title: Dynamic Effects of Topoisomerase I Inhibition on R-Loops and Short Transcripts at Active Promoters.
abstract: Topoisomerase I-DNA-cleavage complexes (Top1cc) stabilized by camptothecin (CPT) have specific effects at transcriptional levels. We recently reported that Top1cc increase antisense transcript (aRNAs) levels at divergent CpG-island promoters and, transiently, DNA/RNA hybrids (R-loop) in nuclear and mitochondrial genomes of colon cancer HCT116 cells. However, the relationship between R-loops and aRNAs was not established. Here, we show that aRNAs can form R-loops in N-TERA-2 cells under physiological conditions, and that promoter-associated R-loops are somewhat increased and extended in length immediately upon cell exposure to CPT. In contrast, persistent Top1ccs reduce the majority of R-loops suggesting that CPT-accumulated aRNAs are not commonly involved in R-loops. The enhancement of aRNAs by Top1ccs is present both in human colon cancer HCT116 cells and WI38 fibroblasts suggesting a common response of cancer and normal cells. Although Top1ccs lead to DSB and DDR kinases activation, we do not detect a dependence of aRNA accumulation on ATM or DNA-PK activation. However, we showed that the cell response to persistent Top1ccs can involve an impairment of aRNA turnover rather than a higher synthesis rate. Finally, a genome-wide analysis shows that persistent Top1ccs also determine an accumulation of sense transcripts at 5'-end gene regions suggesting an increased occurrence of truncated transcripts. Taken together, the results indicate that Top1 may regulate transcription initiation by modulating RNA polymerase-generated negative supercoils, which can in turn favor R-loop formation at promoters, and that transcript accumulation at TSS is a response to persistent transcriptional stress by Top1 poisoning.
authors.affiliation: Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy.
authors.forename: Jessica
authors.initials: J
authors.lastname: Marinello
authors.affiliation: Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Bertoncini
authors.affiliation: Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy.
authors.forename: Iris
authors.initials: I
authors.lastname: Aloisi
authors.affiliation: Cancer Research Center of Toulouse, INSERM UMR1037, Toulouse, France.
authors.forename: Agnese
authors.initials: A
authors.lastname: Cristini
authors.affiliation: Center for Genome Research, Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy.
authors.forename: Guidantonio
authors.initials: G
authors.lastname: Malagoli Tagliazucchi
authors.affiliation: Center for Genome Research, Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy.
authors.forename: Mattia
authors.initials: M
authors.lastname: Forcato
authors.affiliation: Cancer Research Center of Toulouse, INSERM UMR1037, Toulouse, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Sordet
authors.affiliation: Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Capranico
mesh_terms: D002166:Camptothecin / Q000494:pharmacology*; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics; D045325:HCT116 Cells; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D059004:Topoisomerase I Inhibitors / Q000494:pharmacology*; D014158:Transcription, Genetic
chemical_list: D059004:Topoisomerase I Inhibitors; D004264:DNA Topoisomerases, Type I; C528909:TOP1 protein, human; D002166:Camptothecin