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.

血红蛋白病、镰状细胞病和地中海贫血是世界范围内最常见的人类遗传性疾病。血红蛋白A2（HbA 2，2 2）和胎儿血红蛋白（HbF，2 2）均能抑制血红蛋白S的聚合而导致红细胞镰状化。红系Kruppel样因子（EKLF）和GATA 1的表达对于将血红蛋白从HbF转变为血红蛋白A（HbA，2 2）和HbA 2至关重要。较低水平的-珠蛋白的表达与-珠蛋白的表达在成年期看到的可能是由于缺乏一个EKLF结合基序在-珠蛋白近端启动子。为了上调β-珠蛋白以增加HbA 2表达，我们创建了一系列由EKLF的反式激活结构域和GATA 1的DNA结合结构域组成的EKLF-GATA 1融合构建体，然后测试它们对血红蛋白表达的影响。EKLF-GATA 1融合蛋白激活K562细胞中的-、-和-珠蛋白启动子，并显著上调K562细胞中的-和-珠蛋白RNA转录和蛋白表达。我们发现长型EKLF-GATA 1的表达在转染后24小时分别增加了-、-和-珠蛋白启动子活性的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）。中等形式EKLF-GATA 1表达对珠蛋白启动子活性的影响不如长形式EKLF-GATA 1深刻，在24小时时β-珠蛋白启动子活性增加1.9倍，在48小时时β-珠蛋白和β-珠蛋白启动子活性分别增加2.5倍和3.2倍。在24或48小时后，短形式EKLF-GATA 1和载体仅对珠蛋白启动子活性没有明显影响。GATA 1表达增加β-珠蛋白启动子活性约2倍，在24小时和4.3倍，在48小时; EKLF诱导β-珠蛋白启动子活性约2倍，在24和48小时。这些结果表明，长和中等形式的EKLF-GATA 1融合蛋白，其中包含的GATA 1结合域的N-指和C-指，可以很好地结合和激活的-珠蛋白启动子。相反，缺乏完整C指的EKLF-GATA 1融合蛋白的短形式不能结合珠蛋白启动子，因此对珠蛋白表达没有影响。在CD 34+细胞中，长型EKLF-GATA 1上调β-珠蛋白表达1.7倍，β-珠蛋白基因表达2.7倍，β-珠蛋白基因表达1.9倍。中等形式的EKLF-GATA 1上调-珠蛋白基因表达2.2倍和-珠蛋白1.3倍，但对-珠蛋白基因表达没有影响。我们还观察到，仅EKLF转导的CD 34+细胞表达1.4倍高水平的β-珠蛋白表达，仅GATA 1转导的细胞表达1.5倍高水平的β-珠蛋白和1.3倍高水平的β-珠蛋白。相反，EKLF-GATA 1的短形式对珠蛋白表达没有显著影响。 Western Blot分析证实了K562和CD 34+细胞中基因表达的结果。通过染色质免疫沉淀试验证实了EKLF-GATA 1融合蛋白在-珠蛋白启动子中GATA 1共有位点的结合。总之，我们提出了两个功能性EKLF-GATA 1融合蛋白含有GATA 1的主要结合结构域，可以结合并激活-珠蛋白启动子，并显着增加-珠蛋白在K562细胞和CD 34+骨髓细胞中的表达。虽然长型EKLF-GATA 1融合蛋白也增加了CD 34+细胞中的β-珠蛋白表达，但其主要作用是对β-珠蛋白和β-珠蛋白的诱导;中型EKLF-GATA 1升高了β-珠蛋白和β-珠蛋白的表达，而对β-珠蛋白的表达没有影响。诱导β-珠蛋白和β-珠蛋白表达可能有利于抗镰状效应和补偿受损的β-珠蛋白产生。这些EKLF-GATA 1融合蛋白可能被证明是有用的SCD和地中海贫血的遗传治疗工具，并保证进一步的临床前体内评价。