Camptothecin effects on transcription-related events.

Top1 has long been shown to play a role in transcription. However the molecular functions in living human cells have remained elusive. We have determined the early effects of camptothecin on genomic DNA-binding sites of RNA polymerase II (RNAPII), TATA-binding protein (TBP), DNA topoisomerase I (Top1), and histone components in human transcribed loci by chromatin-immunoprecipitation (ChIP). Camptothecin induced a specific reduction of RNAPII density at promoter pause sites and histone modifications suggesting an increased chromatin accessibility. Interestingly, RNAPII reduction at promoter pause sites occurred within 5-10min of camptothecin treatment, and was not a response to replication-dependent DNA breaks. ChIP analyses of RNAPII along transcribed genes indicated that RNAPII levels were transiently increased at internal exons, and that camptothecin effects could be fully reversed by DRB, a cdk inhibitor. Top1 was found to be enriched in active chromatin, therefore suggesting that Top1 inhibition at the transcribed template and/or adjacent regulating regions immediately affects RNAPII at active genes. We suggested that Top1 inhibition can increase RNAP escape from promoter-proximal pausing sites. To further understand the transcriptional functions of Top1, we have then investigated the effects of CPT on transcript maturation of selected genes in human cancer cells with the endogenous TOP1 gene down-regulated by siRNA. The studied genes include cMYC, GAPD, and HIF-1alpha. 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 treatments, and CPT effects on RNAP were 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 intron. Drug effects on splicing 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. Other findings will be presented and discussed at the meeting. In summary, our results demonstrate that Top1 inhibitors can increase RNAP escape from promoter-proximal pausing sites along with an impairment of intron splicing. Top1 may therefore regulate the coupling of transcription pausing and mRNA maturation, and Top1 inhibition may interfere with this process, thereby altering the expression of specific genes