R-ChIP for genome-wide mapping of R-loops by using catalytically inactive RNASEH1
R-ChIP for genome-wide mapping of R-loops by using catalytically inactive RNASEH1
复制标题
利用催化失活的核糖核酸酶H1（RNASEH1）进行R环全基因组定位的R - 染色质免疫沉淀（R-ChIP）
DOI：
10.1038/s41596-019-0154-6
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发表时间：
2019-05-01
期刊：
影响因子：
14.8
通讯作者：
Chen, Liang
中科院分区：
文献类型：
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作者：
Chen, Jia-Yu;Zhang, Xuan;Chen, Liang
新生的RNA可能与DNA形成三链结构，称为R-环，它与基本的生物过程有关，如转录、复制和基因组不稳定性。在这里，我们提供了一个新开发的策略的详细协议，称为R-CHIP，用于在全基因组范围内稳健地捕获R-环。与识别RNA-DNA杂合结构的基于单抗S9.6的R-环作图法不同，R-CHIP涉及在细胞中表达外源催化失活的RNASEH1，以结合RNA-DNA杂交物，但不能分解它们。随后是染色质免疫沉淀(CHIP)标记的RNASEH1和构建用于深度测序的链特异性文库。建立一个稳定表达突变酶的细胞系需要大约3周的时间，而进行R-芯片方案则需要5天的时间。原则上，R-ChIP既适用于细胞系，也适用于动物，只要能够表达催化活性不高的RNASEH1，就可以研究R环的形成和分解的动力学，以及它对基因组功能的影响。在我们最近对R-CHIP的研究中，我们发现R环与RNA聚合酶II暂停/暂停释放之间存在密切的时空关系，并将增强的R环形成与与骨髓增生异常综合征(MDS)相关的关键剪接因子的驱动突变所引起的DNA损伤反应联系起来。
Nascent RNA may form a three-stranded structure with DNA, called an R-loop, which has been linked to fundamental biological processes such as transcription, replication and genome instability. Here, we provide a detailed protocol for a newly developed strategy, named R-ChIP, for robust capture of R-loops genome-wide. Distinct from R-loop-mapping methods based on the monoclonal antibody S9.6, which recognizes RNA-DNA hybrid structures, R-ChIP involves expression of an exogenous catalytically inactive RNASEH1 in cells to bind RNA-DNA hybrids but not resolve them. This is followed by chromatin immunoprecipitation (ChIP) of the tagged RNASEH1 and construction of a strand-specific library for deep sequencing. It takes similar to 3 weeks to establish a stable cell line expressing the mutant enzyme and 5 more days to proceed with the R-ChIP protocol. In principle, R-ChIP is applicable to both cell lines and animals, as long as the catalytically inactive RNASEH1 can be expressed to study the dynamics of R-loop formation and resolution, as well as its impact on the functionality of the genome. In our recent studies with R-ChIP, we showed an intimate spatiotemporal relationship between R-loops and RNA polymerase II pausing/pause release, as well as linking augmented R-loop formation to DNA damage response induced by driver mutations of key splicing factors associated with myelodysplastic syndrome (MDS).