Olga P Zoueva1, Griffin P Rodgers. 1. Molecular and Clinical Hematology Branch, National Institute of Diabetes & Digestive & Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Abstract
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.
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 humanerythroleukemia 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.