Zoueva Olga P, Rodgers Griffin P
Molecular and Clinical Hematology Branch, National Institute of Diabetes & Digestive & Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Exp Hematol. 2004 Aug;32(8):700-8. doi: 10.1016/j.exphem.2004.05.024.
In this paper, we report new observations related to the mechanism of the negative regulation of the important adult beta-globin gene in the erythroid cells at the embryonic-fetal stage of their development. We focused on the role of the silencer II region located upstream of the beta-globin gene, which along with its cognate binding protein BP1, negatively regulates beta-globin transcription.
We prepared plasmid constructs containing the wild-type silencer II sequence, a mutated silencer II sequence, or a mutated control sequence in the beta-globin promoter 690-bp insert, which in turn was linked to an enhanced green fluorescent protein (EGFP) reporter gene. A human erythroleukemia cell line (K562) with embryonic-fetal phenotype was transfected with these EGFP constructs.
Flow cytometry and fluorescence digital imaging showed about threefold increase in the beta-globin promoter activity of the mutated silencer II construct. Introduction of a small interfering RNA (siRNA) complementary to BP1 into the cells caused a 75% decrease in BP1 expression and a simultaneous approximately 40% elevation of beta-globin promoter activity as well as an increase in beta-globin mRNA levels, as compared with controls. We detected no changes in the mRNA levels of positive regulators of hemoglobin transcription such as EKLF and GATA-1.
Our results support the involvement of BP1 in the mechanism of the negative regulation of beta-globin transcription. A better understanding of this mechanism may lay the groundwork for novel gene therapy approaches to inhibit the expression of abnormal structural variants of adult beta globin, such as sickle hemoglobin.
在本文中，我们报告了与重要的成人β-珠蛋白基因在红系细胞胚胎-胎儿发育阶段负调控机制相关的新观察结果。我们重点关注了位于β-珠蛋白基因上游的沉默子II区域的作用，该区域与其同源结合蛋白BP1一起对β-珠蛋白转录进行负调控。
我们制备了在β-珠蛋白启动子690 bp插入片段中包含野生型沉默子II序列、突变的沉默子II序列或突变的对照序列的质粒构建体，该插入片段又与增强型绿色荧光蛋白（EGFP）报告基因相连。用这些EGFP构建体转染具有胚胎-胎儿表型的人红白血病细胞系（K562）。
流式细胞术和荧光数字成像显示，突变的沉默子II构建体的β-珠蛋白启动子活性增加了约三倍。与对照相比，将与BP1互补的小干扰RNA（siRNA）导入细胞导致BP1表达降低75%，同时β-珠蛋白启动子活性约升高40%，β-珠蛋白mRNA水平也增加。我们未检测到血红蛋白转录正调控因子如EKLF和GATA-1的mRNA水平有变化。
我们的结果支持BP1参与β-珠蛋白转录负调控机制。更好地理解这一机制可能为抑制成人β珠蛋白异常结构变体（如镰状血红蛋白）表达的新型基因治疗方法奠定基础。
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