Syncytiotrophoblasts are a major placental component responsible for feto-maternal exchange and secretion of pregnancy-specific hormones. Syncytia formation is a hallmark of placental cell differentiation, accompanied by dramatic activation of the chorionic somatomammotropin (CS) gene. CS, growth hormone, and prolactin comprise a gene family required for normal growth, metabolism, and reproduction. We demonstrated that placental-specific human CS gene I expression is mediated by cooperative action of enhancers (CSEn) located downstream of each of the three CS genes. These enhancers stimulate the CS promoter in placental cells, but silence the promoter in pituitary cells. Members of the transcription enhancer factor (TEF) family bind to several GT-IIC and Sphl/Sphll elements in CSEn and mediate the CS enhancer/silencer functions. We isolated two differential splicing isoforms TEF-1beta and TEF-1gamma, and found that their limited sequence divergence is associated with stronger CSEn binding and trans-repression function than TEF-la. We cloned a new TEF family member, TEF-5, that trans-activates CSEn in placental cells. We are now addressing TEF actions on endogenous hCS gene expression and placental cell differentiation using primary cultures of human placental trophoblasts. We discovered that fusogenic membrane glycoprotein (FMG)-mediated fusion of choriocarcinoma cells (BeWo) results in activation of CSEn, providing another possible physiologic model that will facilitate the study of TEF regulation on placental cell function. Using these cell models and the newly developed TEF antibodies, we will define the function of individual TEF members by hCS/TEF expression correlation, TEF antisense experiments, and chromatin immunoprecipitation assays (Aim 1). Our preliminary studies demonstrate several fundamental insights with direct bearing on the mechanism of TEF action: 1) TEF control of GT-IIC enhancer is subject to PKC phosphorylation regulation; 2) TEF molecules bind cooperatively to direct repeats of GT-IIC sequences separated by 3 nucleotides, suggesting that TEF homo- and hetero-dimerization may be involved in their action; 3) two TEF-5 differentially spliced cDNA clones containing gross structural alterations are present in placenta and may play distinct roles in CSEr regulation. We will perform mechanistic studies on how PKC phosphorylation (Aim 2) and homo- and hetero-dimerization (Aim 3) regulate TEF DNA binding and transcriptional activities in cellular environments. We will determine TEF-5 isoform expression levels in placenta and examine their roles in CSEn function (Aim 4). The proposal addresses specific hypotheses that will enhance our understanding of TEF-mediated placental gene expression and trophoblast differentiation that are important for human fetal development.
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