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.
Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham.
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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