Systematic Mapping of RNA-Chromatin Interactions In Vivo

Sridhar, Bharat; Rivas‐Astroza, Marcelo; Nguyen, Tri C.; Chen, Weizhong; Yan, Zhangming; Cao, Xiaoyi; Hebert, L. P.; Zhong, Sheng

doi:10.1016/j.cub.2017.01.011

Cited by 153 publications

(156 citation statements)

References 20 publications

(18 reference statements)

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“…RADICL-seq introduces substantial improvements over similar RNA-chromatin proximity ligation approaches 7,8,9 . Compared to MARGI, RADICL-seq minimizes the frequency of spurious interactions in the dataset by performing the in situ ligation in intact nuclei.…”

Section: Comparison Of Radicl-seq With Existing Technologies That Tarmentioning

confidence: 99%

“…A few technologies have emerged to assess genome-wide RNA-chromatin interactions 7,8,9 which nevertheless present limitations. Mapping RNA-Genome Interactions (MARGI) is a proximity ligation-based technology that requires a high number of input cells (i.e.…”

Section: Introductionmentioning

confidence: 99%

“…Mapping RNA-Genome Interactions (MARGI) is a proximity ligation-based technology that requires a high number of input cells (i.e. hundreds of millions) and the disruption of the nuclear structure 7 , which can result in detection of a large number of spurious interactions 10 and therefore offers a limited pan-cellular application to investigate cell type-specific RNA-chromatin interactions. Chromatin associated RNA sequencing (CHAR-seq) and global RNA Interaction with DNA by deep-sequencing (GRID-seq) utilize in situ approaches to detect genomewide RNA-chromatin contacts 8,9 .…”

Section: Introductionmentioning

confidence: 99%

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RADICL-seq identifies general and cell type-specific principles of genome-wide RNA-chromatin interactions

Bonetti

Agostini

Hashimoto

et al. 2019

Preprint

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Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding transcripts exhibit nuclear localization and several have been shown to play a role in the regulation of gene expression and chromatin remodelling. To investigate the function of such RNAs, methods to massively map the genomic interacting sites of multiple transcripts have been developed. However, they still present some limitations. Here, we introduce RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq), a technology that maps genome-wide RNA-chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation-based methodology that reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency compared to existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of transcripts as well as cell type-specific RNA-chromatin interactions, and emphasizes the role of transcription in the establishment of chromatin structure.RADICL-seq reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency. Application of RADICL-seq to mouse embryonic stem cells (mESCs) and mouse oligodendrocyte progenitor cells (mOPCs) reveals distinct genome occupancy patterns for specific classes of transcripts and uncovers cell-type specific RNA-chromatin interactions.Furthermore, our results highlight the role of transcription in the establishment of the threedimensional (3D) structure of chromatin. Results RADICL-seq technologyWe developed RADICL-seq by using R08, a Mus musculus male embryonic stem cell line with a deeply characterized transcriptome 11 , to identify genome-wide RNA-chromatin (or RNA-DNA) interactions in preserved nuclei (Fig. 1a). We crosslinked cells with two different formaldehyde (FA) concentrations (1% and 2%) to test whether captured interactions were dependent on the amount of crosslinking agent. After crosslinking we isolated the nuclei, partially digested the genomic DNA with DNase I and ends-prepared the chromatin. During technical development of RADICL-seq we evaluated different enzymes that specifically act on RNA to generate a 3'-hydroxyl end, compatible with RNA ligation (Supplementary Fig. 1a). Sequencing data of test RADICL-seq libraries showed that RNase H treatment increased the percentage of uniquely mapped RNA-chromatin interactions by decreasing the ribosomal RNA (rRNA) content, when compared to nuclease S1, RNase V1 or absence of treatment. RNase H enzymatic treatment is known to target RNA-DNA hybrids and, therefore, it could potentially digest nascent RNA bound to its transcription locus, including the highly transcribed rRNA. Indeed, we observed a 2.5-fold reduction in the number of RNA-DNA interactions occurring at a distance below 1 kb between RNAse H-treated and untreated samples (Supplementary Fig. 1b).After enzymatic treatment of the RNA, we introduced a bridge adapter to specifically ligate proximal RNA and DNA (Supplementary Fig. 1c). The adapter is a 5'...

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Section: Comparison Of Radicl-seq With Existing Technologies That Tarmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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RADICL-seq identifies general and cell type-specific principles of genome-wide RNA-chromatin interactions

Bonetti

Agostini

Hashimoto

et al. 2019

Preprint

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“…The development of tools for unbiased discovery and mapping of chromatin-associated RNAs will undoubtedly clarify the prevalence of trans -acting mechanisms, identify classes of RNAs acting by these mechanisms, and begin to dissect their modes of chromatin recognition [25,26]. …”

Section: Non-coding Rnas That Control Facultative Heterochromatinmentioning

confidence: 99%

RNA-mediated regulation of heterochromatin

Johnson

Straight

2017

Current Opinion in Cell Biology

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The formation of condensed, transcriptionally repressed heterochromatin is essential for controlling gene expression throughout development, silencing parasitic DNA elements, and for genome stability and inheritance. Cells employ diverse mechanisms for controlling heterochromatin states through proteins that modify DNA and histones. An emerging theme is that chromatin-associated RNAs play important roles in regulating heterochromatin proteins by controlling their initial recruitment to chromatin, their stable association with chromatin, their spread along chromatin, or their enzymatic activity. Major challenges for the field include not only identifying regulatory RNAs, but understanding the underlying biochemical mechanisms for how RNAs associate with chromatin, the specificity of interactions between heterochromatin proteins and RNA, and how these binding events manifest in cells to orchestrate RNA-mediated regulation of heterochromatin.

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“…What RNAs localize near my DNA region(s) of interest? Results from ChAR-seq and other recently published methods that identify RNA-DNA interactions (Li et al, 2017;Sridhar et al, 2017;Quinodoz et al, 2018) can also be used to ask more sophisticated questions about chromatin-RNA biology relating to enhancer-promoter contacts, splicing regulation, nascent transcription, and the three-dimensional organization of nucleic acids in the nucleus.…”

Section: Introductionmentioning

confidence: 99%

Chromatin‐Associated RNA Sequencing (ChAR‐seq)

Jukam

Limouse

Smith

et al. 2019

CP Molecular Biology

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RNA is a fundamental component of chromatin. Noncoding RNAs (ncRNAs) can associate with chromatin to influence gene expression and chromatin state; many also act at long distances from their transcriptional origin. Yet we know almost nothing about the functions or sites of action for most ncRNAs. Current methods to identify sites of RNA interaction with the genome are limited to the study of a single RNA at a time. Here we describe a protocol for ChAR‐seq, a strategy to identify all chromatin‐associated RNAs and map their DNA contacts genome‐wide. In ChAR‐seq, proximity ligation of RNA and DNA to a linker molecule is used to construct a chimeric RNA‐DNA molecule that is converted to DNA for sequencing. In a single assay, ChAR‐seq can discover de novo chromatin interactions of distinct RNAs, including nascent transcripts, splicing RNAs, and long noncoding RNAs (lncRNAs). Resulting “maps” of genome‐bound RNAs should provide new insights into RNA biology. © 2019 by John Wiley & Sons, Inc.

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Systematic Mapping of RNA-Chromatin Interactions In Vivo

Cited by 153 publications

References 20 publications

RADICL-seq identifies general and cell type-specific principles of genome-wide RNA-chromatin interactions

RADICL-seq identifies general and cell type-specific principles of genome-wide RNA-chromatin interactions

RNA-mediated regulation of heterochromatin

Chromatin‐Associated RNA Sequencing (ChAR‐seq)

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