The POU Homeodomain Transcription Factor Oct-1 Is Essential for Activity of the Gonadotropin-Releasing Hormone Neuron-Specific Enhancer

Abstract

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 proteinbinding 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.

TALE Homeodomain Proteins Regulate Gonadotropin-releasing Hormone Gene Expression Independently and via Interactions with Oct-1

“…We further predicted their function as activators since most of the Oct-1-and TALEbinding sites within the GnRH enhancer and promoter are essential for transcriptional activation (7,11,35). Indeed, the combination of Prep1 or Pbx1b with Oct-1 significantly increased GnRH reporter gene expression (Fig.…”

“…Each probe (1 fmol) was incubated with 2 g of GT1-7 crude nuclear extract in 20-l reactions. Reactions were incubated at room temperature for 5 min and separated on 5% polyacrylamide gel in 0.25ϫ Tris borate/EDTA as described previously (7). Competitions were performed by preincubating the reactions with a 100 -200-fold excess of unlabeled oligonucleotide for 5 min on ice prior to adding the probe.…”

“…Probes were diluted in 50 mM NaCl. Binding reactions were carried out under conditions described previously (7). Each probe (1 fmol) was incubated with 2 g of GT1-7 crude nuclear extract in 20-l reactions.…”

“…In particular, the POU homeodomain protein Oct-1 was identified as an essential factor regulating basal and hormone-induced transcription of the GnRH gene (7,9,12,13). Oct-1 binds to five elements within the rat GnRH regulatory region and is necessary for enhancer activity in GT1-7 cells (7).…”

Identification of a gene selectively expressed in the brain, which encodes a putative transmembrane protein and a soluble cytoplasmic isoform

“…However, neither a CCAAT box nor binding sites for the ubiquitous transcription factor SP-1 were identified, which is consistent with the described specificity of gene expression in porcine brain. Nevertheless, the potential promoter region I contains many putative binding sites for factors known to exist in the brain, such as IK2 [50], NF-1 [51], NFκB [52,53] and OCT-I/Brn-2 [54,55]. Since their consensus sequences are very similar, further experimental data are required to distinguish between binding sites for IK2 and NFκB [50].…”