EXCEED THE SPACE PROVIDED. Syncytiotrophoblasts are a major placental component responsible for feto-maternal exchange and secretion oi 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 ol 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-113 and TEF-1y, and found thal 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 _ctions on endogenous hCS gene expression and placental cell differentiation using primary cultures of human placenta] irophoblasts. 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 egulation on placental cell function. Using these cell models and the newly developed TEF antibodies, we will defin_ the function of individual TEF members by hCSFFEF expression correlation, TEF antisense experiments, and chromatir immunoprecipitation assays (Aim 1). Our preliminary studies demonstrate several fundamental insights with direcl bearing on the mechanism of TEF action: 1) TEF control of GT-IIC enhancer is subject to PKC phosphorylatior 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 splicec 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 (Aims 2) and homo- anc '_etero-dimerization (Aim 3) regulate TEF DNA binding and transcriptional activities in cellular environments. We wil :letermine TEF-5 isoform expression levels in placenta and examine their roles in CSEn function (Aim 4). The proposal _ddresses specific hypotheses that will enhance our understanding of TEF-mediated placental gene expression and Irophoblast differentiation that are important for human fetal development. PERFORMANCE SITE ========================================Section End===========================================
Showing the most recent 10 out of 18 publications
