Ultra-deep Coverage Single-molecule R-loop Footprinting Reveals Principles of R-loop Formation

• Published in: Journal of molecular biology 2020-03, Vol.432 (7), p.2271-2288
• Main Authors: Malig, Maika, Hartono, Stella R., Giafaglione, Jenna M., Sanz, Lionel A., Chedin, Frederic
• Format: Article
• Language: English
• Subjects: DNA - chemistry; DNA:RNA immunoprecipitation; Embryonal Carcinoma Stem Cells - cytology; Embryonal Carcinoma Stem Cells - metabolism; Humans; Nondenaturing bisulfite conversion; R-Loop Structures; R-loops; RNA - chemistry; S9.6 antibody; Single-Cell Analysis - methods; SMRT-sequencing; Transcription, Genetic
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2020.02.014

R-loops are a prevalent class of non-B DNA structures that have been associated with both positive and negative cellular outcomes. DNA:RNA immunoprecipitation (DRIP) approaches based on the anti-DNA:RNA hybrid S9.6 antibody revealed that R-loops form dynamically over conserved genic hotspots. We have developed an orthogonal approach that queries R-loops via the presence of long stretches of single-stranded DNA on their looped-out strand. Nondenaturing sodium bisulfite treatment catalyzes the conversion of unpaired cytosines to uracils, creating permanent genetic tags for the position of an R-loop. Long-read, single-molecule PacBio sequencing allows the identification of R-loop ‘footprints’ at near nucleotide resolution in a strand-specific manner on long single DNA molecules and at ultra-deep coverage. Single-molecule R-loop footprinting coupled with PacBio sequencing (SMRF-seq) revealed a strong agreement between S9.6-based and bisulfite-based R-loop mapping and confirmed that R-loops form over genic hotspots, including gene bodies and terminal gene regions. Based on the largest single-molecule R-loop dataset to date, we show that individual R-loops form nonrandomly, defining discrete sets of overlapping molecular clusters that pileup through larger R-loop zones. R-loops most often map to intronic regions and their individual start and stop positions do not match with intron-exon boundaries, reinforcing the model that they form cotranscriptionally from unspliced transcripts. SMRF-seq further established that R-loop distribution patterns are not simply driven by intrinsic DNA sequence features but most likely also reflect DNA topological constraints. Overall, DRIP-based and SMRF-based approaches independently provide a complementary and congruent view of R-loop distribution, consolidating our understanding of the principles underlying R-loop formation. [Display omitted] •Methodologies to profile R-loops independently of S9.6 antibody are desirable.•Single-molecule R-loop footprinting coupled with PacBio sequencing (SMRF-seq) allows deep single-molecule R-loop footprinting on long amplicons.•SMRF-seq and S9.6-based DRIPc-seq show striking agreement at all tested loci.•Individual R-loops form from unspliced transcripts over discrete molecular clusters.•R-loop distribution is not simply defined by DNA sequence.