title: Temporal recruitment of transcription factors at the 3',5'-cyclic adenosine 5'-monophosphate-response element of the human GnRH-II promoter.
abstract: GnRH-II is a potent GnRH subtype involved in modulating OVCAR-3 cell proliferation and the invasive properties of JEG-3 cells, and an atypical cAMP-response element (CRE) in the human GnRH-II promoter influences its activation. We demonstrated that the GnRH-II promoter is activated by 8-bromoadenosine-cAMP in several cell lines including alphaT3, TE671, JEG-3, and OVCAR-3 cells and that cAMP enhances GnRH-II mRNA levels in JEG-3 and OVCAR-3 cells. Moreover, 8-bromoadenosine-cAMP increases cAMP response element-binding protein (CREB) phosphorylation in JEG-3 and OVCAR-3 cells and augments CBP and CCAAT/enhancer-binding protein (C/EBP)-beta coimmunoprecipitation with phosphorylated CREB (p-CREB) in a temporally defined manner from nuclear extracts. When CREB, CBP, and C/EBPbeta levels were knocked down by small interfering RNA, reductions in any of these transcription factors reduced cAMP-enhanced GnRH-II promoter activity and GnRH-II mRNA levels in JEG-3 and OVCAR-3 cells. Importantly, chromatin immunoprecipitation assay showed that p-CREB bound the CRE within the endogenous GnRH-II promoter within 1 h and that p-CREB association with C/EBPbeta occurs within 2 h of cAMP stimulation, coincident with the first appearance of C/EBPbeta at the CRE. By contrast, maximum interactions between p-CREB and CBP do not occur until at least 4 h after cAMP stimulation, and this is reflected in the progressive loading of CBP at the CRE at 2-4 h, as demonstrated by chromatin immunoprecipitation. Taken together, these data suggest that p-CREB, C/EBPbeta, and CBP are recruited to the CRE of the GnRH-II promoter in a temporarily defined manner to enhance its transcription in JEG-3 and OVCAR-3 cells in response to cAMP.
authors.affiliation: Department of Obstetrics and Gynaecology, University of British Columbia, Room 2H-30, 4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5.
authors.forename: Song Ling
authors.initials: SL
authors.lastname: Poon
authors.forename: Beum-Soo
authors.initials: BS
authors.lastname: An
authors.forename: Wai-Kin
authors.initials: WK
authors.lastname: So
authors.forename: Geoffrey L
authors.initials: GL
authors.lastname: Hammond
authors.forename: Peter C K
authors.initials: PC
authors.lastname: Leung
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000230:Adenocarcinoma; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism; D050882:CREB-Binding Protein / Q000378:metabolism; D045744:Cell Line, Tumor; D002822:Choriocarcinoma; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000502:physiology; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives* / Q000235:genetics; D006801:Humans; D008527:Medulloblastoma; D010051:Ovarian Neoplasms; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D022782:CCAAT-Enhancer-Binding Protein-beta; D017362:Cyclic AMP Response Element-Binding Protein; D012333:RNA, Messenger; D014157:Transcription Factors; D015124:8-Bromo Cyclic Adenosine Monophosphate; D007987:Gonadotropin-Releasing Hormone; C047717:LHRH, His(5)-Trp(7)-Tyr(8)-; D000242:Cyclic AMP; D050882:CREB-Binding Protein; C494363:CREBBP protein, human; D017868:Cyclic AMP-Dependent Protein Kinases