title: Oct-1 and nuclear factor Y bind to the SURG-1 element to direct basal and gonadotropin-releasing hormone (GnRH)-stimulated mouse GnRH receptor gene transcription.
abstract: 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 the SURG-1 element by EMSA using nuclear extracts from the gonadotrope-derived alphaT3-1 and LbetaT2 cell lines. Serial mutagenesis and supershift assays identified nuclear factor Y (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 chromatin immunoprecipitation 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 alphaT3-1 cells using a 1.2-kb mGnRHR (-1164/+62) gene promoter-luciferase reporter construct with selective mutations of the Oct-1, NF-Y, and/or the previously characterized activating protein 1 (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. Overexpression 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 small interfering RNA 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.
authors.affiliation: Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, 221 Longwood Avenue, Boston, Massachusetts 02115, USA.
authors.forename: Kyung-Yoon
authors.initials: KY
authors.lastname: Kam
authors.forename: Kyeong-Hoon
authors.initials: KH
authors.lastname: Jeong
authors.forename: Errol R
authors.initials: ER
authors.lastname: Norwitz
authors.forename: Elisa M
authors.initials: EM
authors.lastname: Jorgensen
authors.forename: Ursula B
authors.initials: UB
authors.lastname: Kaiser
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D023081:CCAAT-Binding Factor / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D047369:Chromatin Immunoprecipitation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology*; D051379:Mice; D009154:Mutation / Q000235:genetics; D050811:Octamer Transcription Factor-1; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011966:Receptors, LHRH / Q000235:genetics*; D020218:Response Elements / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects* / Q000235:genetics
chemical_list: D023081:CCAAT-Binding Factor; D004268:DNA-Binding Proteins; D050811:Octamer Transcription Factor-1; C494166:Pou2f1 protein, mouse; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011966:Receptors, LHRH; D014157:Transcription Factors; C402244:nuclear factor Y; D007987:Gonadotropin-Releasing Hormone