Figure 2.

The CCAAT box is important for basal expression and the CRE site is critical for GnRH induction of c-Jun. A, Different lengths of the c-Jun promoter linked to luciferase reporter in pGL3 backbone were transfected into LβT2 cells with Herpes virus thymidine kinase-driven β-galactosidase gene as an internal control for transfection efficiency. After overnight starvation, cells were treated with vehicle or GnRH for 5 hours, after which the luciferase and β-galactosidase values were obtained. # indicates a significant drop in luciferase expression from the previous truncation, while * indicates significant decrease in GnRH fold induction. B, 10 base pair internal deletions that encompass the CCAAT box and the CRE site were created in the -1000 c-Jun-luciferase reporter (WT) and the experiment was performed as above. C, Three and four base pair mutations were created in the residues that were previously determined to be critical for CCATT and CRE sites function, respectively. # indicates a significant decline in luciferase expression from the wild type control (WT), while * indicates significant decrease in GnRH fold induction. D, Four tandem copies of CRE (TGACGTCA) or TRE (TGAGTCA) sites were linked to the minimal heterologous promoter (ctrl) and luciferase reporter, and co-transfected with β-galactosidase internal control. * indicates significant induction by GnRH.