To determine the role of Sp family members in trophoblast-specific and cAMP-responsive transcription of the rhesus monkey growth hormone variant gene. RESULTS We wished to further define rhesus monkey GH-V transcription which is cAMP-respon-sive in differentiated trophoblasts. Mutagenesis of the GH-V gene promoter and subsequent binding analysis showed that Sp1 and Sp3 transcription factors bind to two distinct elements at -136/-131 and -65/-60 in the mGH-V promoter. Three distinct complexes containing either Sp1 or Sp3 formed at each element, however none of the complexes contained both Sp1 and Sp3. To investigate the molec-ular basis for the presence of these multiple complexes, we used Western blots to characterize Sp1 and Sp3 immunoreactive proteins in trophoblasts. We determined that each factor exists as a population of molecules of varied sizes, including full length 95-105 kDa species, and 70, 64 and 46 kDa species as well. Since the antibodies were raised to small Sp1 or Sp3 peptides in the zinc finger region (Sp1) or the carboxy terminus (Sp3), these are unlikely to represent degradation fragments, and more likely represen t specific splice variants of these transcription factors. Heating or incubation at room temperature did not increase the proportion of these smaller variants relative to full-length species. We examined whether treatment with cAMP altered the amount or expression pattern of these variants. However, we could document no effects of cAMP treatment on the amounts of Sp factors, or their distribution among the different molecular weights. Scatchard analysis of complex formation at these sites indicated that treatment with cAMP for 48 h had no effect on affinity of any of the complexes for binding sites. FUTURE DIRECTIONS We will explore the role of post-translational modification of pre-existing Sp1 and Sp3 proteins and co-activators in stimulating gene transcription through the Sp1/Sp3 sites. KEY WORDS placenta, gene transcription, placental growth hormone, transcription factors FUNDING NIH R01 HD26458 PUBLICATIONS Schanke, J.T., K.J. Johnson, M. Durning, L.K. Bennett and T.G. Golos. 1998. Sp1/Sp3 binding sites and adjacent elements contribute to basal and cAMP-stimulated transcriptional activation of the rhesus growth hormone-variant gene in trophoblasts. Molecular Endocrinology. 12:405-417.
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