DNA Topoisomerase I differentially modulates R loop across long genes and at human replication origins

Co-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. Here, we performed high-resolution strand-specific R-loop mapping in human cells depleted for Top1 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 origins. Interestingly, Top1 depletion coincided with a block of the cell cycle in G0/G1 phase and a trend towards global replication delay. Our findings reveal new properties of Top1 in regulating R-loop homeostasis and suggest a potential role for Top1 in controlling replication origin via R-loop formation. DNA-RNA immunoprecipitation (DRIP-seq) and DNA-RNA immunoprecipitation strand-specific (DRIPc-seq) were performed on control, scramble, and two Top1 KD HEK293 cell lines. RNA Polymerase II ChIP-seq, and total RNA-seq were performed on two scramble and two Top1 KD HEK293 cell lines. All samples (control, scrambles, Top1 KD cells) are of different passages (i.e. control of DRIP did not come from the same population as DRIPc, et cetera.)