Abstract
Human β-globin disorders are relatively common genetic diseases cause by mutations in the β-globin gene. Increasing the expression of the γ-globin gene has great benefits in reducing complications associated with these diseases. The Oct-1 transcription factor is involved in the transcriptional regulation of the γ-globin gene. The human γ-globin genes (both Aγ and Gγ-globin genes) carry three Oct-1 transcription factor consensus sequences within their promoter regions. We have studied the possibility of inducing γ-globin gene expression using decoy oligonucleotides that target the Oct-1 transcription factor consensus sequence. A double-stranded 22 bp decoy oligonucleotide containing the Oct-1 consensus sequence was synthesized. The results obtained from our in vitro binding assay revealed a strong competitive binding of the decoy oligonucleotide for the Oct-1 transcription factor. When K562 human erythroleukemia cells were treated with the Oct-1 decoy oligonucleotide, significant increases in the level of the γ-globin mRNA were observed. The results of our western blots further demonstrated significant increases of the fetal hemoglobin (HbF, α2γ2) in the Oct-1 decoy oligonucleotide-treated K562 cells. The results of our immunoprecipitation (IP) studies revealed that the treatment of K562 cells with the Oct-1 decoy oligonucleotide significantly reduced the level of the endogenous γ-globin gene promoter region DNA co-precipitated with the Oct-1 transcription factor. These results suggest that the decoy oligonucleotide designed for the Oct-1 transcription factor consensus sequence could induce expression of the endogenous γ-globin gene through competitive binding of the Oct-1 transcription factor, resulting in activation of the γ-globin genes. Therefore, disrupting the bindings of the Oct-1 transcriptional factors with the decoy oligonucleotide provides a novel approach for inducing expression of the γ-globin genes. It also provides an innovative strategy for the treatment of many disease conditions, including sickle cell anemia and β-thalassemia.
Highlights
Human β-hemoglobin disorders, such as sickle cell anemia and β-thalassemia, are relatively common genetic diseases that affect millions of people worldwide
We have studied the possibility of using a decoy oligonucleotide targeting the Oct-1 transcription factor to induce expression of the endogenous γ-globin gene
The results obtained from our Oct-1 decoy oligonucleotide treatment studies demonstrate that the treatment of the K562 cells with the Oct-1 decoy oligonucleotide results in an increase in transcriptions of the γ-globin genes and an increase in accumulation of the HbF in the K562 cells
Summary
Human β-hemoglobin disorders, such as sickle cell anemia and β-thalassemia, are relatively common genetic diseases that affect millions of people worldwide. It is well documented that these diseases are caused by mutations in the β-globin gene: a T-to-A mutation at the sixth amino acid codons of the β-globin gene causes sickle cell anemia and various deletions that occur (page number not for citation purposes). When γglobin genes are highly expressed, the presence of high levels of fetal hemoglobin (HbF, α2γ2) in erythrocytes (~20–30%) can compensate for the defective β-globin product and significantly reduce disease symptoms [1]. The γ-globin genes are developmentally regulated and normally expressed at high levels only during the fetal stage of human development [2,3,4,5]. Several strategies have been developed to induce expression of γ-globin genes for the treatment of sickle cell anemia and β-thalassemia [69]. New strategies still need to be developed so that more effective treatments can be provided for these patients
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