DNA topoisomerase I inhibition by camptothecin induces escape of RNA polymerase II from promoter-proximal pause site, antisense transcription and histone acetylation at the human HIF-1α gene locus

Abstract

Top1 inhibition by camptothecin (CPT) perturbs RNA polymerase II (Pol II) density at promoters and along transcribed genes suggesting an involvement of Top1 in Pol II pausing. Here, we demonstrate that Top1 inhibition favors Pol II escape from a promoter-proximal pausing site of the human HIF-1α gene in living cells. Interestingly, alternative splicing at exon 11 was markedly altered in nascent HIF-1α mRNAs, and chromatin structure was also affected with enhanced histone acetylation and reduced nucleosome density in a manner dependent on cdk activity. Moreover, CPT increases transcription of a novel long RNA (5′aHIF1α), antisense to human HIF-1α mRNA, and a known antisense RNA at the 3′-end of the gene, while decreasing mRNA levels under normoxic and hypoxic conditions. The effects require Top1, but are independent from Top1-induced replicative DNA damage. Chromatin RNA immunoprecipitation results showed that CPT can activate antisense transcription mediated by cyclin-dependent kinase (cdk) activity. Thus, Top1 inhibition can trigger a transcriptional stress, involving antisense transcription and increased chromatin accessibility, which is dependent on cdk activity and deregulated Pol II pausing. A changed balance of antisense transcripts and mRNAs may then lead to altered regulation of HIF-1α activity in human cancer cells.