Reduction of DNA binding activity of the GATA-1 transcription factor in the apoptotic process induced by overexpression of PU.1 in Murine erythroleukemia cells

Previously we have shown that overexpression of PU.1, an Ets family transcription factor, in murine erythroleukemia (MEL) cells results in apoptotic cell death in the presence of the differentiation-inducing reagent dimethyl sulfoxide (DMSO). In this study, we examined the dynamics of GATA-1 and NF-E2 hematopoietic transcription factors during the induction of apoptosis, because GATA-1 has been shown to be implicated in survival of erythroid cells. Formation of the GATA- 1-DNA complex as judged by EMSA was markedly reduced when apoptosis was induced, although subcellular localization of the GATA-1 protein and expression levels of the GATA-1 mRNA and protein were not changed during the apoptotic process. Complex formation was not reduced when apoptosis was avoided by adding 30% serum in culture medium and when mutant PU. 1 proteins with the deletion of the DNA-binding (Ets) or transactivation domain were expressed. Complex formation in nuclear extracts of parental MEL cells was reduced when they were mixed with those of apoptotic cells, suggesting that apoptotic cells may contain a factor(s) preventing GATA-1 from binding to DNA. In contrast to GATA-1, formation of the NF-E2-DNA complex was not changed during the process of apoptosis, although the expression level of the NF-E2 p45 gene was reduced in the process. These results suggest that reduction of the DNA-binding activity of GATA-1 may partly account for PU.1-mediated apoptosis in MEL cells.