Wang Duncheng, Li Yan, Shen Beifen
Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
Chin Med J (Engl). 2002 Nov;115(11):1701-5.
To further characterize the differentiation inducing properties of EDRF1 and demonstrate its functional pathway involved in regulation of globin gene expression.
By transfecting EDRF1 sense and antisense constructs into HEL cells, we identified the expression of globin and erythropoietin receptor genes by Northern blot analysis. RT-PCR and EMSA (electrophoresis mobility shift assay) were performed to monitor the expression and DNA-binding activity of erythroid specific transcription factors GATA-1 and NF-E2.
It was shown that when EDRF1 was overexpressed, production of alpha-globin increased. In antisense EDRF1, overexpression of HEL cells, significant loss of alpha-, gamma-globin mRNA synthesis was observed. The transcription of endogenous GATA-1 and NF-E2 mRNA expression were maintained at the same levels compared with control experiments. However, the transcription activity of GATA-1 was severely impaired. Expression of erythropoietin receptor gene was not influenced by EDRF1 gene overexpression.
The results suggested that EDRF1 regulated alpha- and gamma-globin gene synthesis by modulating DNA-binding activity of GATA-1 transcription factor.
进一步表征内皮舒张因子1（EDRF1）的分化诱导特性，并证明其参与珠蛋白基因表达调控的功能途径。
通过将EDRF1正义和反义构建体转染到人红白血病细胞（HEL细胞）中，我们通过Northern印迹分析鉴定珠蛋白和促红细胞生成素受体基因的表达。进行逆转录聚合酶链反应（RT-PCR）和电泳迁移率变动分析（EMSA）以监测红系特异性转录因子GATA-1和NF-E2的表达及DNA结合活性。
结果显示，当EDRF1过表达时，α-珠蛋白的产量增加。在反义EDRF1过表达的HEL细胞中，观察到α-、γ-珠蛋白mRNA合成显著减少。与对照实验相比，内源性GATA-1和NF-E2 mRNA表达的转录维持在相同水平。然而，GATA-1的转录活性严重受损。促红细胞生成素受体基因的表达不受EDRF1基因过表达的影响。
结果表明，EDRF1通过调节GATA-1转录因子的DNA结合活性来调控α-和γ-珠蛋白基因的合成。
