Abstract
Gonadotropin-releasing hormone-1 (GnRH1) controls reproduction by stimulating the release of gonadotropins from the pituitary. To characterize regulatory factors governing GnRH1 gene expression, we employed biochemical and bioinformatics techniques to identify novel GnRH1 promoter-binding proteins from the brain of the cichlid fish, Astatotilapia burtoni (A. burtoni). Using an in vitro DNA-binding assay followed by mass spectrometric peptide mapping, we identified two members of the purine-rich element-binding (Pur) protein family, Puralpha and Purbeta, as candidates for GnRH1 promoter binding and regulation. We found that transcripts for both Puralpha and Purbeta colocalize in GnRH1-expressing neurons in the preoptic area of the hypothalamus in A. burtoni brain. Furthermore, we confirmed in vivo binding of endogenous Puralpha and Purbeta to the upstream region of the GnRH1 gene in A. burtoni brain and mouse neuronal GT1-7 cells. Consistent with the relative promoter occupancy exhibited by endogenous Pur proteins, overexpression of Purbeta, but not Puralpha, significantly downregulated GnRH1 mRNA levels in transiently transfected GT1-7 cells, suggesting that Purbeta acts as a repressor of GnRH1 gene transcription.