Activation of delta-globin gene expression by erythroid Krupple-like factor: a potential approach for gene therapy of sickle cell disease.
Hemoglobin A2 (HbA2; alpha 2 delta 2) is a powerful inhibitor of HbS (alpha 2 beta 2(3)) polymerization. However, HbA2 levels are normally low in sickle cell patients. We show that a major reason for low delta-globin gene expression is the defective CACCC box at -90 in the delta-globin promoter. When the CACCC box defect in delta is corrected, expression of an HS2 delta /Luciferase reporter is equivalent to HS2 beta /Luciferase. Erythroid Krupple-like factor (EKLF), which binds to the CACCC box of the beta-globin gene and activates high-level expression, does not bind to the normal delta-globin promoter. Our goal is to design a modified EKLF that binds to the defective delta-globin promoter and enhances delta-globin gene expression. To test the feasibility of this strategy, we inserted the beta-globin CACCC box at -90 of the delta-globin gene promoter to produce an HS2 delta CAC-beta construct and quantitated human delta- and beta-globin mRNA in stably transformed murine erythroleukemia (MEL) cells. delta- Globin mRNA in these cells was 22.0% +/- 9.0% of total human globin mRNA (delta/delta + beta) as compared with 3.0% +/- 1.3% in the HS2 delta-beta control. In a second set of experiments a GAL4 DNA-binding site was inserted at -90 of the delta-globin gene to produce an HS2 delta GAL4-beta construct. This construct and a GAL4(1-147)/EKLF expression vector were stably transfected into MEL cells. delta-Globin mRNA in these cells was 27.8% +/- 7.1% of total human globin mRNA as compared with 9.9% +/- 2.5% in the HS2 delta GAL4-beta plus GAL4(1-147) control. These results show that delta-globin gene expression can be significantly increased by a modified EKLF. Based on these results, we suggest that modified EKLFs, which contain zinc fingers designed to bind specifically to the defective delta-globin CACCC box, may be useful in gene therapy approaches to increase HbA2 levels and inhibit HbS polymerization.
Donze
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Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham.
Jeancake
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Townes
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D000755:Anemia, Sickle Cell / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005240:Feasibility Studies; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D015316:Genetic Therapy; D005914:Globins / Q000235:genetics*; D006443:Hemoglobin A2 / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D051741:Kruppel-Like Transcription Factors; D004915:Leukemia, Erythroblastic, Acute / Q000473:pathology; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D012333:RNA, Messenger / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D016415:Sequence Alignment; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D051741:Kruppel-Like Transcription Factors; D012333:RNA, Messenger; D014157:Transcription Factors; C080774:erythroid Kruppel-like factor; D005914:Globins; D004247:DNA; D006443:Hemoglobin A2; D008156:Luciferases