Genome-wide homeostatic regulation of R-loops by the dual RNA-DNA topoisomerase, topoisomerase III-β (TOP3B) II

We report the mapping of R-loops across the human genome in wild-type and TOP3B-KO HCT116 and HT1080 cells by performing DNA: RNA Immunoprecipitation followed by high-throughput sequencing (DRIP-seq). Cellular depletion of TOP3B globally increases the intensity of pre-existing R-loops. The enriched R-loop peaks in TOP3B-KO cells are both in intergenic and genic regions, including protein-coding and non-coding RNA genes. Long genes and genes with high exon-intron junctions accumulate more R-loops. Global transcription analyses indicate that increased R-loop signals do not significantly change transcript level in TOP3B-KO cells. However, increased R-loops in TOP3B-KO cells cause stabilization of G4-quadruplexs and increased gH2AX foci. Altogether, these results demonstrate that TOP3B is needed for the removal of deleterious R-loops that lead to genomic breaks. To investigate the roles of topoisomerase III-beta (TOP3B) in regulating genomic R-loop structures and global transcription process, we generated TOP3B-KO HCT116 cells using CRISPR technique and guide RNA targeting Exon 2 of topoisomerase III-beta. We isolated RNA from two replicates of wild type HCT116 and two replicates of E2-4 TOP3B-KO HCT116 cells and performed ribo-depleted total RNA-seq. We then performed gene expression profiling analysis using data obtained from total RNA-seq of pair of replicates of wild type HCT116 and E2-4 TOP3B-KO HCT116 cells.