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

Abstract

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 cAMPstimulated 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 trophoblastspecific cAMP-responsiveness of the mGH-V proximal promoter.