Figure 1

Effects of ERα on the transcriptional activity of Bmi1 promoter. (A) The composition of the Bmi1 core promoter. The transcription element E-box is in italics, AP-1 is in boldface, several Sp-1 s are in the shadow box, and the putative ERα response elements (ERE) are underlined. +1 indicates the transcription start. (B) Luciferase reporter construction. A series of reporters including pGL2-1200, pGL2-900, pGL2-460, pGL2-240 and pGL2-152 were constructed spanning the sequence +36 nt to −1158 nt of the Bmi1 promoter, and the two putative EREs were in black box. (C) The transcriptional activity of the Bmi1 gene promoter in ERα-positive or –negative breast cancer cells. MCF-7 and MDA-MB-231 cells were cultured in phenol red free medium containing 10% charcoal stripped FBS and transiently transfected with 200 ng each of empty pGL2-basic, pGL2-1200, pGL2-900, pGL2-460. pGL2-240 or pGL2-152 in the absence or presence of 10⁻⁸ M E2, respectively. Cells were harvested 48 h after transfection and assayed for luciferase activity. (D) The transfection of ERα enhanced transcriptional activity of the Bmi1 promoter. MCF-7 cells were co-transfected with 200 ng each of reporter plasmids and 200 ng of ERα expression plasmid (pcDNA3.1-ERα) or pcDNA3.1 empty vector. Cells were harvested 48 h after transfection and assayed for luciferase activity. (E) The reactivation of Bmi1 promoter in ERα-restored ERα-negative cells. ERα-restored MDA-MB-231 cells (231/ERα) or their control 231/vec cells were transfected with 200 ng of each of the reporter plasmids. The relative luciferase activity values are corrected for co-transfected Renilla activity. And the experiments were repeated at least three times independently and all data are shown by bars as means ± SD (^*P < 0.05, ^**P < 0.01, ^***P < 0.001 when compared with the control groups, respectively).