R-ChIP for genome-wide mapping of R-loops by using catalytically inactive RNASEH1

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-...

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Vydáno v:	Nature protocols Ročník 14; číslo 5; s. 1661 - 1685
Hlavní autoři:	Chen, Jia-Yu, Zhang, Xuan, Fu, Xiang-Dong, Chen, Liang
Médium:	Journal Article
Jazyk:	angličtina
Vydáno:	England Nature Publishing Group 01.05.2019
Témata:	Animals Biological activity Cell Line Cell lines Chromatin Chromatin Immunoprecipitation - methods Chromosome Mapping - methods Deoxyribonucleic acid DNA DNA - chemistry DNA - genetics DNA damage DNA structure DNA-directed RNA polymerase Gene expression Gene mapping Genomes Genomic instability Hybrid structures Hybrids Immunoprecipitation Mapping Monoclonal antibodies Mutation Myelodysplastic syndrome R-loops Ribonuclease H - chemistry Ribonuclease H - genetics Ribonuclease H - metabolism Ribonucleic acid RNA RNA - chemistry RNA - genetics RNA polymerase RNA polymerase II Splicing Splicing factors Stability Transcription
ISSN:	1754-2189, 1750-2799, 1750-2799
On-line přístup:	Získat plný text
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Shrnutí:	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 ~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).
Bibliografie:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23
ISSN:	1754-2189 1750-2799 1750-2799
DOI:	10.1038/s41596-019-0154-6