Developmental silencing of human ζ-globin gene expression is mediated by the transcriptional repressor RREB1

The mammalian embryonic ζ-globin genes, including that of humans, are expressed at the early embryonic stage and then switched off during erythroid development. This autonomous silencing of the ζ-globin gene transcription is probably regulated by the cooperative work of various protein-DNA and protein-protein complexes formed at the ζ-globin promoter and its upstream enhancer (HS-40). We present data here indicating that a protein-binding motif, ZF2, contributes to the repression of the HS-40-regulated human ζ-promoter activity in erythroid cell lines and in transgenic mice. Combined site-directed mutagenesis and EMSA suggest that repression of the human ζ-globin promoter is mediated through binding of the zinc finger factor RREB1 to ZF2. This model is further supported by the observation that human ζ-globin gene transcription is elevated in the human erythroid K562 cell line or the primary erythroid culture upon RNA interference (RNAi)² knockdown of RREB1 expression. These data together suggest that RREB1 is a putative repressor for the silencing of the mammalian ζ-globin genes during erythroid development. Because ζ-globin is a powerful inhibitor of HbS polymerization, our experiments have provided a foundation for therapeutic up-regulation of ζ-globin gene expression in patients with severe hemoglobinopathies.