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

The role of testosterone (T) and 17 beta-estradiol (E(2)) in the contr ol of chinook salmon gonadotropin II beta subunit (sGTHII beta) gene w as examined. Both E(2) and T specifically stimulated GTHII beta gene e xpression in cultured juvenile rainbow trout pituitary cells. 5'-Flank ing regions of the sGTHII beta gene linked to the chloramphenicol acet yltransferase (CAT) expression vector were transfected into these pitu itary 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. B inding specificity of pERE was confirmed by mobility shift and DNA met hylation interference assays using the DNA binding domain of the human estrogen receptor. Interestingly, the pERE functioned to derepress th e 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 tande mly linked half-ERE palindromes was located from -2736 to -2659 [dista l ERE (dERE)] of the sGTHII beta gene. Distal ERE might be responsible for the steroid responsiveness of the longest sGTHII beta/CAT constru ct (-3500CAT) observed in the pituitary cells of maturing fish. The fu nction 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 o f pERE severely impaired its function. When the sequences between pERE and dERE were deleted, a 200-fold increase in CAT activity was observ ed in response to E(2). A model is proposed to describe the regulation of GTHII beta gene expression at different reproductive stages.