Human (h) growth hormone (GH), chorionic somatomammotropin (CS) and prolactin (PRL) comprise a family of related genes that are essential for normal growth and development. Knowledge of the regulation of these genes is important for our understanding their physiologic and pathophysiologic functions. Cell-specific regulation of the hGH and hCS genes is of interest because these genes are nearly identical (93-96 percent nucleotide sequence homology), are closely linked (hGH/CS locus spans ca. 55 kbp), and their expression is tightly controlled in pituitary (GH) and placenta (CS), respectively. The proposed studies will extend our previous investigations of an hCS enhancer/silencer, designated CSEn, which is involved in mediating cell-specific expression of the hCS genes. We have presented evidence that the three copies of CSEn present with the hGH/CS locus function as a composite element which synergistically stimulates hCS gene expression in syncytiotrophoblasts and silences hCS gene expression in the pituitary. We have cloned a member of the human transcription enhancer factor-1 (TEF) family, designated TEF-P, which is involved in mediating CSEn enhancer function. Four members of this family have been identified to date: TEF-1, TEF-3/RTEF-1, TEF-4, and TEF-P/DTEF-1. TEF-P is preferentially expressed in placental tissue, choriocarcinoma cells (BeWo), and cardiac and skeletal muscle. We have demonstrated that TEF-P is a potent transactivator in BeWo cells, which distinguishes it from TEF-1, which acts as a transrepressor in these cells. TEF-1 binding to the enhancer has been implicated in mediating CSEn silencing activity in cultured rat pituitary GC cells. In the current studies we will perform structure/ function analyses of TEF-P and TEF-1 to identify the transactivation and transrepressor domains of the proteins (Aim 1). We will quantitate the mRNA and protein levels of all the TEF-1 family members in pituitary and placental cells. This information will be coupled with functional studies and with studies that will modulate the expression of each family member to ascertain which family members regulate the enhancer and silencer activities in placental and pituitary cells, respectively (Aim 2). The chromosomal TEF-P locus will be cloned and characterized, including chromosomal localization and promoter characterization (Aim 3).
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