The POU homeodomain transcription factor Oct-1 is essential for activity of the gonadotropin-releasing hormone neuron-specific enhancer.
The mechanisms of specification of gene expression in a complex tissue such as the brain remain poorly understood. To provide a model system for the study of gene regulation in a specific subpopulation of differentiated neurons, we have derived cell lines from tumors created in transgenic mice by targeting simian virus 40 T antigen expression by using the regulatory regions of the gene for gonadotropin-releasing hormone (GnRH), a decapeptide released from specialized neurons in the hypothalamus. Transfections into the cultured GnRH-secreting hypothalamic neuronal cell line GT1 have identified a neuron-specific enhancer, 1.5 kb upstream of the GnRH gene, which binds multiple GT1 nuclear proteins. In particular, one AT-rich protein-binding region, AT-a, is critical for enhancer activity. In this study, we used electrophoretic mobility shift assays to detect a GT1 nuclear protein complex that binds the AT-a region. Close inspection of the AT-a bottom-strand sequence revealed homology to the octamer motif, a sequence known to bind members of the POU homeodomain transcription factor family. Although we demonstrate expression of a number of POU homeodomain genes in GT1 cells, a supershift assay with Oct-1 antibody demonstrates that Oct-1 is the protein binding the enhancer. Finally, specific mutations in the AT-a region that affected Oct-1 binding were correlated with decreased transcription. Thus, Oct-1 binds to the GnRH enhancer in vitro, and this binding is critical to the transcriptional activity of this neuron-specific enhancer in GT1 cells.
Clark
M E
ME
Department of Reproductive Medicine, University of California, San Diego, La Jolla 92093-0674, USA.
Mellon
P L
PL
D000818:Animals; D001483:Base Sequence; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000235:genetics; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D007987:Gonadotropin-Releasing Hormone / Q000235:genetics*; D018398:Homeodomain Proteins / Q000235:genetics*; D051863:Host Cell Factor C1; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000502:physiology*; D050811:Octamer Transcription Factor-1; D012333:RNA, Messenger / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C497693:Hcfc1 protein, mouse; D018398:Homeodomain Proteins; D051863:Host Cell Factor C1; D050811:Octamer Transcription Factor-1; C494166:Pou2f1 protein, mouse; D012333:RNA, Messenger; D014157:Transcription Factors; D007987:Gonadotropin-Releasing Hormone