Activation of antisense transcription by Top1cc in human colon cancer cells

Camptothecin (CPT) and derivatives are effective anti-cancer agents exerting their activity through the inhibition of DNA topoisomerase I (Top1). The cascade of events following the collision of a replication fork with CPT-trapped Top1 (TopIcc) has been deeply studied. Recently, strong evidence demonstrated that Top1cc affects specific transcriptional mechanisms as well. In particular, Top1ccs specifically favor RNA polymerase II escape from promoter-proximal pausing sites, and can alter chromatin structure and accessibility. We have also demonstrated that the CPT-induced transcriptional stress determines a changed balance between sense and antisense transcripts at the human HIF-1α locus in cancer cells [Capranico et al., BBA 2010]. Here, we have further investigated the Top1cc-related antisense transcriptional activation at the single gene locus and genome-wide. CPT treatments promote the transcription, in normal as well as hypoxic conditions, of a novel long non-coding antisense RNA at the 5’ of the human HIF-1α mRNA (5’aHIF1α), which is 5’-capped and polyadenylated, indicating that is transcribed by RNA polymerase II. The 5’aHIF1α RNA localizes preferentially in the nuclear compartment, in particular RNA FISH experiments show that it accumulates mainly in the perinuclear region of human HCT116 cells. Thus, the data may suggest that it may have a regulatory role in membrane RNA/protein trafficking. To investigate whether the described effect at HIF-1α locus is a general event after Top1cc, we have carried out a genome-wide analyses of cellular transcripts. rRNA-depleted total RNA samples from CPT-treated and untreated HCT116 cells were subjected to bisulfite C>T conversion to maintain the information of the strand polarity of transcripts, and then sequenced on the Illumina platform (Solexa). Initial analyses of the ratio between sense and antisense sequence tags indicate that Top1cc induces the activation of antisense transcription at several gene loci. The results thus indicate that activation of antisense transcription may be a general response to the transcriptional stress caused by Top1ccs, possibly contributing to the pharmacological activity of camptothecins.