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: 8557970
• 负责人: GRIFFIN RODGERS
• 金额: $ 70.74万
• 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/8557970
• 关键词: Adult; Affect; Affinity; Binding; Binding Sites; Biological Assay; Blood; Bone Marrow Cells; Boxing; CD34 gene; Cell membrane; Cells; Characteristics; Chimeric Proteins; Clinical; Consensus; DNA Binding; DNA Binding Domain; DNA Sequencing Facility; DNA-Binding Proteins; Development; Disease; Erythrocytes; Erythroid Cells; Erythropoiesis; Fetal Hemoglobin; Fingers; GATA1 gene; Gene Expression; Genes; Genetic; Globin; Hemoglobin; Hemoglobin A; Hemoglobin A2; Hemoglobinopathies; Hemolytic Anemia; Hereditary Disease; Hour; Human Genetics; K-562; K562 Cells; Lead; Link; MEL Gene; Minor; Mutate; Mutation; Point Mutation; Population; Production; Promoter Regions; Public Health; RNA; Recombinants; Reporting; Series; Severity of illness; Sickle Cell; Sickle Cell Anemia; Sickle Hemoglobin; Site; Testing; Thalassemia; Therapeutic; Transactivation; Transcript; Transfection; Transfusion; Western Blotting; Zinc Fingers; base; cellular transduction; chromatin immunoprecipitation; erythroid Kruppel-like factor; flexibility; global health; hemoglobin A2' ; hydroxyurea; in vivo; mortality; polymerization; preclinical evaluation; prevent; promoter; protein expression; restoration; sickle deoxyhemoglobin; tool; transcription factor; vector

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.

β-血红蛋白病、镰状细胞病和β-地中海贫血是全世界最常见的人类遗传性疾病。血红蛋白 A2（HbA2、α2δ2）和胎儿血红蛋白（HbF、α2γ2）均能抑制血红蛋白 S 的聚合，从而导致红细胞镰状化。红系 Kruppel 样因子 (EKLF) 和 GATA1 的表达对于血红蛋白从 HbF 转变为血红蛋白 A (HbA、α2β2) 和 HbA2 至关重要。与成年期观察到的 β-珠蛋白表达相比，δ-珠蛋白表达水平较低，可能是由于 δ-珠蛋白近端启动子中缺乏 EKLF 结合基序。为了上调δ-珠蛋白以增加HbA2表达，我们创建了一系列由EKLF的反式激活结构域和GATA1的DNA结合结构域组成的EKLF-GATA1融合构建体，然后测试它们对血红蛋白表达的影响。 EKLF-GATA1融合蛋白激活K562细胞中的δ-、γ-和β-球蛋白启动子，并且显着上调K562中的δ-和γ-球蛋白RNA转录物和蛋白质表达。我们发现，长型 EKLF-GATA1 的表达使 δ-、γ- 和 β-珠蛋白启动子活性在转染后 24 小时分别增加 1.7-、2.2- 和 6.8 倍，以及 5.4-、2.9- 倍。转染后 48 小时分别为 、 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 深刻，24 小时时 γ-珠蛋白启动子活性增加了 1.9 倍，2.5 倍和 3.2 倍48 小时时，δ- 和 γ- 珠蛋白启动子活性分别增加。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 表达使 δ-珠蛋白启动子活性在 24 小时时增加约 2 倍，在 48 小时时增加 4.3 倍； EKLF 在 24 小时和 48 小时诱导 β-珠蛋白启动子活性大约增加 2 倍。这些结果表明长型和中型EKLF-GATA1融合蛋白含有GATA1结合结构域的N指和C指，可以很好地结合并激活δ-珠蛋白启动子。相比之下，EKLF-GATA1融合蛋白的短形式缺乏完整的C指，无法与δ-珠蛋白启动子结合，因此对珠蛋白表达没有影响。在 CD34+ 细胞中，长型 EKLF-GATA1 将 β-珠蛋白表达上调 1.7 倍，δ-珠蛋白基因表达上调 2.7 倍，γ-珠蛋白基因表达上调 1.9 倍。中等形式的EKLF-GATA1将δ-珠蛋白基因表达上调2.2倍，将γ-珠蛋白基因表达上调1.3倍，但对β-珠蛋白基因表达没有影响。我们还观察到，仅转导 EKLF 的 CD34+ 细胞的 β-珠蛋白表达水平高出 1.4 倍，仅转导的 GATA1 细胞表达的 δ-珠蛋白水平高出 1.5 倍，β-珠蛋白水平高出 1.3 倍。相反，EKLF-GATA1的短形式对珠蛋白表达没有显着影响。 Western Blot 分析证实了 K562 和 CD34+ 细胞中基因表达的结果。通过染色质免疫沉淀测定证实了 EKLF-GATA1 融合蛋白在 δ-珠蛋白启动子中 GATA1 共有位点的结合。总之，我们提出了两种含有 GATA1 主要结合域的功能性 EKLF-GATA1 融合蛋白，它们可以结合并激活 δ-珠蛋白启动子，并显着增加 K562 细胞和 CD34+ 骨髓细胞中 δ-珠蛋白的表达。尽管长型 EKLF-GATA1 融合蛋白也增加了 CD34+ 细胞中 β-珠蛋白的表达，但其主要作用是对 δ- 和 γ-珠蛋白的诱导。中等形式的 EKLF-GATA1 升高了 δ- 和 γ- 珠蛋白的表达，而不影响 β- 珠蛋白的表达。 δ-和γ-珠蛋白表达的诱导可能有利于抗镰状化作用并补偿受损的β-珠蛋白产生。这些 EKLF-GATA1 融合蛋白可能被证明可用作 SCD 和 β-地中海贫血的遗传治疗工具，并值得进一步的体内临床前评估。