Mechanisms in the transcriptional regulation of bradykinin B1 receptor gene expression. Identification of a minimum cell-type specific enhancer.
To investigate the mechanisms of bradykinin B1 (BKB1) receptor gene expression, transient DNA transfection analyses of human BKB1 receptor gene promoter were performed in SV-40 transformed IMR90 cells. A positive regulatory element (PRE) located at position -604 to -448 base pair (bp) upstream of the transcription start site consistently exhibited, by far, the highest level of relative luciferase activity. A negative regulatory element, at position -682 to -604 bp, was able to completely ablate the function of the PRE. Transfection combined with deletion and mutation analyses illustrated that the PRE contains a classic, powerful enhancer. This enhancer was minimized to a 100-bp element at position -548 to -448 bp. A 78-bp fragment of negative regulatory element functioned as a silencer. Transient transfection of the enhancer construct, driven by heterologous herpes simplex thymidine kinase promoter, into a variety of cell types, showed that this enhancer presents a cell-type specific feature. In the characterization of the enhancer, motifs A (-548 to -532) and B (-483 to -477) were found to be essential for full enhancer activity. Motif D (-472 to -467) played a smaller role in enhancer activation. Gel shift and antibody supershift assays determined that an AP-1 factor binds with motif B. The nuclear protein which binds to motif A has yet to be identified. Both factors are the critical regulators for this enhancer activation.
Yang
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Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Taylor
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Polgar
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D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051381:Rats; D043783:Receptor, Bradykinin B1; D018002:Receptors, Bradykinin / Q000235:genetics* / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D043783:Receptor, Bradykinin B1; D018002:Receptors, Bradykinin; D004247:DNA