The POU homeodomain protein Oct-1 binds Cis-regulatory element essential for the human GnRH upstream promoter activity in JEG-3 cells.
In previous studies, we have localized four specific nuclear protein-binding elements in the human GnRH upstream promoter. To test whether these four elements are reproductive tissue specific, we placed the four elements upstream to a thymidine kinase (TK) promoter/luciferase reporter gene, and transfected the constructs into human placental choriocarcinoma (JEG-3) cells. The 272-bp fragment (-994 to -723) containing the four elements can drive heterologous TK promoter expression in JEG-3 cells about 15 times more than that of basal TK promoter activity. Deletion of element 4 (E4, -987/-968) significantly decreased (4-fold) the luciferase activity. Further deletion of the other elements (E3 individual, -960/-940 or E3 and E2 in combination, -919/-896) only slightly decreased the luciferase activity. In contrast, deletion of element 1 (E1, -876/-851) caused a 2-fold loss of luciferase activity and elimination of E2 and E3 only lost less than 2-fold of the luciferase activity. Study performed with 5' end deletion of this region confirmed these observations. Furthermore, E4 DNA-protein complex can be supershifted by Oct-1 antibody, indicating that Oct-1 binds to E4. These results clearly demonstrated that all four elements are required to confer tissue-specific expression of the hGnRH gene in JEG-3 cells. However, the E4 is the most important for the tissue-specific expression of the hGnRH gene in JEG-3 cells. Oct-1 factor binds with E4 element and may be involved in the mediation of the human GnRH upstream promoter activity.
Dong
K W
KW
Department of Obstetrics and Gynecology, Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia 23507, USA. DONGKW@EVMS.EDU
Zheng
H M
HM
Wen
Z Y
ZY
Chen
Z G
ZG
D004268:DNA-Binding Proteins / Q000378:metabolism*; D017353:Gene Deletion; D015870:Gene Expression / Q000502:physiology; D005809:Genes, Regulator / Q000502:physiology; D007987:Gonadotropin-Releasing Hormone / Q000235:genetics*; D018398:Homeodomain Proteins / Q000378:metabolism*; D051863:Host Cell Factor C1; D006801:Humans; D050811:Octamer Transcription Factor-1; D011401:Promoter Regions, Genetic / Q000502:physiology*; D013237:Stereoisomerism; D013937:Thymidine Kinase / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; D018398:Homeodomain Proteins; D051863:Host Cell Factor C1; D050811:Octamer Transcription Factor-1; C494165:POU2F1 protein, human; D014157:Transcription Factors; D007987:Gonadotropin-Releasing Hormone; D013937:Thymidine Kinase