Context-dependent EKLF responsiveness defines the developmental specificity of the human ε-globin gene in erythroid cells of YAC transgenic mice

We explored the mechanism of definitive-stage epsilon -globin transcriptional inactivity within a human beta -globin YAC expressed in transgenic mice. We focused on the globin CAC and CAAT promoter motifs, as previous laboratory and clinical studies indicated a pivotal role for these elements in globin gene activation. A high-affinity CAC-binding site for the erythroid kruppel-like factor (EKLF) was placed in the E-globin promoter at a position corresponding to that in the adult beta -globin promoter, thereby simultaneously ablating a direct repeat (DR) element. This mutation led to EKLF-independent epsilon -globin transcription during definitive erythropoiesis. A second 4-bp substitution in the epsilon -globin CAAT sequence, which simultaneously disrupts a second DR element, further enhanced ectopic definitive erythroid activation of epsilon -globin transcription, which surprisingly became EKLF dependent. We finally examined factors in nuclear extracts prepared from embryonic or adult erythroid cells that bound these elements in vitro, and we identified a novel DR-binding protein (DRED) whose properties are consistent with those expected for a definitive-stage epsilon -globin repressor. We conclude that the suppression of epsilon -globin transcription during definitive erythropoiesis is mediated by the binding of a repressor that prevents EKLF from activating the epsilon -globin gene.