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

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

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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 Online:2024-08-20 Published:2024-07-10
Contact: Qing Li, Tingzheng Zhan E-mail:wjlshuaifeng@163.com;qing_napier@126.com;ztznn@163.com
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)

Abstract

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

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.

Figures/Tables 7

Fig. 1

Table 1

Comparison of enhancer research methods"

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]

Table 1

Fig. 2

Fig. 3

Fig. 4

Table 2

Fig. 5

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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]

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

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