Figure 2.

CPT induces increased levels of unknown tag clusters mainly at CGI promoters of genes in a Top1-dependent manner. (A) Pie charts representing the effect of CPT on unknown clusters at promoters of the indicated cell lines. Tag clusters were long <5000 nucleotides, with no annotation at the corresponding genomic region and localized from the TSS to 5000 bases upstream. Light gray, black and grey show unknown clusters that were increased, decreased and unchanged by CPT, respectively. (B) Common promoter-associated antisense transcripts (33) between HCT116 and HCT116-shRNATop1 cells. (C) Antisense transcripts were determined upstream the TSS of selected genes by rtPCR in the indicated cell lines. The selected genes showed CPT-increased tag clusters as determined with RNA-seq data, with the exception of TNIK gene that had an antisense transcript reduced by CPT. (D) Antisense transcripts were evaluated by rtPCR in HCT116 after transient silencing of Top1 protein (black bars). In (C) and (D), PCR determinations were normalized to cytochrome b mRNA and to untreated cells (dotted line). Values are means +/− SEM of at least four determinations of two independent experiments. Genomic coordinates of analyzed antisense transcripts are reported in Supplementary Data Set. Primers are reported in Supplementary Table SV. (E) Correlation between expression levels in CPT-treated and control cells of the genes with CPT-increased promoter-associated antisense transcripts in HCT116 (256) and HCT116-shRNATop1 (84) cell lines.