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Submitted on January 22, 2004
Accepted on September 16, 2004
Departments of Medicine (K.-Y.K., K.-H.J., E.M.J., U.B.K.) and Obstetrics, Gynecology and Reproductive Biology (E.R.N.), Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115
* To whom correspondence should be addressed. E-mail: UKaiser{at}partners.org.
The cis-regulatory element localized to position -292/-285 of the mouse GnRH receptor (mGnRHR) gene promoter, designated Sequence Underlying Responsiveness to GnRH-1 (SURG-1), has been shown previously to contribute to stimulation of mGnRHR gene expression by GnRH. We have identified three specific protein-DNA complexes on this element by electrophoretic mobility shift assay (EMSA) using nuclear extracts from the gonadotrope-derived 
T3-1 and L
T2 cell lines. Serial mutagenesis and supershift assays identified NF-Y binding to -288/-284 and Oct-1 binding to a TAAT sequence at -290/-287. Binding of these two transcription factors was confirmed in vivo by ChIP assay and increased in response to GnRH stimulation. To define the functional significance of these sequences in the regulation of mGnRHR gene transcription, transient transfection assays were performed in T3-1 cells using a 1.2 kb mGnRHR (-1164/+62) gene promoter-luciferase reporter construct with selective mutation of the Oct-1, NF-Y and/or the previously characterized AP-1 binding site (-274/-268). Individual mutations in the Oct-1, NF-Y, and AP-1 sites decreased both basal expression and stimulation by GnRH agonist, and the combined mutation of the Oct-1 and AP-1 binding sites further reduced basal transcriptional activity and abolished GnRH stimulation. Over-expression of NF-YA increased GnRHR promoter activity, whereas expression of a dominant negative NF-YA mutant decreased activity, further supporting a role of NF-Y in regulation of mGnRHR gene transcription. In addition, knockdown of Oct-1 by siRNA confirmed that Oct-1 is important for mGnRHR gene expression. In conclusion, NF-Y and Oct-1 bind to the SURG-1 element to direct basal and GnRH-stimulated expression of the mGnRHR gene.
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