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 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.
Schanke
J T
JT
Wisconsin Regional Primate Research Center and Department of Obstetrics and Gynecology, University of Wisconsin Medical School, Madison 53715-1299, USA.
Durning
M
M
Johnson
K J
KJ
Bennett
L K
LK
Golos
T G
TG
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism* / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005260:Female; D013006:Growth Hormone / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D008253:Macaca mulatta; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014327:Trophoblasts / Q000187:drug effects / Q000502:physiology*
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D013006:Growth Hormone; D000242:Cyclic AMP