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The POU Homeodomain Protein Oct-1 Binds Cis-Regulatory Element Essential for the Human GnRH Upstream Promoter Activity in JEG-3 Cells¹

Department of Obstetrics and Gynecology, Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia 23507

Address all correspondence and requests for reprints to: Ke-Wen Dong, Ph.D., Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, 601 Colley Avenue, Norfolk, Virginia 23507. E-mail: DONGKW{at}EVMS.EDU

Abstract

In previous studies, we have localized four specific nuclear protein-binding elements in the human GnRH upstream promoter. To test whether these four elements are reproductive tissue specific, we placed the four elements upstream to a thymidine kinase (TK) promoter/luciferase reporter gene, and transfected the constructs into human placental choriocarcinoma (JEG-3) cells. The 272-bp fragment (-994 to -723) containing the four elements can drive heterologous 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, deletion of element 1 (E1, -876/-851) caused a 2-fold loss of luciferase activity and elimination of E2 and E3 only lost less than 2-fold of the luciferase activity. Study performed with 5' end deletion of this region confirmed these observations. Furthermore, E4 DNA-protein complex can be supershifted by Oct-1 antibody, indicating that Oct-1 binds to E4. These results clearly demonstrated that all four elements are required to confer tissue-specific expression of the hGnRH gene in JEG-3 cells. However, the E4 is the most important for the tissue-specific expression of the hGnRH gene in JEG-3 cells. Oct-1 factor binds with E4 element and may be involved in the mediation of the human GnRH upstream promoter activity.

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