II.

The orphan nuclear receptor, steroidogenic factor-1 (SF-1), is expressed in the pituitary and in the gonadotrope precursor cell line, ␣T3-1, where it is believed to enhance expression of the common gonadotropin ␣-subunit gene through transactivation of the gonadotropespecific element (GSE). Sequence analysis of the rat luteinizing hormone ␤-subunit (LH␤) gene promoter revealed the presence of a consensus GSE at ؊127 to ؊119 (TGACCTTGT). We have demonstrated the ability of SF-1 to bind specifically to this putative GSE sequence by electrophoretic mobility shift assay, utilizing both ␣T3-1 nuclear extracts and in vitro translated SF-1. In addition, mutation of the putative LH␤-GSE (TGAAATTGT) eliminated specific DNA binding. To examine the ability of SF-1 to enhance LH␤ promoter activity, CV-1 cells, which lack endogenous SF-1, were cotransfected with an SF-1-containing expression vector and an LH␤-luciferase reporter construct. When cotransfected with ؊209/؉5 of the LH␤ promoter, SF-1 increased luciferase activity by 56-fold. SF-1 responsiveness was markedly diminished with loss of the putative GSE region in deletion constructs and in the presence of a two base pair mutation, analogous to the mutation which eliminated DNA binding. Finally, the LH␤-GSE was able to confer SF-1 responsiveness on a heterologous minimal growth hormone promoter, GH50 (57-fold). We conclude that SF-1 both binds to and transactivates the rat LH␤ promoter. These data suggest that SF-1 may participate in the expression of the LH␤ gene by the gonadotrope.