A 3'-flanking NF-kappaB site mediates developmental silencing of the human zeta-globin gene.

The central developmental event in the human (h)alpha-globin gene cluster is selective silencing of the zeta-globin gene as erythropoiesis shifts from primitive erythroblasts in the embryonic yolk sac to definitive erythroblasts in the fetal liver. Previous studies have demonstrated that full developmental silencing of the hzeta-globin gene in transgenic mice requires the proximal 2.1 kb of its 3'-flanking region. In the current report, we localize this silencing activity to a 108 bp segment located 1.2 kb 3' to the zeta-globin gene. Protein(s) in nuclear extracts from cell lines representing the fetal/adult erythroid stage bind specifically to an NF-kappaB motif located at this site. In contrast, this binding activity is lacking in the nuclear extract of an embryonic-stage erythroid line expressing zeta-globin. This complex is quantitatively recognized by antisera to the NF-kappaB p50 and to a lesser extent to p65 subunits. A two-base substitution that disrupts NF-kappaB site protein binding in vitro also results in the loss of the developmental silencing activity in vivo. The data suggest that NF-kappaB complex formation is a crucial component of hzeta-globin gene silencing. This finding expands the roles of this widely distributed transcriptional complex to include negative regulation in mammalian development.