(Scanned from the applicant's description): Fetal development is a complex process in which multiple placental genes are specifically regulated. However, molecular mechanisms for placenta-specific gene regulation are largely unknown. Leukemiainhibitory factor receptor (LIFR) has recently been shown to maintain normal placental function and is specifically regulated in placenta during fetal development. This proposal aims to study the mechanism of placenta-specific gene regulation of human LIFR, which I have found to possess both a placenta-specific enhancer and a placenta-specific promoter. I also identified a novel placenta-specific element (PSE) in LIFR placenta-specific enhancer, and now propose to characterize its corresponding placenta-specific element binding protein (PSEB) and their interactions on placenta-specific LIFR gene regulation. The PSEB cDNA will he cloned by screening a lambda expression library or DNA-affinity chromatography purification coupled with RT-PCR cloning. The DNA-binding domain and transactivation domain of PSEB will be localized by serial deletions. electrophoretic mobility shift assay (EMSA), transfection and reporter assays. The critical amino acid residues in these two domains will also be pinpointed by site-directed mutagenesis. Consensus PSEB binding motif(s) will be determined by PCR-assisted binding-site selection. Meanwhile, transgenic mice with dual reporter luciferase and enhanced green fluorescent protein (EGFP) cDNAs under the control of the cassette of the cloned human LIFR gene placenta-specific enhancer and promoter will be produced, and the cell type specific expression patterns and levels of these reporter genes in placenta and other tissues will be analyzed in both founder mice and their Fl generation mice. These studies will thus unravel regulatory mechanisms of placenta-specific gene expression and could provide an outstanding placenta-specific target molecule for regulating placental functions and implicating fetal development and/or pregnancy.
