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

Naama Rave-Harel,Marjory L Givens,Shelley B Nelson,Hao A Duong,Djurdjica Coss,Melody E Clark,Sara Barth Hall,Mark P Kamps,Pamela L Mellon

doi:10.1074/jbc.m402960200

Abstract

Gonadotropin-releasing hormone (GnRH) is the central regulator of reproductive function. Expression of the GnRH gene is confined to a rare population of neurons scattered throughout the hypothalamus. Restricted expression of the rat GnRH gene is driven by a multicomponent enhancer and an evolutionarily conserved promoter. Oct-1, a ubiquitous POU homeodomain transcription factor, was identified as an essential factor regulating GnRH transcription in the GT1-7 hypothalamic neuronal cell line. In this study, we conducted a two-hybrid interaction screen in yeast using a GT1-7 cDNA library to search for specific Oct-1 cofactors. Using this approach, we isolated Pbx1b, a TALE homeodomain transcription factor that specifically associates with Oct-1. We show that heterodimers containing Pbx/Prep1 or Pbx/Meis1 TALE homeodomain proteins bind to four functional elements within the GnRH regulatory region, each in close proximity to an Oct-1-binding site. Cotransfection experiments indicate that TALE proteins are essential for GnRH promoter activity in the GT1-7 cells. Moreover, Pbx1 and Oct-1, as well as Prep1 and Oct-1, form functional complexes that enhance GnRH gene expression. Finally, Pbx1 is expressed in GnRH neurons in embryonic as well as mature mice, suggesting that the associations between TALE homeodomain proteins and Oct-1 regulate neuron-specific expression of the GnRH gene in vivo.

Highlights

Tissue-specific gene expression can be mediated, in the simplest case, by a transcription factor restricted to a particular cell type
We show that heterodimers containing Pbx/ Prep1 or Pbx/Meis1 TALE homeodomain proteins bind to four functional elements within the Gonadotropin-releasing hormone (GnRH) regulatory region, each in close proximity to an Oct-1-binding site
The Homeodomain Protein Pbx1b Interacts with Oct-1 in a Yeast Two-hybrid Screen—To understand the mechanism by which the ubiquitous transcription factor Oct-1 specifies GnRH expression, we searched for Oct-1 cofactors expressed in the GT1-7 cell line

Summary

The abbreviations used are

GnRH, gonadotropin-releasing hormone; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; dnPrep, dominant-negative Prep; GnRHe, GnRH enhancer; RSVp, Rous sarcoma virus promoter; DAB, 3,3Ј-diaminobenzidine; dpc, days postcoitus; GnRHp, GnRH promoter; X-gal, 5-bromo-4chloro-3-indolyl-␤-D-galactopyranoside. 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]. Oct-1 may interact with other DNA-binding proteins that are neuron-specific [16]. We used a two-hybrid interaction screen in yeast to search for Oct-1 cofactors in GT1-7 cells. This approach led us to the isolation of the TALE homeodomain transcription factor Pbx1b. We demonstrate a role for this interaction in the activation of GnRH gene expression. The findings in GT1-7 cells are likely relevant to the regulation of GnRH expression in vivo since Pbx is expressed in embryonic as well as mature GnRH neurons in mice

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION