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

Abstract

RNA is a critical component of chromatin in eukaryotes, both as a product of transcription, and as an essential constituent of ribonucleoprotein complexes that regulate both local and global chromatin states. Here we present a proximity ligation and sequencing method called Chromatin-Associated RNA sequencing (ChAR-seq) that maps all RNA-to-DNA contacts across the genome. ChAR-seq provides unbiased, de novo identification of targets of chromatin-bound RNAs including nascent transcripts, chromosome-specific dosage compensation ncRNAs, and genome-wide trans-associated RNAs involved in co-transcriptional RNA processing.

Chromatin‐Associated RNA Sequencing (ChAR‐seq)

“…We thank Nikki Teran and Whitney Johnson for contributions during development of the original version of ChAR‐seq as described in Bell et al. (), and thank Ali Shariati for assistance with hESC culture. This work was supported by a Stanford Center for Systems Biology (NIH P50 GM107615 to James E. Ferrell) Seed Grant Award to DJ, VIR, and JCB, by the Katharine McCormick Advanced Postdoctoral Fellowship to VIR, by an NSF‐GRFP to OKS, and by NIH R01 HG009909 to AFS.…”

“…To fill this gap, we developed ChAR‐seq to identify chromatin‐associated RNAs transcriptome‐ and genome‐wide (Bell et al., ). This method relies on in situ proximity ligation of RNA and DNA ends that either directly interact or are in close proximity in nuclear space.…”

“…In principle, ChAR‐seq can map the chromosomal contact sites of all chromatin‐associated RNAs. ChAR‐seq can thus be thought of as a multiplexed de novo RNA‐DNA mapping assay capable of generating hundreds, or even thousands, of individual RNA‐binding maps (Bell et al., ). With such information in hand, experimenters can answer simple, but important, questions such as: Where does my RNA(s) of interest associate with DNA across the genome?…”

“…Cartoon shows three different RNAs binding the genome at distinct sites. More complex, genome‐wide interaction maps can be generated for the entire transcriptome and genome (Bell et al., ).…”

RNA-mediated regulation of heterochromatin

“…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]. …”