The primate syncytiotrophoblast expresses placental growth hormone- variant and chorionic somatomammotropin members of the growth hormone gene locus. The genetic mechanisms which underlie placenta-specific expression of these hormones remain poorly understood. To define these mechanisms in vitro, the regulation of monkey placental growth hormone- variant (mGH-V) transcription by cAMP (an important determinant of trophoblast hormone gene expression) will be studied during cytotrophoblast to syncytiotrophoblast differentiation in primary cultures. We hypothesize that cAMP stimulation and trophoblast differentiation induce or activate placenta-specific DNA-binding proteins or co-activators. Novel elements mediating transcription- activating effects of cAMP will be identified by block mutagenesis of the proximal (-140 to -66 bp) 5'-flanking DNA of-the mGH-V gene, shown to confer basal and cAMP-sensitive/placenta-specific transcriptional activity. Transcription factors which interact with these elements will be characterized by gel mobility-shift assays, uv cross-linking analyses and affinity chromatography. These transcription factors will be cloned by screening placental syncytiotrophoblast cDNA expression libraries with mGH-V regulatory element DNA probes, by using a yeast hybrid approach to screen for interacting proteins, or by differential display analysis to identify placenta-specific and cAMP-induced mRNAs. Additional distal enhancers will be located in upstream 5' and 3'- flanking gene regions. These studies will provide direct information on how transcription factors and their expression underlie the cell- specific and developmentally regulated expression of placental hormone genes at the maternal-fetal interface during trophoblast differentiation.
