Early effects of topoisomerase I inhibition on RNA polymerase II along transcribed genes in human cells.
We have determined the early effects of camptothecin and alpha-amanitin 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). The two agents caused notably different alterations in active chromatin. Camptothecin induced a specific reduction of RNAPII density at promoter pause sites and histone modifications suggesting an increased chromatin accessibility. alpha-Amanitin caused an accumulation of RNAPII at transcribed genes, a reduction of TBP bound to chromatin and a less accessible chromatin structure. 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. The findings are novel in vivo evidence of camptothecin effects on RNAPII bound to transcribing genomic regions, and are consistent with the hypothesis that Top1 activity can be involved in transcription regulation at the level of promoter clearance.
Khobta
Andriy
A
Department of Biochemistry, University of Bologna School of Pharmacy, via Irnerio 48, 40126 Bologna, Italy.
Ferri
Francesca
F
Lotito
Luca
L
Montecucco
Alessandra
A
Rossi
Rossella
R
Capranico
Giovanni
G
D000546:Amanitins / Q000494:pharmacology; D001665:Binding Sites; D002166:Camptothecin / Q000494:pharmacology; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism*; D035181:TATA-Box Binding Protein / Q000378:metabolism; D059004:Topoisomerase I Inhibitors; D014158:Transcription, Genetic
D000546:Amanitins; D002843:Chromatin; D004791:Enzyme Inhibitors; D035181:TATA-Box Binding Protein; D059004:Topoisomerase I Inhibitors; D018844:Cyclin-Dependent Kinases; D012319:RNA Polymerase II; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin