A direct-repeat sequence of the human BiP gene is required for A23187-mediated inducibility and an inducible nuclear factor binding.
We have recently isolated a functional promoter encoding the human polypeptide-binding protein (BiP) gene from Burkitt's lymphoma cells by polymerase chain reaction (The EMBL Data Library accession number X59969, 1991). This promoter DNA segment (termed BiP670) was fused to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene and expressed in NIH3T3 cells. BiP670 retains basal and Ca2+ ionophore A23187-inducible activities. Using 5' deletion assay, we found three basal expression elements (BEE) in the BiP670. Removal of the distal BBE (BBE3), which is contained in a segment spanning -368/-170, caused a 50% loss of the basal activity; removal together with the middle BBE (BBE2), which is contained in a segment spanning -170/-107, resulted in a further 30% loss of the activity. Further removal of the proximal BBE (BBE1), which spans -107/-39, abolished greater than 95% of the basal expression. In addition, an A23187-inducible element (AIE) appeared to be associated with the BBE1. At least a six-fold inducibility remained as long as the BiP promoter retained the sequences -107/-39. Using an in vitro gel mobility shift assay, an A23187-inducible nuclear factor (AINF) was detected from NIH3T3 cells. DNA binding competition experiments indicate that the -107/-39 segment contains a sequence motif which interacts with this cellular factor. Further analysis showed that the two direct repeats, ranging -108/-73 and -72/-40, are the target for AINF binding. A 3-4 fold increase of the AINF binding to both repeated sequences was detected from induced cells. Similar results were also demonstrated in HeLa cells, suggesting that transcriptional control of BiP gene expression in mammalian cells is conserved. These findings also imply that the identified nuclear factor may be important in mediating transcriptional activation of the BiP gene.
Chao
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Department of Biochemistry, Chang Gung Medical College, Taoyuan, Taiwan, China.
Lin-Chao
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D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D000001:Calcimycin / Q000494:pharmacology*; D002352:Carrier Proteins / Q000235:genetics*; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D006367:HeLa Cells; D006360:Heat-Shock Proteins; D006801:Humans; D051379:Mice; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000096:biosynthesis*; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection
D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D018832:Molecular Chaperones; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D000001:Calcimycin; D015500:Chloramphenicol O-Acetyltransferase