Figure 3.

Two regions of the rat PACAP gene promoter are critical for full GnRH responsiveness. Gonadotrope LβT2 cells were transiently transfected with various lengths of the rat PACAP gene promoter fused to a luciferase reporter followed by treatment with 100 nm GnRH for 6 h. A, Serial 5′-truncation analysis of the rat PACAP promoter across region −1916 to +906 relative to the transcriptional start site. *, P < 0.001 vs. the full-length construct. B, Effect of single and combinatorial site-directed mutations in the putative AP-1 and CRE identified by sequence homology in region −402 to −77 (−275 AP-1, −205 CRE, −179 CRE). All mutations resulted in a statistically significant loss of GnRH responsiveness at P < 0.001 vs. the wild-type construct. C, Detailed serial 5′-truncation analysis between positions −915 and −402. *, P < 0.001 vs. next longer construct. D, Site-directed mutation of the putative AP-1 element at position −448 and combinatorial site-directed mutagenesis of the putative −275 AP-1 and −205 CRE within region −915 to −77 identified as critical for GnRH responsiveness. All experiments were performed a minimum of three times with data expressed as the mean ± sem.