Abstract 637: Antisense transcripts and R-loops caused by DNA topoisomerase I inhibition by camptothecin at human active CpG island promoters.

Abstract

Abstract DNA Topoisomerase I (Top1) is specifically inhibited by camptothecin (CPT), a natural product with effective anticancer activity. CPT can stabilize a DNA-Top1 DNA cleavage complex that can lead to irreversible DNA breakage at replication forks, and transcription-dependent genome instability. The bulk of cellular Top1 activity is required at transcribing regions to modulate DNA supercoils generated by elongating RNA polymerases. However, the molecular effects of CPT inhibition of Top1 at transcriptional levels are not fully established. By next generation sequencing analyses of bisulfite-treated RNAs extracted from human HCT116 cancer cells, we here show that CPT promoted high levels of antisense tags specifically at active promoters containing CpG islands (CGI). We found several not annotated transcripts close to Refseq genes, the majority of which were promoter-associated antisense transcripts increased by CPT. We identified 256 and 84 of such transcripts in HCT116 cells and Top1-silenced HCT116-siRNATop1 cells, respectively. We validated CPT-stimulated antisense transcripts by PCR in both cell lines, and found that several of them could form R-loops at corresponding genomic regions by affinity purification with a recombinant inactive mutant RNaseH1. Drug effects were independent from replication, and required both Top1 and ongoing transcription. In addition, CPT induced an immediate burst and a subsequent rapid reduction of Top1-DNA cleavage complexes at active, but not inactive, promoters indicating a partial, time-dependent removal of Top1 from chromatin. Our findings demonstrate that Top1 activity prevents accumulation of antisense R-loops and RNAs at human active CGI promoters by relaxing negative DNA supercoils. The transcriptional CPT effects can contribute to drug therapeutic activity in cancer and other diseases. We will discuss our findings in relation to transcription regulation and genome instability caused by interference with Top1 activity. Citation Format: Jessica Marinello, Giovanni Chillemi, Susana Bueno, Stefano G. Manzo, Giovanni Capranico. Antisense transcripts and R-loops caused by DNA topoisomerase I inhibition by camptothecin at human active CpG island promoters. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 637. doi:10.1158/1538-7445.AM2013-637