Figure 4.

E2F binds the promoters of the paralogous miRNA polycistrons and induces their transcription. (A) E2F1 binds conserved E2F sites upstream of each of the paralogous miRNA polycistrons. ChIP analysis was performed on U2OS cells with an anti-E2F1 antibody (IP: E2F1) and a control antibody against HA (IP: HA). The precipitated DNA was measured using QRT–PCR. The β-actin gene serves as a negative control for E2F1 binding. Values were normalized to the levels of β-tubulin. For schematic representation of the polycistrons' genomic organization and corresponding E2F sites, see Supplementary Figure S3A. (B) E2F activation by E1A induces the polycistronic miRNAs. WI-38 cells were infected with the oncoprotein E1A or a control vector (Con) and selected with puromycin. QRT–PCR revealed upregulation of the known E2F1 target, Cyclin E, as well as of host transcripts and miRNAs, representatives of the three paralogous polycistrons (miRs-106b/93/25, miR-17-92 and miR-106a-92). (C) E2F1 activation results in rapid induction of the polycistronic miRNAs. WI-38 cells were stably infected with ER-E2F1 and treated with 4-OHT (300 nM) for the indicated time periods. QRT–PCR analysis was performed to measure the levels of miRNAs and mRNAs. QRT–PCR data are represented as mean±s.d.