Multiple regulatory elements in the 5′-flanking sequence of the human ε-globin gene

We have previously reported, on the basis of transfection experiments, the existence of a silencer element in the 5'-flanking region of the human embryonic (epsilon) globin gene, located at -270 base pairs 5' to the cap site, which provides negative regulation for this gene. Experiments in transgenic mice suggest the physiological importance of this epsilon-globin silencer, but also suggest that downregulation of epsilon-globin gene expression may involve other negative elements flanking the epsilon-globin gene. We have now extended the analysis of epsilon-globin gene regulation to include the flanking region spanning up to 6 kilobase pairs 5' to the locus control region using reporter gene constructs with deletion mutations and transient transfection assays. We have identified and characterized other strong negative regulatory regions, as well as several positive regions that affect transcription activation. The negative regulatory regions at -3 kilobase pairs (epsilon NRA-I and epsilon NRA-II), flanked by a positive control element, has a strong effect on the epsilon-globin promoter both in erythroid K562 and nonerythroid HeLa cells and contains several binding sites for transcription factor GATA-1, as evidenced from DNA-protein binding assays. The GATA-1 sites within epsilon NRA-II are directly needed for negative control. Both epsilon NRA-I and epsilon NRA-II are active on a heterologous promoter and hence appear to act as transcription silencers. Another negative control region located at -1.7 kilobase pairs (epsilon NRB) does not exhibit general silencer activity as epsilon NRB does not affect transcription activity when used in conjunction with an epsilon-globin minimal promoter, The negative effect of epsilon NRB is erythroid specific, but not stage-specific as it can repress transcription activity in both K562 erythroid cells as well as in primary cultures of adult erythroid cells. Phylogenetic DNA sequence comparisons with other primate and other mammalian species show unusual degree of flanking sequence homology for the epsilon-globin gene, including in several of the regions identified in these functional and DNA-protein binding analyses, providing alternate evidence for their potential importance. We suggest that the down-regulation of epsilon-globin gene expression as development progresses involves complex, cooperative interactions of these negative regulatory elements, epsilon NRA-I/epsilon NRA-II, epsilon NRB, the epsilon-globin silencer and probably other negative and positive elements in the 5'-flanking region of the epsilon-globin gene.