DNA topoisomerase I (Top1) can regulate the coupling of RNA polymerase II (RNAP) pausing at promoter-proximal sites and transcript maturation in human cells.

Top1 has long been shown to play a role in transcription. However the molecular functions in living human cells have remained elusive. Recently, we have reported unexpected effects of camptothecin (CPT), a specific antitumor inhibitor of Top1, on RNAP distribution along transcribed genes in human cancer cells (JMB, 357: 127, 2006). We suggested that Top1 inhibition can increase RNAP escape from promoter-proximal pausing sites. To further understand the transcriptional functions of Top1, we have investigated the effects of CPT on chromatin-bound RNAP and transcript maturation for a number of genes in human cancer cells with the endogenous TOP1 gene down-regulated by siRNA. The studied genes include cMYC, GAPD, H3B, HIF-1alpha, Actin, and others. Chromatin-bound RNAP levels were determined by chromatin-immunoprecipitation (ChIP) and specific splicing was determined by qrt-PCR. CPT, but not VM-26 and cisplatin, increased the RNAP escape from the pause sites at low, sub-toxic concentrations after few minutes of drug treatment. The CPT effect on RNAP was reduced in cells with siRNA-silenced Top1 gene. Interestingly, CPT treatments impaired splicing of the first intron of the examined genes, in particular those with a long first intron. Drug effects were independent from DNA replication and checkpoint activation, as suggested by co-treatments with aphidicolin and caffeine (specific inhibitors of DNA polymerase and checkpoint kinases). In addition, analyses of chromatin-bound pre-mRNA (with a modified ChIP technique) showed that CPT is able to increase RNA synthesis downstream of RNAP pausing sites. We are currently investigating topotecan, a clinical relevant CPT analog, and data will be presented at the meeting. Our findings thus demonstrate that Top1 inhibitors can increase RNAP escape from promoter-proximal pausing sites along with an impairment of first intron splicing. We conclude that Top1 regulates the coupling of transcription pausing and mRNA maturation, and that Top1 inhibition may interfere with this process; thereby altering the expression of specific genes.