POSITIVE AND NEGATIVE REGULATORY ELEMENTS OF THE RABBIT EMBRYONIC EPSILON-GLOBIN GENE REVEALED BY AN IMPROVED MULTIPLE ALIGNMENT PROGRAM AND FUNCTIONAL-ANALYSIS

The epsilon-globin genes of mammals are expressed in early embryos, bu t are silenced during fetal and adult erythropoiesis. As a guide to de fining the regulatory elements involved in this developmental switch, we have searched the sequences of epsilon-globin genes from different mammals for highly conserved segments. The search was facilitated by t he development of a new program, called yama, to generate a multiple a lignment of very long sequences using an improved scoring scheme. This allowed us to generate a multiple alignment of sequences from a more divergent group than previously analyzed, as demonstrated here for rep resentatives of four mammalian orders. In parallel experiments, we con structed a series of deletion mutations in the 5' flank of the rabbit epsilon-globin gene and tested their effect on an epsilon-globin-lucif erase hybrid reporter gene. These results show that 121 bp of 5' flank , containing CACC, CCAAT and ATA motifs, is sufficient for expression in erythroid K562 cells. Both positive and negative cis-acting control sequences are located between 218 and 394 bp 5' to the cap site, in a region previously proposed to be a silencer. The positive regulatory sequence contains conserved binding sites for the nuclear protein YY1 adjacent to another highly conserved sequence. The negative element co ntains a conserved sequence followed by a purine-rich segment. This an alysis maps the upstream control sequences more precisely and points t o a very complex regulatory scheme for this gene.