Figure 1.

ZEB1 represses GnRH- and/or EGR1-stimulated murine Lhb expression/transcription. (A) LβT2 cells were transduced with GFP or ZEB1 expressing adenovirus. Cells were treated with pulsatile GnRH. Lhb mRNA was measured by qPCR. N = 3 independent experiments. (B) LβT2 cells were transfected with 225 ng/well −232/+5 mLhb-luc reporter, and 25 ng/well ZEB1 and/or 50 ng/well EGR1 expression vector. Cells were treated with 100 nM GnRH for 6 hours. N = 4 independent experiments. (C) Alignment of the murine Lhb and human LHB promoters. In both cases, +1 refers to the transcription start site (TSS). Consensus ZEB1 cis-elements (E- and Z-box motifs shown) are outlined in red boxes. Positions of murine Lhb probes and human LHB probes used in electrophoretic mobility shift assays (EMSAs) are outlined in filled blue boxes and pink boxes, respectively. (D) LβT2 cells were transfected with 225 ng/well of the indicated −232/+5 mLhb-luc reporter and 25 ng/well ZEB1 expression vector.

Abbreviations: WT, wild-type; −30Z/-18E MUT, mutated site −30Z, and −18E. Cells were treated with GnRH as in panel B. N = 3 independent experiments. In B and D, protein lysates were collected and reporter activity measured by luciferase assays. White circles represent experimental replicates. Percent repression of GnRH-induced promoter-reporter activity by ZEB1 is shown in red. Data were analyzed by two-way ANOVA followed by Holm–Sidak multiple comparisons test. Bars with different letters differ significantly.