Identification and characterization of cis- and trans-acting factors involved in the expression of the rat placental lactogen II gene

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Sun, Yuxiang
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The rat and mouse placental lactogen I and II genes (PLI and PLII) are members of a large family of prolactin-related proteins that are expressed in a developmentally specific manner by the placenta during pregnancy. There is an intriguing switch in expression between PL-I and PL-II at midpregnancy; the factors that control this switc are unknown. PLI and PLII are expressed in the placental trophoblast giant cells according to different but overlapping developmental patterns, suggesting there may be both common and unique factors which regulate the expression of these genes. DNA binding sites for these transcription factors are conserved in the 5' flanking region of the rat PLI gene (rPLI), suggesting a similar regulation. Little is known about the regulation of PLII gene. The work in this thesis focuses on the molecular mechanisms of rat PLII (rPLII) gene regulation. The rat choriocarcinoma cell line, Rcho, which differentiates in culture into the trophoblast giant cell type, has proven to be a good modelsystem for studying gene expression in this cell. Transfection studies with the luciferase reporter gene indicated a 3031 bp rPLII 5' flanking sequence DNA is active in the Rcho cell line, but not in the non-placental pituitary GC cell line. Transgenic mouse studies also indicated that this fragment was efficient in targeting reporter gene expression to the placenta 'in vivo'. Deletion analysis of this 3031 bp 5' flanking fragment showed that the proximal 1435 bp of rPLII 5' flanking DNA is required for minimal expression of the reporter gene in Rcho cells. DNAse I protection analysis of this 65 bp enhancer fragment identified two regions which were protected by placental and Rcho nuclear extracts from DNAse I digestion, but not by GC nuclear extracts. Sequence analysis showed that the two protected regions corresponded to putative binding sites for the Ets and AP-1 families of transcription factors. Site-directed mutagenesis of the individual binding sites led to a partial loss of enhancing activity; a double Ets/AP-1 mutation led to a complete loss of activity. This study is the first to identify a specific regulatory element on the rPLII gene. The identification of the Ets family member that interacts with the rPLII enhancer and investigation of other potential regulatory elements on the rPLII gene will lead to a better understanding of the genetic factors involved in complete developmental regulation of rPLII. (Abstract shortened by UMI.)