Prevalent, Dynamic, and Conserved R-Loop Structures Associate with Specific Epigenomic Signatures in Mammals

Abstract

R-loops are three-stranded nucleic acid structures formed upon annealing of an RNA strand to one strand of duplex DNA. We profiled R-loops using a high-resolution, strand-specific methodology in human and mouse cell types. R-loops are prevalent, collectively occupying up to 5% of mammalian genomes. R-loop formation occurs over conserved genic hotspots such as promoter and terminator regions of poly(A)-dependent genes. In most cases, R-loops occur co-transcriptionally and undergo dynamic turnover. Detailed epigenomic profiling revealed that R-loops associate with specific chromatin signatures. At promoters, R-loops associate with a hyper-accessible state characteristic of unmethylated CpG island promoters. By contrast, terminal R-loops associate with an enhancer- and insulator-like state and define a broad class of transcription terminators. Altogether, this suggests that the retention of nascent RNA transcripts at their site of expression represents an abundant, dynamic, and programmed component of the mammalian chromatin that impacts chromatin patterning and the control of gene expression.

Fragility Extraordinaire: Unsolved Mysteries of Chromosome Fragile Sites

“…DRIPc-seq detected ~70,000 R-loop peaks in the human embryonic carcinoma Ntera2 cells, which is comparable to the number of R-loops detected by RDIP-seq (~64,000 and 39,000 in IMR-90 and HEK 293 T cells, respectively). Consistent with R-loops being co-transcriptional structures, DRIPc-seq peaks were found predominantly in RNA polymerase II-transcribed genes, with two- to three-fold enrichment at the promoter and terminator regions (Sanz et al 2016). Interestingly, this co-transcriptional model of R-loop formation was contended by the RDIP-seq study, at least at the ribosomal DNA loci (Nadel et al 2015).…”

“…Variations of this methodology include DRIPc-seq (DNA/RNA immunoprecipitation followed by cDNA conversion coupled with high-throughput sequencing, an improved version of the previous DRIP-seq method) (Sanz et al 2016) and RDIP (RNA/DNA immunoprecipitation, implementing key technical modifications in RNase I pretreatment and DNA fragmentation by sonication) (Nadel et al 2015). DRIPc-seq detected ~70,000 R-loop peaks in the human embryonic carcinoma Ntera2 cells, which is comparable to the number of R-loops detected by RDIP-seq (~64,000 and 39,000 in IMR-90 and HEK 293 T cells, respectively).…”

The Affinity of the S9.6 Antibody for Double-Stranded RNAs Impacts the Accurate Mapping of R-Loops in Fission Yeast

“…To map R-loops in fission yeast, we used the recently described DRIPc-seq method, where RNA:DNA hybrids are immuno-precipitated with the S9.6 antibody and their associated RNA strands are sequenced in a strand-specific manner to map R-loops at near base-pair resolution [8] (Fig. S1).…”

“…This is true in organisms or genotypes where significant dsRNA loads are produced. Recent DRIPc-seq maps in mammalian cells were strictly directional even in the absence of RNase III treatment [8], suggesting that dsRNA formation was not an issue, at least in the conditions tested. Based on our in vitro results, it is also conceivable that even when DRIP is followed by direct DNA sequencing or qPCR, such as in classic DRIP-seq or DRIP-qPCR, competition from dsRNAs could affect the efficiency of the S9.6 immunoprecipitation (Fig.…”

“…Here we sought to identify R-loops in the fission yeast Schizosaccharomyces pombe using the recently described DRIPc-seq approach, where RNA:DNA hybrids are immuno-precipitated with the S9.6 antibody and their RNA strands are sequenced in a strand-specific manner to map R-loops at near base-pair resolution [8]. This strategy is well suited to mapping R-loops in compact genomes such as fission yeast where genes frequently overlap.…”