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

Stefano G Manzo,Andrea Cossarizza,Jessica Marinello,Stella R Hartono,Giovanni Capranico,Frederic Chedin,Sara De Biasi,Lionel A Sanz

doi:10.1186/s13059-018-1478-1

Abstract

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.ResultsHere, we perform high-resolution strand-specific R-loop mapping in human cells depleted for Top1 and find that Top1 depletion results in both R-loop gains and losses at thousands of transcribed loci, delineating two distinct gene classes. R-loop gains are 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, occur in gene-rich regions overlapping H3K27me3-marked active replication initiation regions. Interestingly, Top1 depletion coincides with a block of the cell cycle in G0/G1 phase and a trend towards replication delay.ConclusionsOur findings reveal new properties of Top1 in regulating R-loop homeostasis in a context-dependent manner and suggest a potential role for Top1 in modulating the replication process via R-loop formation.

Highlights

Co-transcriptional R-loops are abundant non-B DNA structures in mammalian genomes
Top1 depletion causes subtle R-loop gains and losses To investigate how global R-loop patterns change upon Top1 depletion, we used siRNA transfection to efficiently silence Top1 in human HEK293 cells (Fig. 1a) and quantified global R-loop levels using dot blots, taking advantage of the anti-DNA:RNA hybrid S9.6 antibody [29, 30]
R-loop structures were observed over 69,066 peaks using a standard peak calling algorithm, (Additional file 2: Table S1), covering ~ 200 megabases (Mb) of genomic space, which is in close agreement with previous data [4]

Summary

Introduction

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 Top regulates R-loop structures at a global level is unknown Biological processes such as transcription and replication generate torsional stress on the DNA double helix that, if not properly dealt with, can lead to genome instability [1]. R-loop structures, a prevalent non-B DNA structure in mammalian genomes, have been linked to genomic instability by causing interference between the replication and transcription machineries [2, 3]. While it is clear that Top regulates R-loops and prevents R-loop-induced genomic instability, the range of loci that are sensitive to R-loop modulation by Top is not known Addressing this gap in knowledge is important given rising evidence that R-loops are abundant in mammalian genomes and participate in important biological processes [19,20,21]. Our work reveals that Top modulates R-loop structures differently according to genomic context and provide new evidence that R-loops may play a role in the replication process

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