The equine luteinizing hormone beta-subunit promoter contains two functional steroidogenic factor-1 response elements

The requirements for basal expression of the LH beta-subunit promoter in pi tuitary gonadotropes are largely unknown. We have used the equine (e) LH be ta subunit promoter as a model to unravel the combinatorial code required f or gonadotrope expression. Through the use of 5'-deletion mutagenesis, a re gion between -185 and -100 of the eLH beta promoter was shown to play a cri tical role in maintaining basal promoter activity in alpha T3-1 and L beta T2 cells. This region encompasses the steroidogenic factor-1 (SF-1) binding site that has been reported to have a functional role in expression of the LH beta promoter in other species. We have also identified an additional S F-1 site at -55 to -48. Binding of SF-1 to both sites was confirmed by elec trophoretic mobility shift assays. Mutations within these sites, either ind ividually or in combination, did not attenuate basal activity of the eLH be ta promoter in alpha T3-1 cells, but did diminish promoter activity in L be ta T2 cells. Interestingly, cotransfection with an expression vector encodi ng SF-1 induced eLH beta promoter activity, and this induction was abrogate d by mutations within the SF-1 sites in alpha T3-1 cells. Block replacement mutagenesis was performed on the -185/-100 region of the eLH beta promoter to identify DNA response elements responsible for maintaining basal promot er activity. From this analysis, two regions emerged as being important: a distal 31-bp segment (-181 to -150) and an element located immediately 3' t o the distal SF-1 site (-119 to -106). It is hypothesized that these two re gions as well as the SF-1 sites represent regulatory elements that contribu te to a combinatorial code involved in targeting expression of the eLH beta promoter to gonadotropes.