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.


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.


CONCLUSIONS
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.
Zoueva
Olga P
OP
Molecular and Clinical Hematology Branch, National Institute of Diabetes & Digestive & Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Rodgers
Griffin P
GP
D004268:DNA-Binding Proteins / Q000235:genetics; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D020868:Gene Silencing; D015316:Genetic Therapy; D005914:Globins / Q000235:genetics*; D018398:Homeodomain Proteins / Q000502:physiology*; D006801:Humans; D020014:K562 Cells; D051741:Kruppel-Like Transcription Factors; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012333:RNA, Messenger / Q000032:analysis*; D012097:Repressor Proteins / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*
C105304:DLX4 protein, human; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D018398:Homeodomain Proteins; D051741:Kruppel-Like Transcription Factors; D012333:RNA, Messenger; D012097:Repressor Proteins; D014157:Transcription Factors; C080774:erythroid Kruppel-like factor; D005914:Globins