Two papers present novel techniques for more precisely mapping R loops

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Two papers present novel techniques for more precisely mapping R loops — hybrid nucleic acid structures in which RNA is hybridized to one strand of DNA while the other strand of DNA is displaced and 'loops out'. Chen et al. describe a chromatin immunoprecipitation (ChIP) technique, termed R-ChIP, which uses a catalytically inactive RNase H to immunoprecipitate R loops. Dumelie and Jaffrey report a technique called bisulfite DNA–RNA immunoprecipitation sequencing (bisDRIP-seq), in which bisulfite converts cytosines to uracils in genomic regions of single-stranded DNA before DRIP-seq.

In developing R-ChIP, Chen et al. expressed a V5-tagged catalytically inactive RNase H1 — which has high affinity for R loops in vivo — in HEK293T cells and carried out ChIP with a V5-targeted antibody. The single-stranded DNA hybridized to RNA in the captured DNA–RNA hybrids was then converted to double-stranded DNA, and the resulting R-ChIP libraries were subjected to deep sequencing. Relative to the established DRIP-seq approach, in which RNA–DNA hybrids are directly captured by the S9.6 antibody prior to sequencing, initial analysis revealed that R loops identified using R-ChIP have a stronger GC skew (a high GC ratio in the non-template DNA strand, which is typical of R loops), greater linkage with transcription and increased association with open chromatin. Thus, R-ChIP seems to be more robust than DRIP-seq. Further analysis revealed that 59.3% of R loops map to promoter-proximal regions, and, using an inhibitor of transcription in combination with R-ChIP, the authors showed that the induction of R loops correlates with transcriptional pausing at transcription start sites (TSSs). Finally, after observing that the majority of R-ChIP-mapped R loops are associated with a nearby free RNA end, the authors used engineered ribozymes to show that a free RNA end, coupled with an R-loop-promoting sequence, is sufficient to generate R loops.

In bisDRIP-seq, Dumelie and Jaffrey lysed MCF7 cells in the presence of bisulfite, which converts cytosine to uracil in the single-stranded DNA portion of R loops, followed by DRIP-seq. A computational pipeline identified single-stranded regions with high concentrations of uracil; a prominence of cytosine–uracil conversions on one strand of DNA indicates the presence of an R loop. bisDRIP-seq scores were calculated for individual nucleotides, and the authors found that, in line with DRIP-seq scores, these scores were enriched in the regions of active promoters. Interestingly, bisDRIP-seq data suggested that TSSs, which surround R loops, are the 5' boundary of promoter-associated R loops and that the first exon–intron junction of a gene acts as the 3' boundary of R loops. As promoter regions of intronless genes were strongly associated with R loops, the exon–intron junction must not promote, or be required for, R-loop formation. R loops in intronless histone genes were all bordered at the 5' end by the TSS; however, as some R loops were restricted to the initial portion of the gene and others covered nearly the whole gene, 3' R-loop boundaries in intronless genes are variable across genes.

These new techniques for detecting R loops will enable researchers to study the formation of these structures, and their roles in transcription, chromatin structure and genomic instability, in more detail.