DIFFERENTIAL PHYLOGENETIC FOOTPRINTING AS A MEANS TO IDENTIFY BASE CHANGES RESPONSIBLE FOR RECRUITMENT OF THE ANTHROPOID GAMMA-GENE TO A FETAL EXPRESSION PATTERN

Expression of the anthropoid (simian) gamma gene in fetal life contras ts with the exclusively embryonic expression pattern of the gamma-like genes of other eutherian mammals. To elucidate the factors responsibl e for this change in expression pattern, we utilized a strategy called differential phylogenetic footprinting (DPF). This strategy entails t he following: (a) identification, within regulatory regions, of the ga mma promoter, of individual nucleotides that differ between human (fet al expression), and galago (embryonic expression) gamma genes, (b) ana lysis of the effect of these nucleotide differences on the binding of nuclear proteins to human and galago sequences, and (c) assessment of the functional consequences of these binding changes in expression ass ays. The DPF analysis revealed several proteins that bind upstream fro m the CCAAT motif in the galago gamma promoter but do not bind to the corresponding region of the human gamma promoter. In transfection assa ys, binding of these proteins is associated with erythroid-specific re pression of promoter strength. Binding sites for these proteins also o ccur near the CCAAT box of other embryonically expressed genes, includ ing rabbit, mouse, and dwarf lemur gamma genes and the human epsilon g lobin gene. These data are consistent with the hypothesis that sequenc e changes near the proximal CCAAT box in the ancestral simian gamma ge ne may have facilitated a novel expression pattern by reducing the bin ding of repressors that act in the fetal stage.