Inhibition of beta protein 1 expression enhances beta-globin promoter activity and beta-globin mRNA levels in the human erythroleukemia (K562) cell line.
OBJECTIVE: In this paper, we report new observations related to the mechanism of the negative regulation of the important adult beta-globin gene in the erythroid cells at the embryonic-fetal stage of their development. We focused on the role of the silencer II region located upstream of the beta-globin gene, which along with its cognate binding protein BP1, negatively regulates beta-globin transcription. MATERIALS AND METHODS: We prepared plasmid constructs containing the wild-type silencer II sequence, a mutated silencer II sequence, or a mutated control sequence in the beta-globin promoter 690-bp insert, which in turn was linked to an enhanced green fluorescent protein ( EGFP) reporter gene. A human erythroleukemia cell line (K562) with embryonic-fetal phenotype was transfected with these EGFP constructs. RESULTS: Flow cytometry and fluorescence digital imaging showed about threefold increase in the beta-globin promoter activity of the mutated silencer II construct. Introduction of a small interfering RNA (siRNA) complementary to BP1 into the cells caused a 75% decrease in BP1 expression and a simultaneous approximately 40% elevation of beta-globin promoter activity as well as an increase in beta-globin mRNA levels, as compared with controls. We detected no changes in the mRNA levels of positive regulators of hemoglobin transcription such as EKLF and GATA-1. CONCLUSION: Our results support the involvement of BP1 in the mechanism of the negative regulation of beta-globin transcription. A better understanding of this mechanism may lay the groundwork for novel gene therapy approaches to inhibit the expression of abnormal structural variants of adult beta globin, such as sickle hemoglobin.[1]References
- Inhibition of beta protein 1 expression enhances beta-globin promoter activity and beta-globin mRNA levels in the human erythroleukemia (K562) cell line. Zoueva, O.P., Rodgers, G.P. Exp. Hematol. (2004) [Pubmed]
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