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Jones Institute for Reproductive Medicine (K.-W.D., Z.-G.C.), Eastern Virginia Medial School, Norfolk, Virginia 23507; Department of Zoology (K.-L.Y., Y.-D.C.), The University of Hong Kong, Pokfulam Road, Hong Kong; The Dr. Arthur M. Fishberg Research Center for Neurobiology (J.L.R.), Mount Sinai School of Medicine, New York, New York 10029
Address all correspondence and requests for reprints to: Ke-Wen Dong, Ph.D., Eastern Virginia Medical School, Jones Institute for Reproductive Medicine, 601 Colley Avenue, Norfolk, Virginia 23507.
Two promoters directing tissue-specific expression of GnRH gene in neuronal and reproductive tissues were characterized by functional analyses of GnRH promoter-luciferase (LUC) constructs in transfected placental cells (JEG) and hypothalamic neuronal cells (GT1–7). Results indicate that the downstream promoter directs the expression in a neuronal cell-specific manner, whereas the upstream promoter is fully active in the nonneural placental cell line. Transfection studies carried out in several tumor cell lines derived from human reproductive tissues verified that the upstream GnRH promoter construct was much more active in directing luciferase expression in reproductive tissue. The use of both upstream and downstream promoters in various human tumor cell lines derived from reproductive tissues were demonstrated by RT-PCR. Our studies also demonstrate that the reproductive tissue-specific messenger RNA transcribed from upstream promoter is capable of directing synthesis of preproGnRH protein.
Serial deletion studies localized a cell-specific upstream promoter region that directs reproductive tissue expression. DNase I footprint analysis using nuclear extract obtained from the JEG cells indicated DNA/protein interactions in four specific sequence elements of the upstream promoter region. The interaction between nuclear binding proteins present in the JEG cells (but not the GT1–7 cells) and the four specific sequences in the upstream promoter region was confirmed by gel mobility shift analysis.
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