Sanz, L. A. | Search bioRxiv

bioRxiv2019

High-Throughput Single-Molecule R-loop Footprinting Reveals Principles of R-loop Formation

R-loops are a prevalent class of non-B DNA structures that form during transcription upon reannealing of the nascent RNA to the template DNA strand. R-loops have been profiled using the S9.6 antibody to immunoprecipitate DNA:RNA hybrids. S9.6-based DNA:RNA immunoprecipitation (DRIP) techniques revealed that R-loops form dynamically over conserved genic hotspots. We developed an orthogonal profiling methodology that queries R-loops via the presence of long stretches of single-stranded DNA on the looped-out strand. Non-denaturing 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 single DNA molecules and at ultra-deep coverage. Single-molecule R-loop footprinting (SMRF-seq) revealed a strong agreement between S9.6-and bisulfite-based R-loop mapping and confirmed that R-loops form from unspliced transcripts over genic hotspots. Using the largest single-molecule R-loop dataset to date, we show that individual R-loops generate overlapping sets of molecular clusters that pile-up through larger R-loop-prone zones. SMRF-seq further established that R-loop distribution patterns are driven by both intrinsic DNA sequence features and DNA topological constraints, revealing the principles of R-loop formation.

genetics↗

bioRxiv2019

RNA strand invasion activity of the Polycomb complex PRC2

Epigenetic regulation is conveyed through information encoded by specific chromatin features. Non-canonical nucleic acid structures could in principle also convey biological information but their role(s) in epigenetic regulation is not known. Polycomb Group (PcG) proteins form memory of transient transcriptional repression events that is necessary for development. In Drosophila, PcG proteins are recruited to specific DNA sequences, Polycomb Response Elements (PREs). PREs are switchable memory elements that can exist in repressed, active, or unengaged states 1,2. How PcG activities are targeted to PREs to maintain repressed states only in appropriate developmental contexts has been difficult to elucidate. Biochemically, PcG protein complexes modify chromatin to maintain gene repression 1,3,4. However, PcG proteins also interact with both RNA and DNA, and RNA is implicated in the targeting of PcG function. We find that R-loops, three-stranded nucleic acid structures formed when an RNA hybridizes to its complementary DNA and displaces the other DNA strand 5, form at many PREs in Drosophila embryos, and correlate with the repressive state. R-loops are recognized by the PcG complex PRC1 in vitro. Unexpectedly, we find that the PcG complex PRC2 has RNA strand invasion activity, which can drive formation of RNA-DNA hybrids, the key component of R-loops. Our results suggest a new mechanism for targeting PcG function through R-loop formation by PRC2 and recognition by PRC1. More generally, our findings suggest formation and recognition 6 of non-canonical nucleic acid structures as an epigenetic mechanism.

biochemistry↗