Antisense EDRF1 gene inhibited GATA-1 transcription factor DNA-binding activity in K562 cells

Our previous studies showed that EDRF1 influenced expression of a-globin mRNA and synthesis of hemoglobin in K562 cells and modulated self-renewal of K562 cells. To illuminate the function of EDRF1 in K562 cells, sense and antisense EDRF1 constructs were prepared and transfected into K562 cells. By using microarray and dot blot assay, 60 cytokine receptors and some oncogenes sharing important functions in cell proliferation and differentiation were investigated. The results of this study demonstrated that IL-6 receptor, GM-CSF receptor, c-Jun/c-Fos, c-Myc and c-kit genes were regulated by antisense EDRF1 expression. The regulation was confirmed by RNA blot assay. GATA-1 mRNA expression was modulated by EDRF1 gene transfection. Electrophoretic mobility shift assay suggested that the DNA-binding activity of GATA-1 was remarkably inhibited in K562 cells expressing EDRF1 antisense gene. DNA binding activity of NF-E2 was at the same level as control experiment. Therefore EDRF1 may play a role in erythroid pro     

Antisense EDRF1 gene inhibited GATA-1 transcription factor DNA-binding activity in K562 cells

Our previous studies showed that EDRF1 influenced expression of α-globin mRNA and synthesis of hemoglobin in K562 cells and modulated self-renewal of K562 cells. To illuminate the function of EDRF1 in K562 cells, sense and antisense EDRF1 constructs were prepared and transfected into K562 cells. By using microarray and dot blot assay, 60 cytokine receptors and some oncogenes sharing important functions in cell proliferation and differentiation were investigated. The results of this study demonstrated that IL-6 receptor, GM-CSF receptor, c-Jun/c-Fos, c-Myc and c-kit genes were regulated by antisense EDRF1 expression. The regulation was confirmed by RNA blot assay. GATA-1 mRNA expression was modulated by EDRF1 gene transfection. Electrophoretic mobility shift assay suggested that the DNA-binding activity of GATA-1 was remarkably inhibited in K562 cells expressing EDRF1 antisense gene. DNA binding activity of NF-E2 was at the same level as control experiment. Therefore EDRF1 may play a role in erythroid proliferation and differentiation by affecting the interaction between GATA-1 and its cis-elements.     

Phosphorylation of GATA-1 increases its DNA-binding affinity and is correlated with induction of human K562 erythroleukaemia cells.

We have investigated by electrophoretic mobility shift assay (EMSA) the level of GATA-1 DNA-binding activity in nuclear extracts prepared from the human erythroleukaemic cell line, K562, after erythroid induction by hemin, sodium butyrate (NaB) or Trichostatin A or treatment with N -acetylcysteine (NAC). Relative to extract from untreated cells, GATA-1 binding activity increased markedly in all cases. However, immunoblot analysis revealed unchanged levels of GATA-1 protein after induction. Incubation of induced but not uninduced K562 extracts with phosphatase prior to EMSA weakened the binding activity, suggesting that the increase in GATA-1 binding following induction of K562 cells was a consequence of phosphorylation. When the mouse erythroleukaemic cell line MEL was induced with dimethylsulphoxide (DMSO), NaB or NAC, GATA-1 binding activity fell with DMSO, rose significantly with NaB and remained at about the same level in NAC-induced cells. In this case immunoblotting revealed that GATA-1 protein levels were in accord with the EMSA data. The DNA-binding activities of induced and uninduced MEL cell nuclear extracts were decreased by incubation with phosphatase, showing that phosphoryl-ation and DNA binding of GATA-1 are already optimalin these cells. The DNA-binding activity of affinity-purified GATA-1 from MEL cells was also reduced by phosphatase treatment, showing that phosphorylation/dephosphorylation is directly affecting the factor. Furthermore, when a comparison was made by EMSA of nuclear extracts prepared from K562 and MEL cells untreated or incubated with okadaic acid, a phosphatase inhibitor, GATA-1 binding was seen to increase with K562 cells, whereas with MEL cells there was no change in GATA-1 binding. Overall the results suggest that the level of GATA-1 phosphorylation increases after the induction of K562, but not MEL cells, where GATA-1 is already highly phosphorylated. Furthermore, phosphorylation increases the binding affinity of GATA-1 for a canonical binding site.