DIFFERENTIAL RECRUITMENT OF STEROID-HORMONE RESPONSE ELEMENTS MAY DICTATE THE EXPRESSION OF THE PITUITARY GONADOTROPIN II-BETA SUBUNIT GENE DURING SALMON MATURATION

The role of testosterone (T) and 17 beta-estradiol (E(2)) in the control of chinook salmon gonadotropin II beta subunit (sGTHII beta) gene was examined. Both E(2) and T specifically stimulated GTHII beta gene expression in cultured juvenile rainbow trout pituitary cells. 5'-Flanking regions of the sGTHII beta gene linked to the chloramphenicol acetyltransferase (CAT) expression vector were transfected into these pituitary cells, and cultures were treated with steroid hormones. Estrogen-stimulated CAT activity occurred with constructs containing a 13-base pair estrogen responsive element (ERE) sequence [proximal ERE (pERE)] located at -273 to -260 upstream of its transcriptional start site. Binding specificity of pERE was confirmed by mobility shift and DNA methylation interference assays using the DNA binding domain of the human estrogen receptor. Interestingly, the pERE functioned to derepress the activity of the proximal silencer (pSil) only in the pituitary cells of juvenile trout but not in cells derived from maturing and sexually matured fish. Another potential ERE sequence comprised of three tandemly linked half-ERE palindromes was located from -2736 to -2659 [distal ERE (dERE)] of the sGTHII beta gene. Distal ERE might be responsible for the steroid responsiveness of the longest sGTHII beta/CAT construct (-3500CAT) observed in the pituitary cells of maturing fish. The function of pERE and dERE were further examined in the heterologous HeLa cells by mutagenesis and cotransfection with a rainbow trout estrogen receptor expression vector. Disruption of the palindromic structure of pERE severely impaired its function. When the sequences between pERE and dERE were deleted, a 200-fold increase in CAT activity was observed in response to E(2). A model is proposed to describe the regulation of GTHII beta gene expression at different reproductive stages.