A Specific Helical Orientation Underlies the Functional Contribution of the Activin Responsive Unit to Transcriptional Activity of the Murine Gonadotropin-Releasing Hormone Receptor Gene Promoter

Abstract

Activin responsiveness of the murine GnRH receptor (GnRHR) gene promoter requires two spatially distinct regulatory elements termed the GnRH receptor activating sequence or GRAS and the downstream activin regulatory element or DARE. While GRAS interacts with multiple transcription factors, DARE activity requires tandem homeodomain binding motifs (TAAT) and displays specific binding to the LIM homeodomain protein LHX3. Herein, we find that both the murine GnRHR gene promoter and DARE fused to a minimal heterologous promoter are responsive to LHX3 overexpression. A dominant-repressor of LHX3 attenuates transcriptional activity of the murine GnRHR gene promoter but had no impact on activin responsiveness. Thus, LHX3 would not appear to be the protein mediating activin responsiveness of this promoter. Within DARE itself, the tandem TAAT motifs are separated by 4 bp. Although this arrangement differs from the prototypical P2 or P3 binding sites characterized for paired-like homeodomain proteins and from the directly abutting TAAT motifs found for LHX3, a LIM-class homeodomain protein, we find that separation of the TAAT sites by 5 and 10 bp decreases GnRHR promoter activity to a level similar to promoters containing loss of function mutations in either the proximal or distal TAAT motif. Thus, the juxtaposition of the TAAT sites is critical for the functional activity of DARE. That activin responsiveness of the GnRHR promoter requires both GRAS and DARE suggests that these elements may be both functionally and structurally coupled. As to the latter, GRAS and DARE are separated by 20 bp, thus placing the elements on the same side of the helical backbone. To determine if this spatial organization is functionally relevant, multiples of 5 bp were inserted or deleted between GRAS and DARE. Any insertion or deletion that resulted in a half-turn alteration in the helical positioning between the two elements reduced promoter activity. Thus, an important spatial relationship underlies functional cooperation between GRAS and DARE and the emergence of a complex activin responsive unit (ARU) within the mouse GnRHR promoter.