"Repair' of the chorionic somatomammotropin-A "enhancer' region reveals a novel functional element in the chorionic somatomammotropin-B enhancer.
Human chorionic somatomammotropin (CS) synthesis results from the independent expression of two homologous genes, CS-A and CS-B. A transcription enhancer factor-1 (TEF-1) element and an upstream 81 bp modulatory domain, containing repressor (RF-1) and derepressor (DF-1) activities, are important for efficient CS-B enhancer function in transfected placental JEG-3 cells. The equivalent region of the CS-A gene is not active. Although the TEF-1 element is conserved between the CS-A and CS-B genes, a single base substitution is present in the DF-1 element and two more are located between the RF-1 and DF-1 sites in a region we term AF-1. Repair of the DF-1 site increased CS-A enhancer function approximately 70-fold, but repair of previously uncharacterized AF-1 sequences was also required for full (CS-B like) enhancer activity. A 5 bp disruption of AF-1 sequences in the CS-B enhancer region, resulted in a 97% loss of stimulatory activity. The AF-1 sequences showed no intrinsic enhancer activity, however, they were able to significantly repress heterologous promoter activity stimulated by a TEF-1 enhancer element. A high affinity/specificity interaction between JEG-3 nuclear protein and AF-1 sequences was confirmed by gel mobility shift assay. By comparison to "wild type' AF-1 sequences, this interaction was competed to a lesser extent by both RF-1 and DF-1 elements, but not by mutated AF-1 sequences. The major protein binding to AF-1 sequences was estimated to be 23 kDa by UV crosslinking. These data indicate that enhancer activity can be generated by modulating binding events proximal to the TEF-1 element in the CS-A "enhancer' region and that coordinated binding of AF-1 and DF-1 are required for efficient (CS-B) enhancer activity.
Lytras
A
A
Department of Physiology, University of Manitoba, Winnipeg, Canada.
Surabhi
R M
RM
Zhang
J F
JF
Jin
Y
Y
Cattini
P A
PA
D001483:Base Sequence; D003432:Cross-Linking Reagents; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D006801:Humans; D008969:Molecular Sequence Data; D010928:Placental Lactogen / Q000235:genetics*; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D004247:DNA; D010928:Placental Lactogen