title: The equine luteinizing hormone beta-subunit promoter contains two functional steroidogenic factor-1 response elements.
abstract: The requirements for basal expression of the LH beta-subunit promoter in pituitary gonadotropes are largely unknown. We have used the equine (e) LHbeta 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 eLHbeta promoter was shown to play a critical role in maintaining basal promoter activity in alphaT3-1 and LbetaT2 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 LHbeta 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 eLHbeta promoter in alphaT3-1 cells, but did diminish promoter activity in LbetaT2 cells. Interestingly, cotransfection with an expression vector encoding SF-1 induced eLHbeta promoter activity, and this induction was abrogated by mutations within the SF-1 sites in alphaT3-1 cells. Block replacement mutagenesis was performed on the -185/-100 region of the eLHbeta 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 eLHbeta promoter to gonadotropes.
authors.affiliation: Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City 66160-7401, USA. mwolfe2@kumc.edu
authors.forename: M W
authors.initials: MW
authors.lastname: Wolfe
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000502:physiology; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051717:Fushi Tarazu Transcription Factors; D005786:Gene Expression Regulation; D018398:Homeodomain Proteins; D006736:Horses; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D007986:Luteinizing Hormone / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000502:physiology; D012689:Sequence Homology, Nucleic Acid; D054339:Steroidogenic Factor 1; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D004268:DNA-Binding Proteins; D051717:Fushi Tarazu Transcription Factors; D018398:Homeodomain Proteins; C515057:NR5A1 protein, human; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D054339:Steroidogenic Factor 1; D014157:Transcription Factors; C515058:steroidogenic factor 1, mouse; D007986:Luteinizing Hormone; D008156:Luciferases