The POU homeodomain protein Oct-1 binds cis-regulatory element essential for the human GnRH upstream promoter activity in JEG-3 cells

In previous studies, we have localized four specific nuclear protein-bindin g elements in the human GnRH upstream promoter. To test whether these four elements are reproductive tissue specific, we placed the four elements upst ream to a thymidine kinase (TK) promoter/luciferase reporter gene, and tran sfected the constructs into human placental choriocarcinoma (JEG-3) cells. The 272-bp fragment (-994 to -723) containing the four elements can drive h eterologous TK promoter expression in JEG-3 cells about 15 times more than that of basal TK promoter activity. Deletion of element 4 (E4, -987/- 968) significantly decreased (4-fold) the luciferase activity. Further deletion of the other elements (E3 individual, -960/-940 or E3 and E2 in combination , -919/-896) only slightly decreased the luciferase activity In contrast, d eletion of element 1 (E1, -876/-851) caused a 2-fold loss of luciferase act ivity and elimination of E2 and E3 only lost less than 2-fold of the lucife rase activity. Study performed with 5' end deletion of this region confirme d these observations. Furthermore, E4 DNA-protein complex can be supershift ed by Oct-1 antibody, indicating that Oct-1 binds to E4. These results clea rly demonstrated that all four elements are required to confer tissue-speci fic expression of the hGnRH gene in JEG-3 cells. However, the E4 is the mos t important for the tissue-specific expression of the hGnRH gene in JEG-3 c ells. Oct-1 factor binds with E4 element and may he involved in the mediati on of the human GnRH upstream promoter activity.