OBJECTIVETo further characterize the differentiation inducing properties of EDRF1 and demonstrate its functional pathway involved in regulation of globin gene expression.

METHODSBy 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.

RESULTSIt 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.

CONCLUSIONThe results suggested that EDRF1 regulated alpha- and gamma-globin gene synthesis by modulating DNA-binding activity of GATA-1 transcription factor.