A novel chimeric promoter that is highly responsive to hypoxia and metals.
To develop a potent hypoxia-inducible promoter, we evaluated the usefulness of chimeric combinations of the (Egr-1)-binding site (EBS) from the Egr-1 gene, the metal-response element (MRE) from the metallothionein gene, and the hypoxia-response element (HRE) from the phosphoglycerate kinase 1 gene. In transient transfection assays, combining three copies of HRE (3 x HRE) with either EBS or MRE significantly increased hypoxia responsiveness. When a three-enhancer combination was tested, the EBS-MRE-3 x HRE (E-M-H) gave a hypoxia induction ratio of 69. The expression induced from E-M-H-pGL3 was 2.4-fold higher than that induced from H-pGL3 and even surpassed the expression from a human cytomegalovirus promoter-driven vector. The high inducibility of E-M-H was confirmed by validation studies in different cells and by expressing other cDNAs. Gel shift assays together with functional overexpression studies suggested that increased levels of hypoxia-inducible factor 1alpha, metal transcription factor-1 and Egr-1 may be associated with the high inducibility of the E-M-H chimeric promoter. E-M-H was also induced by hypoxia mimetics such as Co2+ and deferoxamine (DFX) and by hydrogen peroxide. Gene expression from the E-M-H was reversible as shown by the reduced expression of the transgene upon removal of inducers such as hypoxia and DFX. In vivo evaluation of the E-M-H in ischemic muscle revealed that erythropoietin secretion and luciferase and LacZ expression were significantly higher in the E-M-H group than in a control or H group. With its high induction capacity and versatile means of modulation, this novel chimeric promoter should find wide application in the treatment of ischemic diseases and cancer.
Department of Medicine, Cardiac and Vascular Center, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Kangnam-ku, Seoul, Korea.
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Lee
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Lee
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Kim
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Kim
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Lee
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Jang
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Shin
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Suh
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Jeon
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Byun
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Kim
D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D002678:Chimera; D004277:DNA, Single-Stranded / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005818:Genetic Engineering; D006367:HeLa Cells; D006614:Hindlimb / Q000098:blood supply; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics; D017078:Laser-Doppler Flowmetry; D008668:Metallothionein / Q000235:genetics; D008670:Metals / Q000378:metabolism*; D051379:Mice; D010735:Phosphoglycerate Kinase / Q000235:genetics; D011401:Promoter Regions, Genetic; D012039:Regional Blood Flow; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection / Q000379:methods; D000097809:Transcription Factor MTF-1
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008668:Metallothionein; D008670:Metals; D010735:Phosphoglycerate Kinase; D014157:Transcription Factors; D000097809:Transcription Factor MTF-1; C400152:DNA enzyme ED5; C497442:HIF1A protein, human