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SP1/SP3-Binding Sites and Adjacent Elements Contribute to Basal and Cyclic Adenosine 3',5'-Monophosphate-Stimulated Transcriptional Activation of the Rhesus Growth Hormone-Variant Gene in Trophoblasts

Wisconsin Regional Primate Research Center and Department of Obstetrics and Gynecology University of Wisconsin Medical School University of Wisconsin Madison, Wisconsin 53715-1299

Transcriptional activation of the rhesus monkey GH-variant gene in syncytiotrophoblasts is developmentally regulated by trophoblast-specific and cAMP-responsive mechanisms. Progressive deletions of 5'-flanking DNA defined the most proximal 140 bp as the minimal region retaining full cAMP-stimulated mGH-V transcription. To identify the regions of this promoter critical for transcription, transient transfections of reporter plasmids containing systematic 10 base mutations throughout this proximal region were performed. Mutation of the region from -140/-131 decreased transcription in syncytiotrophoblasts by 50%, and gel mobility-shift analyses demonstrated that Sp1 and Sp3 bound to a region containing a GGGAGG motif at -136/-131. Mutation of the -62/-53 region decreased transcriptional activation by 66–99%, and Sp1 and Sp3 bound to a GGTGGG motif overlapping this region (at -65/-60). Selective mutation of this Sp1/Sp3 site decreased basal transcription by approximately 80%, and cAMP-stimulated transcription by up to 75% (with the greatest effect in primary syncytiotrophoblast cultures), indicating that the Sp1/Sp3 site is critical for transcriptional activation. Mutations in the regions adjacent to the Sp1/Sp3 sites (-130/-111 and -52/-43) also dramatically reduced (by 75%) transcriptional activation in trophoblasts. We conclude that two Sp1/Sp3 sites as well as additional elements directly adjacent to these sites contribute to trophoblast-specific cAMP-responsiveness of the mGH-V proximal promoter.

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