Activation of delta-globin gene expression by erythroid Krupple-like factor: a potential approach for gene therapy of sickle cell disease.

Abstract

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.

• Authors: D Donze, P H Jeancake, T M Townes
• Journal: Blood (Blood) Vol. 88 Issue 10 Pg. 4051-7 (Nov 15 1996) ISSN: 0006-4971 [Print] United States
• PMID: 8916973 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• DNA-Binding Proteins
• Kruppel-Like Transcription Factors
• RNA, Messenger
• Transcription Factors
• erythroid Kruppel-like factor
• Globins
• DNA
• Hemoglobin A2
• Luciferases

Topics

• Anemia, Sickle Cell (genetics)
• Animals
• Base Sequence
• Binding Sites
• DNA (metabolism)
• DNA-Binding Proteins (metabolism)
• Feasibility Studies
• Gene Expression Regulation
• Genes, Reporter
• Genetic Therapy
• Globins (genetics)
• Hemoglobin A2 (biosynthesis, genetics)
• Humans
• Kruppel-Like Transcription Factors
• Leukemia, Erythroblastic, Acute (pathology)
• Luciferases (biosynthesis, genetics)
• Mice
• Mutagenesis, Site-Directed
• Promoter Regions, Genetic (genetics)
• Protein Binding
• RNA, Messenger (biosynthesis)
• Regulatory Sequences, Nucleic Acid
• Sequence Alignment
• Transcription Factors (metabolism)
• Transfection
• Tumor Cells, Cultured
• Zinc Fingers