Identification of the transcription factors regulating the miR-183/-96/-182 cluster. (A) Phylogenetic analysis demonstrated that there were four conserved transcription factor binding sites located within the 1 kb region upstream of the TSS of the miR-183/-96/-182 cluster in vertebrates. (B) Luciferase activities were decreased after mutation of the first ZEB1 and HSF2 transcription factor binding sites. All luciferase activities were normalized to those obtained with the pGL3-Basic vector alone. Error bars represent SD (n = 3). (C) Transfection of ZEB1 and HSF2 transcription factors could elevate the luciferase activity of native upstream 1 kb luciferase reporter but not its mutants. All luciferase activities were normalized to those obtained with the native upstream 1 kb alone. Error bars represent standard deviation (n = 3). (D) Real-time PCR showing that miR-96 and miR-183 levels were increased in ZEB1 and HSF2 overexpressing MCF-7 cell lines. U6 snRNA was used as internal control. Error bars represent SD (n = 3).