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/

Transcriptional activation of the rhesus monkey ON-variant gene in syn cytiotrophoblasts 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 f ull cAMP-stimulated mGH-V transcription. To identify the regions of th is promoter critical for transcription, transient transfections of rep orter 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 mobil ity-shift analyses demonstrated that Spl and Sp3 bound to a region con taining a GGGAGG motif at -136/-131. Mutation of the -62/-53 region de creased transcriptional activation by 66-99%, and Spl and Sp3 bound to a GGTGGG motif overlapping this region (at -65/-60). Selective mutati on of this Sp1/Sp3 site decreased basal transcription by approximately 80%, and cAMP-stimulated transcription by up to 75% (with the greates t 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) als o dramatically reduced (by 75%) transcriptional activation in trophobl asts. We conclude that two Sp1/Sp3 sites as well as additional element s directly adjacent to these sites contribute to trophoblast-specific cAMP-responsiveness of the mGH-V proximal promoter.