Hartono, S. R. | bioRxiv Science

bioRxiv2018

DNA Topoisomerase I differentially modulates R-loops across the human genome

BackgroundCo-transcriptional R-loops are abundant non-B DNA structures in mammalian genomes. DNA Topoisomerase I (Top1) is often thought to regulate R-loop formation owing to its ability to resolve both positive and negative supercoils. How Top1 regulates R-loop structures at a global level is unknown.\n\nResultsHere, we performed high-resolution strand-specific R-loop mapping in human cells depleted for Top 1 and found that Top1 depletion resulted in both R-loop gains and losses at thousands of transcribed loci, delineating two distinct gene classes. R-loop gains were characteristic for long, highly transcribed, genes located in gene-poor regions anchored to Lamin B1 domains and in proximity to H3K9me3-marked heterochromatic patches. R-loop losses, by contrast, occurred in gene-rich regions overlapping H3K27me3-marked active replication initiation regions. Interestingly, Top1 depletion coincided with a block of the cell cycle in G0/G1 phase and a trend towards replication delay.\n\nConclusionsOur findings reveal new properties of Top1 in regulating R-loop homeostasis and suggest a potential role for Top1 in controlling replication initiation via R-loop formation.

genomics↗

bioRxiv2017

The affinity of the S9.6 antibody for double-stranded RNAs impacts the mapping of R-loops in fission yeast.

R-loops, which result from the formation of stable DNA:RNA hybrids, can both threaten genome integrity and act as physiological regulators of gene expression and chromatin patterning. To characterize R-loops in fission yeast, we used the S9.6 antibody-based DRIPc-seq method to sequence the RNA strand of R-loops and obtain strand-specific R-loop maps at near nucleotide resolution. Surprisingly, preliminary DRIPc-seq experiments identified mostly RNase H-resistant but exosome-sensitive RNAs that mapped to both DNA strands and resembled RNA:RNA hybrids (dsRNAs), suggesting that dsRNAs form widely in fission yeast. We confirmed in vitro that S9.6 can immuno-precipitate dsRNAs and provide evidence that dsRNAs can interfere with its binding to R-loops. dsRNA elimination by RNase III treatment prior to DRIPc-seq allowed the genome-wide and strand-specific identification of genuine R-loops that responded in vivo to RNase H levels and displayed classical features associated with R-loop formation. We also found that most transcripts whose levels were altered by in vivo manipulation of RNase H levels did not form detectable R-loops, suggesting that prolonged manipulation of R-loop levels could indirectly alter the transcriptome. We discuss the implications of our work in the design of experimental strategies to probe R-loop functions.

molecular biology↗