Topoisomerase I specifically affects RNA polymerase II paused at promoter-proximal sites in human cells.

We have determined early effects of camptothecin and other drugs on DNA-binding sites of RNAPII in transcribed human genes by chromatin-immunoprecipitation (ChIP). Camptothecin induced a reduction of RNAPII density at promoter pause sites while TBP was not affected in a number of human cell lines. 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 and transcription elongation inhibitor. Decreased RNAPII density was also detected in cells treated with cisplatin or teniposide but at higher concentrations than camptothecin. Moreover, RNAPII increases in the body of genes were observed with camptothecin only. The specific effects of camptothecin were less marked in HCT116 cells with a functional knock-down of the TOP1 gene by siRNA. The results thus strongly support a specific involvement of Top1 in the pausing of RNAPII at promoter-proximal sites. Interestingly, RNAPII reduction at promoter pause sites occurred within 5-10 minutes of camptothecin treatments, and was not a response to replication-dependent DNA breaks. Since Top1 was found to be enriched in active chromatin, we suggest that Top1 inhibition at the active chromatin domain immediately affects RNAPII at transcribed genes. The findings are novel in vivo evidence for a role of Top1 in transcription regulation at the level of promoter clearance, and provide novel insights into the molecular action of the antitumor drug camptothecin.