A TEF-1 binding motif that interacts with a placental protein is important for the transcriptional activity of the hCS-B enhancer.
The transcriptional activity of the human placental lactogen genes (choriosomatomammotropic hormone, hCS) is controlled by tissue-specific enhancers located 4 kb downstream from their respective origins of transcription. The hCS-B enhancer is the strongest; its activity is mediated by synergism between two protein-binding sites (DF-3 and DF-4). The DF-4 site possesses a potential binding sequence for TEF-1, a known transcription factor. In this paper, we show by electrophoretic mobility-shift assays and antibody supershift experiments that TEF-1 does not bind to site DF-4. Mutations in the TEF-1-like binding motif of site DF-4 prevent formation of the DNA-protein complex, called complex f, in the presence of placental JEG-3 cell extracts. When HeLa cell extracts are used, another complex (complex c) is also affected. In transient expression experiments, TKCAT constructs linked to this mutated DF-4 site exhibit greatly reduced transcriptional activity when introduced into JEG-3 cells. Some cell lines contain both protein c and protein f (the proteins forming complexes c and f); when transfected, these lines display reduced DF-4-driven activity, suggesting that the two proteins could compete for the same DF-4 sequence. We conclude that protein f is important for the placenta-specific activity of the hCS-B enhancer. By UV cross-linking, we show that protein f is actually three polypeptides ranging in size from about 12 to 21 kD.
Jacquemin
P
P
Laboratoire de Biologie Moléculaire et de Génie Génétique, Université de Liège, Sart-Tilman, Belgium.
Oury
C
C
Belayew
A
A
Martial
J A
JA
D001483:Base Sequence; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D010455:Peptides / Q000378:metabolism; D010928:Placental Lactogen / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D000091262:TEA Domain Transcription Factors; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D000091262:TEA Domain Transcription Factors; C057182:TEAD1 protein, human; D014157:Transcription Factors; D010928:Placental Lactogen