PHYLOGENETIC FOOTPRINTING REVEALS UNEXPECTED COMPLEXITY IN TRANS FACTOR-BINDING UPSTREAM FROM THE EPSILON-GLOBIN GENE

The human epsilon-globin gene undergoes dramatic changes in transcript ional activity during development, but the molecular factors that cont rol its high expression in the embryo and its complete repression at 6 -8 weeks of gestation are unknown. Although a putative silencer has be en identified, the action of this silencer appears to be necessary but not sufficient for complete repression of epsilon gene expression, su ggesting that multiple control elements may be required. Phylogenetic footprinting is a strategy that uses evolution to aid in the elucidati on of these multiple control points. The strategy is based on the obse rvation that the characteristic developmental expression pattern of th e epsilon gene is conserved in all placental mammals. By aligning epsi lon genomic sequences (from -2.0 kb upstream to the epsilon polyadenyl ylation signal), conserved sequence elements that are likely binding s ites for trans factors can be identified against the background of neu tral DNA. Twenty-one such conserved elements (phylogenetic footprints) were found upstream of the epsilon gene. Oligonucleotides spanning th ese conserved elements were used in a gel-shift assay to reveal 47 nuc lear binding sites. Among these were 8 binding sites for YY1 (yin and yang 1), a protein with dual (activator or repressor) activity; 5 bind ing sites for the putative stage selector protein, SSP; and 7 binding sites for an as yet unidentified protein. The large number of high-aff inity interactions detected in this analysis further supports the noti on that the epsilon gene is regulated by multiple redundant elements.