The human epsilon-globin gene is transcribed in erythroid cells only during the embryonic stages of development. The developmental control of epsilon-globin gene as previously shown, is mediated in part by a transcriptional silencer characterized as the e-globin gene silencer motif (epsilonSm) located at -278 to -257 bp 5' to the cap site. It has been shown that epsilonGs inhibits transcription slightly in embryonic erythroid cells but strongly in human adult erythroid or nonerythroid cells. Within the epsilonGS silencer two overlapping motifs for transcription factors YY1 and GATA-1 have been identified by DNA-binding assays and directed mutagenesis. We expanded the study of epsilonGSm in animals models and showed that mutation in the GATA-1/YY1 sites within eGSm is sufficient to cause expression of the human epsilon-globin gene in transgenic mice. For a further understanding of the role of transcription factor GATA-1 on epsilon- silencing, we have studied the effect of GATA-1 on the epsilon- globin gene expression. We have transiently transfected embryonic erythroid K562 cells with increasing amounts of a expression vector for the human transcription factor GATA-1. We have observed that increasing amounts of GATA-1 transcripts have a negative effect on the level of expression of endogenous epsilon-globin gene, while there is no effect or little in other globin genes. We have also observed that the level of expression of transcription factor GATA-2 decreases as the expression of GATA-1 increases. Those findings suggest that GATA-1 might mediate transcription activation of epsilon-globin gene at low concentrations and transcription repression at high concentrations. Experiments are in progress to create by stable transfections K562 cell lines constitutively expressing high levels of GATA-1 and GATA-2. Stable transfected K562 cells will provide an interesting model to understand how quantitative changes in transcription factors can result in qualitative alteration in target gene expression.
