PARENT SESSION

(106) THE SPATIAL ORIENTATION BETWEEN THE GnRH RECEPTOR ACTIVATING SEQUENCE AND THE DOWNSTREAM ACTIVIN REGULATORY ELEMENT IS CRITICAL FOR GnRH RECEPTOR PROMOTER ACTIVITY.

Clay, Colin¹, Cherrington, Brian¹, Farmerie, Todd¹, ¹ Colorado State University, Fort Collins, CO

ABSTRACT- Activin responsiveness of the GnRH receptor (GnRHR) gene is dependent on a complex enhancer element termed the GnRH receptor activating sequence (GRAS). Recently, we used a series of chimeric promoters to define an additional regulatory element located approximately 19 bp downstream of GRAS that is also necessary for activin responsiveness and basal activity of the GnRHR gene promoter in the gonadotrope derived T3-1 cell line. This new element, the downstream activin regulatory element or DARE consists of paired TAAT motifs that represent the core binding sites of homeodomain DNA binding proteins. Consistent with this, we find that DARE is capable of binding the recombinant LHX2 homeodomain. Collectively, these data suggest that a functional interaction between the protein binding events organized at GRAS and DARE is required for full activity of the murine GnRHR gene promoter. At issue is whether a critical spatial relationship is necessary for the functional interaction between these two enhancers. To address this issue, we altered the spatial orientation of GRAS and DARE by either reducing the spatial separation by 5, 10 or 15 bp or by adding 5, 10, 15 or 20 bp in a region that has no impact on transcriptional activity of the promoter. Our choice of altering the spacing of the two sites by multiples of 5 was to determine if there was a functional consequence of separation by half or full turns of the DNA helix. Each of these spacer mutants was fused to the cDNA encoding luciferase and examined for transcriptional activity after transient transfection of T3-1 cells. Luciferase expression was attenuated by addition or subtraction of 5 or 15 bp but not reduced by a 10 or 20 bp addition or 10 bp subtraction. Thus, transcriptional activity was compromised when GRAS and DARE were separated by half turns but not full turns of the helix. Based on these data, we suggest that the functional cooperation between GRAS and DARE in mediating GnRHR promoter activity is dependent on a critical spatial orientation between the protein binding events organized at these separate regulatory elements.

KEY WORDS: GnRH receptor, activin, transcription, promoter