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PLoS One. 2016 Jan 19;11(1):e0147053. doi: 10.1371/journal.pone.0147053. eCollection 2016.

Dynamic Effects of Topoisomerase I Inhibition on R-Loops and Short Transcripts at Active Promoters.

Author information

1
Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy.
2
Cancer Research Center of Toulouse, INSERM UMR1037, Toulouse, France.
3
Center for Genome Research, Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy.

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.

PMID:
26784695
PMCID:
PMC4718701
DOI:
10.1371/journal.pone.0147053
[Indexed for MEDLINE]
Free PMC Article

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