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 contrasts with the exclusively embryonic expression pattern of the gamma-like genes of other eutherian mammals. To elucidate the factors responsible for this change in expression pattern, we utilized a strategy called differential phylogenetic footprinting (DPF). This strategy entails the following: (a) identification, within regulatory regions, of the gamma promoter, of individual nucleotides that differ between human (fetal expression), and galago (embryonic expression) gamma genes, (b) analysis 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 assays. The DPF analysis revealed several proteins that bind upstream from the CCAAT motif in the galago gamma promoter but do not bind to the corresponding region of the human gamma promoter. In transfection assays, binding of these proteins is associated with erythroid-specific repression of promoter strength. Binding sites for these proteins also occur near the CCAAT box of other embryonically expressed genes, including rabbit, mouse, and dwarf lemur gamma genes and the human epsilon globin gene. These data are consistent with the hypothesis that sequence changes near the proximal CCAAT box in the ancestral simian gamma gene may have facilitated a novel expression pattern by reducing the binding of repressors that act in the fetal stage.