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Originally published In Press as doi:10.1074/jbc.M206571200 on July 26, 2002

J. Biol. Chem., Vol. 277, Issue 40, 37469-37478, October 4, 2002
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Direct Binding of AP-1 (Fos/Jun) Proteins to a SMAD Binding Element Facilitates Both Gonadotropin-releasing Hormone (GnRH)- and Activin-mediated Transcriptional Activation of the Mouse GnRH Receptor Gene*

Errol R. NorwitzDagger §, Shuyun XuDagger , Jian Xu, Lisa B. SpirydaDagger , Joong Shin ParkDagger ||, Kyeong-Hoon Jeong**Dagger Dagger , Elizabeth A. McGee, and Ursula B. Kaiser**

From the Departments of Dagger  Obstetrics, Gynecology and Reproductive Biology and of ** Medicine, Brigham & Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, the  Department of Obstetrics & Gynecology, Magee Womens Research Institute, Pittsburgh, Pennsylvania 15213, and the || Department of Obstetrics & Gynecology, Seoul National University College of Medicine, Seoul 110-744, Korea

The response of pituitary gonadotropes to gonadotropin-releasing hormone (GnRH) correlates directly with the concentration of GnRH receptors (GnRHR) on the cell surface, which is mediated in part at the level of gene expression. Several factors are known to affect expression of the mouse GnRHR (mGnRHR) gene, including GnRH and activin. We have previously shown that activin augments GnRH-mediated transcriptional activation of mGnRHR gene, and that region -387/-308 appears to be necessary to mediate this effect. This region contains two overlapping cis-regulatory elements of interest: GnRHR activating sequence (GRAS) and a putative SMAD-binding element (SBE). This study investigates the role of these elements and their cognate transcription factors in transactivation of the mGnRHR gene. Transfection studies confirm the presence of GnRH- and activin-response elements within -387/-308 of mGnRHR gene promoter. Competition electrophoretic mobility shift assay experiments using -335/-312 as probe and alpha T3-1 nuclear extract or SMAD, Jun, and Fos proteins demonstrate direct binding of AP-1 (Fos/Jun) protein complexes to -327/-322 and SMAD proteins to -329/-328. Further transfection studies using mutant constructs of these cis-regulatory elements confirm that both are functionally important. These data define a novel cis-regulatory element comprised of an overlapping SBE and newly characterized non-consensus AP-1 binding sequence that integrates the stimulatory transcriptional effects of both GnRH and activin on the mGnRHR gene.

* This work was supported in part by the Reproductive Scientist Development Program through the Association of Professors of Obstetricians & Gynecologists and the National Institutes of Health (NIH, Grant K12-HD00840), the Women's Reproductive Health Research Award (NIH Grant K12-HD01255) (both to E. R. N.), and NIH Grant R01-HD19938 (to U. B. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Obstetrics, Gynecology and Reproductive Biology, Division of Maternal-Fetal Medicine, Harvard Medical School, Brigham & Women's Hospital, 75 Francis St., Boston, MA 02115. Tel.: 617-278-0897; Fax: 617-232-6346; E-mail: enorwitz@partners.org.

Dagger Dagger Supported by the Lalor Foundation.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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