DEREPRESSION OF MOUSE BETA-MAJOR-GLOBIN GENE-TRANSCRIPTION DURING ERYTHROID-DIFFERENTIATION

Functional analysis of the mouse beta-major-globin gene promoter has revealed a negative regulatory element (-100 to -250 bp) which represses promoter activity in mouse erythroleukemia (MEL) cells. Promoter activity is induced 14-fold during terminal differentiation of MEL cells. Three major in vitro binding sites for NF1 (-250 bp), GATA-1 (-212 bp), and a sequence at -165 bp (BB1) have been defined in this region. Site-directed mutagenesis of any one of the three sites resulted in a five- to sixfold up-regulation of promoter activity in uninduced MEL cells, but only three- to fourfold stimulation was observed from the mutant promoters during MEL cell terminal differentiation. This finding suggests that all three sites are required for repressor activity in uninduced MEL cells and that derepression occurs during MEL cell differentiation. BB1 DNA-binding activity decreases during MEL cell differentiation, suggesting a central role for this factor in modulating the effects of the repressor element. The BB1-binding factor also competes with the CCAAT-binding protein for binding the CCAAT motif. The fact that a reduced but significant stimulation of promoter activity during differentiation is observed in the absence of the repressor element raises the possibility that the BB1 factor also down-regulates transcription in undifferentiated MEL cells by displacing binding of CCAAT-binding protein to the proximal CCAAT motif.