Oct-1 is involved in the transcriptional repression of the gonadotropin-releasing hormone receptor gene.
Previous deletion analysis of the 5'-flanking region of human GnRH receptor (GnRHR) gene has revealed a powerful negative regulatory element (NRE) located between nucleotide -1017 and -771. In the present study, we demonstrated that this NRE could repress the homologous promoter, irrespective of its position and completely abolish the activity of a heterologous thymidine kinase promoter in an orientation-dependent manner. Progressive 3'-deletion analysis revealed that most of the silencing activity of the NRE resided in a putative octamer regulatory sequence (5'AAGCAAACT3'), which alone could repress the promoter activities by 69-90% in ovarian OVCAR-3, placental JEG-3, and gonadotrope-derived alphaT3-1 cells. Mutation of the AAAC residues of the octamer sequence completely removed its silencing activity. Interestingly, conversion of the octamer sequence into that of the rodent GnRHR promoter (5'AAGCAAAGT3') did not attenuate its silencing effect, indicating that the repressive role of the octamer sequence is evolutionarily conserved. EMSAs showed that common DNA-protein complexes of the same mobility were formed with nuclear extracts from the reproductive cells and gonadotropes, and a consensus octamer transcription factor-1 (Oct-1) binding sequence could dose dependently inhibit the complex formation. Antibody supershift and Southwestern blot assays confirmed that the protein binding to the octamer sequence was the ubiquitously expressed transcription factor Oct-1. Overexpression of Oct-1 augmented the silencing activity of the octamer sequence in alphaT3-1 cells. Taken together, our results clearly indicate a role of Oct-1 in the transcriptional repression of the human GnRHR gene.
Cheng
Chi Keung
CK
Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, Canada V6H 3V5.
Yeung
Chung Man
CM
Hoo
Ruby L C
RL
Chow
Billy K C
BK
Leung
Peter C K
PC
D001665:Binding Sites; D002461:Cell Line, Transformed; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D005260:Female; D017353:Gene Deletion; D006107:Granulosa Cells; D051863:Host Cell Factor C1; D006801:Humans; D008184:Luteal Cells; D016297:Mutagenesis, Site-Directed; D050811:Octamer Transcription Factor-1; D011401:Promoter Regions, Genetic; D011966:Receptors, LHRH / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000502:physiology*; D017422:Sequence Analysis, DNA; D013329:Structure-Activity Relationship; D013937:Thymidine Kinase / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D014162:Transfection
D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; D051863:Host Cell Factor C1; D050811:Octamer Transcription Factor-1; C494165:POU2F1 protein, human; D011966:Receptors, LHRH; D012097:Repressor Proteins; D014157:Transcription Factors; D004247:DNA; D013937:Thymidine Kinase