Recombinant erythroid Kruppel-like factor fused to GATA1 upregulates globin expr

与 GATA1 融合的重组红系 Kruppel 样因子上调珠蛋白 expr

• 批准号: 8149535
• 负责人: GRIFFIN RODGERS
• 金额: $ 69.25万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8149535
• 关键词: Binding; CD34 gene; DNA Binding; DNA-Binding Proteins; Disease; Erythroid Cells; GATA1 gene; Globin; Recombinants; chromatin immunoprecipitation; erythroid Kruppel-like factor; prevent; protein expression

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.

血红蛋白病、镰状细胞病和地中海贫血是世界上最常见的人类遗传疾病。血红蛋白A2 （HbA2, α2δ2）和胎儿血红蛋白（HbF, α2γ2）都抑制血红蛋白S的聚合，导致红细胞镰状细胞。红系kruppel样因子（EKLF）和GATA1的表达对于血红蛋白从HbF转化为血红蛋白A （HbA, α2β2）和HbA2至关重要。与成年期的&#948；-globin表达相比，&#946；-globin表达水平较低，这可能是由于&#948；-globin近端启动子中缺乏eklf结合基序。为了上调&#948；-globin以增加HbA2的表达，我们构建了一系列由EKLF的反激活结构域和GATAl的dna结合结构域组成的EKLF-GATAl融合构建体，并测试了它们对血红蛋白表达的影响。EKLF-GATAl融合蛋白激活K562细胞中的&#948；-、&#947；-和&#946；-球蛋白启动子，并显著上调K562细胞中的&#948；-和&#947；-球蛋白RNA转录本和蛋白表达。我们发现，长形EKLF-GATA1的表达在转染后24小时分别增加了&#948；-、&#947；-和&#946；-球蛋白启动子活性1.7倍、2.2倍和6.8倍，在转染后48小时分别增加了5.4倍、2.9倍和9.4倍（与模拟转染相比）。中型EKLF-GATA1表达对珠蛋白启动子活性的影响不如长型EKLF-GATA1表达的影响深远，在24小时内&#947；-珠蛋白启动子活性增加了1.9倍，在48小时内&#948；-和&#947；-珠蛋白启动子活性分别增加了2.5倍和3.2倍。短型EKLF-GATA1和载体仅在24或48小时后对珠蛋白启动子活性没有明显影响。GATA1的表达使&#948；-球蛋白启动子活性在24小时增加约2倍，在48小时增加约4.3倍；EKLF在24小时和48小时诱导了大约2倍的-珠蛋白启动子活性。这些结果表明，含有gata1结合域n指和c指的EKLF-GATA1融合蛋白的长形和中形可能与&#948；-球蛋白启动子结合并激活。相比之下，缺少完整c指的短型EKLF-GATA1融合蛋白无法与&#948；-球蛋白启动子结合，因此对球蛋白表达没有影响。在CD34+细胞中，长链EKLF-GATA1上调&#946；-globin表达1.7倍，&#948；-globin基因表达2.7倍，&#947；-globin基因表达1.9倍。中型EKLF-GATA1上调&#948；-globin基因表达2.2倍，上调&#947；-globin基因表达1.3倍，但对&#946；-globin基因表达无影响。我们还观察到，仅EKLF转导的CD34+细胞表达的&#946；-globin表达水平高出1.4倍，仅GATA1转导的细胞表达的&#948；-globin表达水平高出1.5倍，&#946；-globin表达水平高出1.3倍。而短链EKLF-GATA1对珠蛋白表达无显著影响。Western Blot分析证实，K562和CD34+细胞均表达了上述基因。通过染色质免疫沉淀法证实了EKLF-GATA1融合蛋白在&#948；-球蛋白启动子中GATA1共识位点的结合。综上所述，我们获得了两个功能性的EKLF-GATA1融合蛋白，它们含有GATA1初级结合域，可以结合并激活&#948；-globin启动子，显著增加K562细胞和CD34+骨髓细胞中&#948；-globin的表达。虽然长链EKLF-GATA1融合蛋白也增加了CD34+细胞中的&#946；-globin表达，但其主要作用是诱导&#948；-和&#947；-globin；中等形式的EKLF-GATA1提高了&#948；-和&#947；-globin的表达，但对&#946；-globin的表达没有影响。诱导&#948；-和&#947；-珠蛋白表达可能有利于抗镰状细胞效应，并补偿受损的&#946；这些EKLF-GATA1融合蛋白可以作为SCD和地中海贫血的遗传治疗工具，并需要进一步的体内临床前评估。