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.

Activin responsiveness of the murine GnRH receptor(GnRHR) gene promoter requires two spatially distinctregulatory elements termed the GnRH receptoractivating sequence or GRAS and the downstream activinregulatory element or DARE. While GRAS interactswith multiple transcription factors, DARE activityrequires tandem homeodomain binding motifs (TAAT)and displays specific binding to the LIM homeodomainprotein LHX3. Herein, we find that both the murineGnRHR gene promoter and DARE fused to a minimalheterologous promoter are responsive to LHX3 overexpression.A dominant-repressor of LHX3 attenuatestranscriptional activity of the murine GnRHR gene promoterbut had no impact on activin responsiveness.Thus, LHX3 would not appear to be the protein mediatingactivin responsiveness of this promoter. WithinDARE itself, the tandem TAAT motifs are separated by4 bp. Although this arrangement differs from the prototypicalP2 or P3 binding sites characterized for pairedlikehomeodomain proteins and from the directlyabutting TAAT motifs found for LHX3, a LIM-class homeodomainprotein, we find that separation of the TAATsites by 5 and 10 bp decreases GnRHR promoter activityto a level similar to promoters containing loss of functionmutations in either the proximal or distal TAATmotif. Thus, the juxtaposition of the TAAT sites is criticalfor the functional activity of DARE. That activinresponsiveness of the GnRHR promoter requires bothGRAS and DARE suggests that these elements may beboth functionally and structurally coupled. As to thelatter, GRAS and DARE are separated by 20 bp, thusplacing the elements on the same side of the helicalbackbone. To determine if this spatial organization isfunctionally relevant, multiples of 5 bp were insertedor deleted between GRAS and DARE. Any insertion ordeletion that resulted in a half-turn alteration in thehelical positioning between the two elements reducedpromoter activity. Thus, an important spatial relationshipunderlies functional cooperation between GRASand DARE and the emergence of a complex activin responsiveunit (ARU) within the mouse GnRHR promoter. [ABSTRACT FROM AUTHOR]