The β-hemoglobinopathies sickle cell disease and β-thalassemia are among the most common human genetic disorders worldwide. Hemoglobin A2 (HbA2, α2δ2) and fetal hemoglobin (HbF, α2γ2) both inhibit the polymerization of hemoglobin S that results in erythrocyte sickling. Expression of erythroid Kruppel-like factor (EKLF) and GATA1 is critical for transitioning hemoglobin from HbF to hemoglobin A (HbA, α2β2) and HbA2. The lower levels of δ-globin expression compared with β-globin expression seen in adulthood are likely due to the absence of an EKLF-binding motif in the δ-globin proximal promoter. In an effort to upregulate δ-globin to increase HbA2 expression, we created a series of EKLF-GATAl fusion constructs composed of the transactivation domain of EKLF and the DNA-binding domain of GATAl and then tested their effects on hemoglobin expression. EKLF-GATAl fusion proteins activated δ-, γ-, and β-globin promoters in K562 cells, and significantly upregulated δ- and γ-globin RNA transcripts and proteins expression in K562. We found that the expression of long-form EKLF-GATA1 increased δ-, γ-, and β-globin promoter activity 1.7-, 2.2-, and 6.8-fold, respectively, at 24 hours after transfection, and 5.4-, 2.9-, and 9.4-fold, respectively, at 48 hours after transfection (when compared with mock transfection). The effect of medium-form EKLF-GATA1 expression on globin promoter activity was less profound than that of long-form EKLF-GATA1, with a 1.9-fold increase for γ-globin promoter activity at 24 hours and a 2.5- and 3.2-fold increase for δ- and γ-globin promoter activity, respectively, at 48 hours. Both the short-form EKLF-GATA1 and vector only had no appreciable effect on globin promoter activity after 24 or 48 hours. GATA1 expression increased δ-globin promoter activity approximately 2-fold at 24 hours and 4.3-fold at 48 hours;EKLF induced β-globin promoter activity approximately 2-fold at both 24 and 48 hours. These results indicate the long- and medium-form of EKLF-GATA1 fusion proteins, which contain the N-finger and C-finger of the GATA1-binding domain, may well bind to and activate the δ-globin promoter. In contrast, the short-form of EKLF-GATA1 fusion protein, which lacked the intact C-finger, was not able to bind to the δ-globin promoter and thus had no impact on globin expression. In CD34+ cells, tThe long-form EKLF-GATA1 upregulated β-globin expression 1.7-fold, δ-globin gene expression 2.7-fold, and γ-globin gene expression 1.9-fold. The medium-form EKLF-GATA1 upregulated δ-globin gene expression 2.2-fold and γ-globin 1.3-fold, but had no effect on β-globin gene expression. We also observed that EKLF only-transduced CD34+ cells expressed 1.4-fold higher levels of β-globin expression, and GATA1 only-transduced cells expressed 1.5-fold higher levels of δ-globin and 1.3-fold higher levels of β-globin. In contrast, the short-form of EKLF-GATA1 had no significant effect on globin expression. The results of gene expression were confirmed in both K562 and CD34+ cells, in Western Blot analysis. The binding of EKLF-GATA1 fusion proteins at the GATA1 consensus site in the δ-globin promoter was confirmed by chromatin immunoprecipitation assay. In summary, we present two functional EKLF-GATA1 fusion proteins containing the GATA1 primary binding domain that could bind to and activate δ-globin promoter and significantly increase δ-globin expression in K562 cells and CD34+ bone marrow cells. Although the long-form EKLF-GATA1 fusion protein also increased β-globin expression in CD34+ cells, its major effects were on δ- and γ-globin induction;the medium-form EKLF-GATA1 elevated δ- and γ-globin expression without an effect on β-globin expression. Induction of both δ- and γ-globin expression may be beneficial for an antisickling effect and compensating for impaired β-globin production. These EKLF-GATA1 fusion proteins could prove useful as a genetic therapeutic tool for SCD and β-thalassemia, and warrant further preclinical evaluation in vivo.

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National Heart, Lung, and Blood Institute
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Zhu, Jianqiong; Chin, Kyung; Aerbajinai, Wulin et al. (2011) Recombinant erythroid Kruppel-like factor fused to GATA1 up-regulates delta- and gamma-globin expression in erythroid cells. Blood 117:3045-52
Vichinsky, Elliott; Rodgers, Griffin P; Rachmilewitz, Eliezer (2008) Hemoglobinopathies. Curr Mol Med 8:591
Kumkhaek, Chutima; Taylor 6th, James G; Zhu, Jianqiong et al. (2008) Fetal haemoglobin response to hydroxycarbamide treatment and sar1a promoter polymorphisms in sickle cell anaemia. Br J Haematol 141:254-9