TRANSCRIPTION OF THE LEYDIG INSULIN-LIKE GENE IS MEDIATED BY STEROIDOGENIC FACTOR-I

The Leydig insulin-like gene (Ley I-L), a member of the insulin-relate d gene family, is specifically expressed in pre-and postnatal Leydig c ells of the testis and in postnatal theca cells of the ovary. To deter mine the functional region of the mouse Ley I-L promoter and factors c ontrolling the Ley I-L gene expression, we used 2.1 kb of the 5'-flank ing region of the mouse Ley I-L gene to generate chimeric constructs w ith the chloramphenicol acetyltransferase gene (CAT). Transient transf ections of MA10 Leydig cells, LTK- fibroblasts, and F9 embryonic cells by a series of 5'-deleted mouse Ley I-L promoter-CAT constructs revea led that the sequence between nucleotides -157 to +4 directs the trans cription of the reporter gene in MA10 but not in LTK- and F9 cells, in dicating that the determinants of Leydig cell-specific expression resi de within this region. Deoxyribonuclease I (DNase I) footprint analysi s revealed that the sequences designated SF-1/1, SF-1/2, and SF-1/3 wi thin three DNase I-protected regions are homologous to the consensus b inding site of the steroidogenic factor-1 (SF-1). Competition and anti body studies showed that the three SF-l-binding sites in the Ley I-L p romoter have similar binding affinities for SF-1. Furthermore, transie nt transfections of MA10 cells with mutant reporter constructs, in whi ch SF-1/1 or both SF-1/2 and SF-1/3 were deleted, demonstrated that al l three SF-1-binding sites are required for SF-l-mediated stimulation of Ley I-L transcription. Cotransfection of an SF-1-containing express ion vector together with a Ley I-L promoter-CAT construct into HeLa ce lls, which lack the endogenous SF-1 protein, resulted in CAT gene tran scription, which indicated that SF-1 can transactivate the Ley I-L pro moter. These data demonstrate an essential role of SF-1 in transcripti onal activation of the Ley I-L promoter.