NeuroD1 and Mash1 temporally regulate GnRH receptor gene expression in immortalized mouse gonadotrope cells

Abstract

Accurate spatial and temporal expression of gonadotrope-specific genes, such as the gonadotropin-releasing hormone receptor (GnRHR) gene, is critical for gonadotrope maturation. Herein, we show that a specific E-box in the mouse GnRHR promoter binds two group A basic-helix-loop-helix (bHLH) transcription factors. Mutation of this E-box decreases expression in mouse gonadotrope-derived alphaT3-1 and LbetaT2 cell lines. Microarray and western blots show that the bHLH transcription factor NeuroD1 is strongly expressed in the gonadotrope progenitor, alphaT3-1, whereas Mash1 is strongly expressed in the more mature gonadotrope, LbetaT2. Over-expression of NeuroD1 or Mash1 increases expression of the GnRHR gene or a multimer of the E-box and this increase is lost upon mutation of the E-box. Electrophoretic mobility shift assays reveal that the GnRHR E-box binds NeuroD1 from alphaT3-1 cells, but binds Mash1 from LbetaT2 cells. The sequential binding of different members of the group A bHLH transcription factor family to mouse GnRHR E-box 3 as the gonadotrope differentiates may represent a mechanism necessary for proper spatial and temporal expression of the GnRHR during gonadotrope development.

Fig. 1

The basic helix-loop-helix transcription factors NeuroD1 and Mash1 are differentially expressed in the gonadotrope-derived αT3-1 and LβT2 cell lines. Western blot analysis of total protein from the gonadotrope-derived αT1-1, αT3-1, and LβT2 cell lines was used to confirm findings in the Affymetrix oligonucleotide arrays. Concentrations of protein in lysates were determined by Bradford assay and sample volume adjusted to ensure equal loading.

Fig. 2

Mutation of E-box 3 significantly decreases basal activity of a mouse GnRHR-Luciferase reporter gene in αT3-1 and LβT2 cells. (A) E-box sequences from the proximal 1000 bp of each gene are shown in comparison to the well-characterized POMC E-box and the Group A consensus (GrpA). Within each gene, both the sense (S) and antisense (AS) strands are shown in the 5’ to 3’ orientation. They are ordered from the most distal (top) to the most proximal (bottom) within each gene. The defining bases (CAnnTG) are in capital letters. The internal base pair matches for the Group A bHLH consensus (GrpA, shown at the top) are underlined. The flanking bp matches for to the well-defined NeuroD1 binding site from the POMC gene (shown second) are in bold. (B) Mutated sequences of E-boxes 3 and 4 from the GnRH gene are shown with the mutated bases underlined. (C) The GnRHR-Luciferase reporter either wild-type or containing mutations in either E-box 4 or E-box 3 were transiently transfected into αT3-1 cells or LβT2 cells.

Fig. 3

Over-expression of NeuroD1 in LβT2 cells or Mash1 in αT3-1 cells activates a wild-type GnRHR-Luciferase reporter gene, but activation is lost upon mutation of E-box 3. NeuroD1 and Mash1 expression vectors or equimolar amounts of an empty expression vector were cotransfected along with the GnRHR-Luciferase reporter gene (GnRHR) or E-box 3 mutant reporter (E-box 3 Mutant) into αT3-1 and LβT2 cells.

Fig. 4

Over-expression of the basic helix-loop-helix proteins NeuroD1 or Mash1 are sufficient to increase expression of a 4X E-box 3 multimer reporter gene. αT3-1 and LβT2 cells were transiently transfected with E-box 3 multimer reporter gene and expression vectors for NeuroD1,Mash1, or empty expression vector, as a control.

Fig.5

NeuroD1 and E47 bind to radiolabeled oligonucleotides containing E-box 3 from the mouse GnRHR gene promoter. EMSAs were performed with radiolabeled oligonucleotides encompassing E-box 3 in the 5' flanking region of the mouse GnRHR gene. Radiolabeled oligonucleotide probe was incubated with nuclear proteins from αT3-1 cells. Specific protein/DNA complexes were identified by competition with 250- or 500-fold excess unlabelled oligonucleotides containing a mutated E-box 3 (µE-box), homologous competitor (wild type Ebox 3), or a positive control E-box from the POMC gene (POMC). Identification of the bHLH transcription factors in specific complexes was determined by inclusion of an antibody directed against NeuroD1 or the ubiquitously expressed bHLH heterodimerization partner, E47.

Fig. 6

Mash1 and E47 bind to radiolabeled oligonucleotides containing E-box 3 from the mouse GnRHR gene promoter. EMSAs were performed with radiolabeled oligonucleotides encompassing E-box 3 in the 5' flanking region of the mouse GnRHR gene. Radiolabeled oligonucleotide probe was incubated with nuclear proteins from LβT2 cells. Specific protein/DNA complexes were identified by competition with 250- or 500-fold excess unlabelled oligonucleotides containing a mutated E-box 3 (µE-box), homologous competitor (wild-type E-box 3), or a positive control (MCK). Identification of the bHLH transcription factors in specific complexes was determined by inclusion of an antibody directed against Mash1 or the ubiquitously expressed bHLH heterodimerization partner, E47.

Fig. 7

The binding of group A bHLH family members NeuroD1 from αT3-1 cells and Mash1 from LβT2 cells to E-box 3 is highly specific and preferential to the binding of group B bHLH family members. EMSAs were performed with radiolabeled oligonucleotides encompassing E-box 3. Radiolabeled oligonucleotide probe was incubated with nuclear proteins from αT3-1 or LβT2 cells. Specificity of binding complexes was determined by inclusion of antibodies directed against NeuroD1, Mash1, and BMAL1, or IgG as a control.