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The Equine Luteinizing Hormone ß-Subunit Promoter Contains Two Functional Steroidogenic Factor-1 Response Elements

Department of Molecular and Integrative Physiology University of Kansas Medical Center Kansas City, Kansas 66160-7401

The requirements for basal expression of the LH ß-subunit promoter in pituitary gonadotropes are largely unknown. We have used the equine (e) LHß subunit promoter as a model to unravel the combinatorial code required for gonadotrope expression. Through the use of 5'-deletion mutagenesis, a region between -185 and -100 of the eLHß promoter was shown to play a critical role in maintaining basal promoter activity in T3–1 and LßT2 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 LHß promoter in other species. We have also identified an additional SF-1 site at -55 to -48. Binding of SF-1 to both sites was confirmed by electrophoretic mobility shift assays. Mutations within these sites, either individually or in combination, did not attenuate basal activity of the eLHß promoter in T3–1 cells, but did diminish promoter activity in LßT2 cells. Interestingly, cotransfection with an expression vector encoding SF-1 induced eLHß promoter activity, and this induction was abrogated by mutations within the SF-1 sites in T3–1 cells. Block replacement mutagenesis was performed on the -185/-100 region of the eLHß promoter to identify DNA response elements responsible for maintaining basal promoter activity. From this analysis, two regions emerged as being important: a distal 31-bp segment (-181 to -150) and an element located immediately 3' to the distal SF-1 site (-119 to -106). It is hypothesized that these two regions as well as the SF-1 sites represent regulatory elements that contribute to a combinatorial code involved in targeting expression of the eLHß promoter to gonadotropes.

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