Functional map of a placenta-specific enhancer of the human leukemia inhibitory factor receptor gene.
We recently reported a placenta-specific enhancer in the human leukemia inhibitory factor receptor (LIFR) gene and now show detailed characterization of the 226-base pair enhancer (-4625/-4400 nucleotides). Four of twenty-two mutants in linker analysis showed reduced promoter activities to 45, 30, 10, and 10%, respectively. Specific binding of region A (-4617/-4602) with nuclear extract was competed by a known Oct-1 oligo and supershifted by Oct-1 antibody. Specific binding of region B (-4549/-4535) was competed by a GATA oligo, but could not be supershifted by four GATA antibodies. Nevertheless, mutagenesis showed that critical bases in region B were identical to the GATA core motif, indicating that region B may bind to a novel GATA family transcription factor. The other two adjacent regions designated as region C (-4464/-4445) showed no known consensus binding sites, and their specific placental JEG-3 nuclear extract binding was not evident in nonplacental nuclear extracts and was not competed by a trophoblast specific element (TSE), indicating that region C is a novel placenta-specific element (PSE, CATTTCCTGAACTAGTTTTT). Footprinting localized the binding boundary of PSE-binding protein (PSEB), and three Gs were found to be important for specific PSE binding. UV cross-linking showed that PSEB had a molecular mass of approximately 160 kDa, substituting the PSE with two previously reported placenta elements TSE or chorionic somatomammotropin enhancer factor 1 (CSEF-1) motifs resulted in markedly different promoter activities, indicating that PSEB is indeed different from TSE binding protein or CSEF-1. These results are the first demonstration that a novel PSE is the major element for placenta-specific enhancer activity in human LIFR gene.
Wang
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Cedars-Sinai Research Institute, UCLA School of Medicine, Los Angeles, California 90048, USA.
Melmed
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D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000032:analysis / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D006131:Growth Inhibitors; D051863:Host Cell Factor C1; D006801:Humans; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D008222:Lymphokines; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009687:Nuclear Proteins / Q000032:analysis; D050811:Octamer Transcription Factor-1; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010920:Placenta / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D018121:Receptors, Cytokine / Q000235:genetics*; D053606:Receptors, OSM-LIF; D016329:Sp1 Transcription Factor; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000235:genetics
D004268:DNA-Binding Proteins; D006131:Growth Inhibitors; C497692:HCFC1 protein, human; D051863:Host Cell Factor C1; D015850:Interleukin-6; C507837:LIF protein, human; C507840:LIFR protein, human; D053605:Leukemia Inhibitory Factor; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D008222:Lymphokines; D009687:Nuclear Proteins; D050811:Octamer Transcription Factor-1; D009838:Oligodeoxyribonucleotides; C494165:POU2F1 protein, human; D018121:Receptors, Cytokine; D053606:Receptors, OSM-LIF; C106703:SNAPC protein complex, human; D016329:Sp1 Transcription Factor; C108209:SP1 protein, human; D014157:Transcription Factors