技术解决了上述缺陷，可在对全基因组增强子高通量挖掘的同时，对其活性进行可靠的验证。自STARR-seq技术发明以来，已被广泛运用于不同物种与细胞中的增强子发现及活性验证研究。本文对传统增强子预测方法以及STARR-seq技术的基本原理、发展历史和具体运用进行了介绍，并对其发展前景进行展望，以期为后续增强子相关领域研究人员提供参考。

关键词: 自转录活性区域测序, 增强子, 转录调控, 单核苷酸多态性

Abstract:

Self-transcribing active regulatory region sequencing (STARR-seq) is a high-throughput sequencing method capable of simultaneously discovering and validating all enhancers within the genome. In this method, candidate sequences are inserted into plasmid vectors and electroporated into cells. Acting as both enhancers and target genes, the self-transcription of these sequences will also be enhanced by themselves. By sequencing the transcriptome and comparing the results with the non-inserted control, the locations and activity of enhancers can be determined. In traditional enhancer discovery strategies, the chromatin open regions and transcription active regions were sequenced and predicted as enhancers. However, the activity of these putative enhancers could only be validated one by one without a high-throughput method. STARR-seq solved this limitation, allowing simultaneous enhancers discovery and activity validation in a high-throughput manner. Since the introduction of STARR-seq, it has been widely used to discover enhancers and validate enhancer activity in a number of organisms and cells. In this review, we present the traditional enhancer prediction methods and the basic principles, development history, specific applications of STARR-seq, and its future prospects, aiming to provide a reference for researchers in related fields conducting enhancer studies.

Key words: STARR-seq, enhancer, transcriptional regulation, single nucleotide polymorphism

王纪龙, 李青, 战廷正. 自转录活性调节区测序技术在增强子发现研究中的应用[J]. 遗传, 2024, 46(8): 589-602.

Jilong Wang, Qing Li, Tingzheng Zhan. Principle and application of self-transcribing active regulatory region sequencing in enhancer discovery research[J]. Hereditas(Beijing), 2024, 46(8): 589-602.

图/表 7

图1

表1

传统增强子研究方法的对比"

基本原理	方法名称	具体方法	运用	优点	缺点	参考文献
检测DNA可及区或染色质开放区域	DNase-seq	通过识别DNase I 超敏位点判断顺式调控元件所在区域后预测增强子	染色质开放区域检测	高通量检测全基因组染色质开放区域	切割偏好度过大，只能预测增强子	[31]
	ATAC-seq	使用链接了测序接头的Tn5转座酶切割染色质获得开放区域文库后进行测序	染色质开放区域检测	高通量，样本需求量小	转座酶切割可能有偏好，只能预测增强子	[32]
	MNase-seq	利用微球核酸酶消化核小体之间裸露区域，富集与核小体结合的DNA片段后测序，绘制核小体位置图谱间接反应染色质开放区域	绘制基因组中核小体位置图谱	分辨率极高可分析单个核小体	间接检测染色质开放区域，工作量大	[36]
	FAIRE-seq	使用甲醛交联染色质互作部位后通过超声进行破碎，通过酚-氯仿抽提分离DNA进行测序	染色质开放区域检测	使用超声破碎，避免酶切偏好性	分辨率较低，背景噪音高	[38]
检测增强子相关组蛋白修饰	ChIP-seq	通过染色质免疫共沉淀特异性富集增强子相关蛋白结合的DNA序列	识别转录因子结合位点以及特异性组蛋白修饰	分辨率高，直接识别转录因子作用位点或组蛋白修饰	需要特异性抗体，只能预测增强子	[32]
	CUT&Run	使用Protein A/G结合的MNase，靶向目的蛋白，并在目的蛋白附近对其互作DNA进行片段化	研究组蛋白修饰和蛋白质-DNA相互作用	对样本需求量较低，高灵敏度低背景噪音	需要特异性抗体	[42]
	CUT&Tag	使用Protein A/G结合的转座酶，靶向目的蛋白，并在目的蛋白结合的DNA区域插入测序接头序列并片段化DNA	高灵敏度低背景噪音的ChIP-seq	对样本需求量极低，高灵敏度低背景噪音	需要特异性抗体	[43]
直接检测增强子RNA	GRO-seq	在体外进行新增强子RNA合成及标记后进行测序	检测增强子RNA	动态实时检测增强子活性，尤其是细胞处于不同环境时	无法直接分辨增强子RNA与mRNA	[45]

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方法名称	具体方法	运用	优点	缺点	参考文献
STARR-seq	将待验证片段克隆至核心启动子下游构建待验证文库，将该文库转染至细胞中，培养细胞并提取RNA。活性增强子将提高自我转录程度并成为最终报告转录本的一部分	全基因组增强子发掘及活性验证	高通量，直接发现并验证全基因组增强子	对文库质量以及转染效率有较高要求	[47]
AAV-STARR-seq	通过AAV传递STARR-seq文库后进行测序	活体动物体内增强子检测	可开展体内增强子检测	无法避免染色质状态对增强子活性的影响	[68]
ChIP-STARR-seq	将ChIP-seq鉴定出的DNA连接至STARR-seq载体后进行测序	转录因子结合的DNA序列增强子活性检测	直接验证转录因子作用位点	无法避免染色质状态对增强子活性的影响	[69]
ATAC-STARR-seq	使用ATAC-seq文库连接STARR-seq载体后进行测序	准确高效的从染色质开放区域鉴定出增强子	可准确检测染色质开放区域增强子	无法避免染色质状态对增强子活性的影响	[66]
FAIRE-STARR-seq	使用FAIRE-seq鉴定出的染色质开放区域连接至STARR-seq载体后进行测序	准确高效的从染色质开放区域鉴定出增强子	可准确检测染色质开放区域增强子	无法避免染色质状态对增强子活性的影响	[67]

自转录活性调节区测序技术在增强子发现研究中的应用

Principle and application of self-transcribing active regulatory region sequencing in enhancer discovery research

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