Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts

Bell, Jason C.; Jukam, David; Teran, Nicole A; Risca, Viviana I.; Smith, Owen K.; Johnson, Whitney L.; Skotheim, Jan M.; Greenleaf, William J.; Straight, Aaron F.

doi:10.7554/elife.27024

Cited by 129 publications

(76 citation statements)

References 55 publications

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“…Xist RNA is one of the well-studied lncRNAs involved in epigenetic regulation and gene silencing. It is a 17-kb lncRNA that is expressed from the inactive Xchromosome (Xi) [53], coating the entire X chromosome to repress gene expression through its ability to recruit repressive complexes such as polycomb complexes PRC1 and PRC2 [54][55][56]. In addition, Xist plays an important role in three-dimensional (3D) conformation and maintains the heterochromatin structure in Xi (Fig.…”

Section: Xist Rna Regulates Gene Silencing and Chromatin Conformationmentioning

confidence: 99%

Functions and properties of nuclear lncRNAs—from systematically mapping the interactomes of lncRNAs

2020

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Protein and DNA have been considered as the major components of chromatin. But beyond that, an increasing number of studies show that RNA occupies a large amount of chromatin and acts as a regulator of nuclear architecture. A significant fraction of long non-coding RNAs (lncRNAs) prefers to stay in the nucleus and cooperate with protein complexes to modulate epigenetic regulation, phase separation, compartment formation, and nuclear organization. An RNA strand also can invade into double-stranded DNA to form RNA:DNA hybrids (R-loops) in living cells, contributing to the regulation of gene expression and genomic instability. In this review, we discuss how nuclear lncRNAs orchestrate cellular processes through their interactions with proteins and DNA and summarize the recent genome-wide techniques to study the functions of lncRNAs by revealing their interactomes in vivo.

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Section: Xist Rna Regulates Gene Silencing and Chromatin Conformationmentioning

confidence: 99%

Functions and properties of nuclear lncRNAs—from systematically mapping the interactomes of lncRNAs

2020

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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%

“…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 . CHAR-seq requires a large number of cells as starting material and the use of Dpn II to digest the chromatin, which has a restricted number of sites across the genome and, hence, a lower coverage for captured RNA-DNA interactions 9 .…”

Section: Introductionmentioning

confidence: 99%

“…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 . CHAR-seq requires a large number of cells as starting material and the use of Dpn II to digest the chromatin, which has a restricted number of sites across the genome and, hence, a lower coverage for captured RNA-DNA interactions 9 . In addition, the technology does not size-select the molecules containing both interacting RNA and DNA, resulting in a large fraction of uninformative sequences in the final library.…”

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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Chromatin‐Associated RNA Sequencing (ChAR‐seq)

Jukam

Limouse

Smith

et al. 2019

CP Molecular Biology

Self Cite

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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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Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts

Cited by 129 publications

References 55 publications

Functions and properties of nuclear lncRNAs—from systematically mapping the interactomes of lncRNAs

Functions and properties of nuclear lncRNAs—from systematically mapping the interactomes of lncRNAs

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

Chromatin‐Associated RNA Sequencing (ChAR‐seq)

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