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Dynamic Effects of Topoisomerase I Inhibition on R-Loops and Short Transcripts at Active Promoters
Jessica Marinello1, Stefania Bertoncini1, Iris Aloisi1
1Department of Pharmacy and Biotechnology, University of Bologna, Bologna, 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.
Insights
Camptothecin (CPT) stabilizes Topoisomerase I-DNA-cleavage complexes (Top1cc), increasing antisense RNAs (aRNAs) and transient R-loops. Persistent Top1cc impairs aRNA turnover, leading to transcript accumulation and truncated transcripts.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- Topoisomerase I-DNA-cleavage complexes (Top1cc) stabilized by camptothecin (CPT) impact cellular transcription.
- Previous studies showed Top1cc increase antisense RNAs (aRNAs) and transient DNA/RNA hybrids (R-loops).
- The direct relationship between aRNAs and R-loops remained unclear.
Purpose of the Study:
- To investigate the relationship between aRNAs and R-loops under Top1cc conditions.
- To determine the cellular response to persistent Top1cc, including transcript accumulation and turnover.
- To explore the genome-wide effects of Top1cc on transcription.
Main Methods:
- Utilized N-TERA-2 cells to study R-loop formation under physiological and CPT-treated conditions.
- Analyzed aRNA and R-loop dynamics in HCT116 colon cancer cells and WI38 fibroblasts.
- Assessed the role of ATM and DNA-PK activation in aRNA accumulation.
- Investigated aRNA turnover mechanisms and performed genome-wide transcript analysis.
Main Results:
- aRNAs can form R-loops under physiological conditions; CPT initially increases promoter-associated R-loops.
- Persistent Top1cc reduces most R-loops, indicating aRNAs are not commonly involved in long-term R-loops.
- aRNA enhancement by Top1cc occurs in both cancer and normal cells.
- aRNA accumulation is not dependent on ATM or DNA-PK activation.
- Persistent Top1cc impairs aRNA turnover rather than increasing synthesis.
- Genome-wide analysis reveals accumulation of sense transcripts at 5'-ends, suggesting truncated transcripts.
Conclusions:
- Topoisomerase I (Top1) may regulate transcription initiation via RNA polymerase-generated supercoils, influencing R-loop formation.
- Persistent Top1cc leads to transcript accumulation at transcription start sites (TSS) as a response to transcriptional stress.
- The findings provide insights into the mechanisms of Top1cc-induced transcriptional dysregulation in cancer and normal cells.
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