Abstract
The mammalian embryonic zeta-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 zeta-globin gene transcription is probably regulated by the cooperative work of various protein-DNA and protein-protein complexes formed at the zeta-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 zeta-promoter activity in erythroid cell lines and in transgenic mice. Combined site-directed mutagenesis and EMSA suggest that repression of the human zeta-globin promoter is mediated through binding of the zinc finger factor RREB1 to ZF2. This model is further supported by the observation that human zeta-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 zeta-globin genes during erythroid development. Because zeta-globin is a powerful inhibitor of HbS polymerization, our experiments have provided a foundation for therapeutic up-regulation of zeta-globin gene expression in patients with severe hemoglobinopathies.
Highlights
The human ␣-like (5Ј--␣2-␣1-1-3Ј) and -like (5Ј-⑀-G␥-A␥-␦--3Ј) globin gene clusters each extend over 50 kb on chromosomes 16 and 11, respectively
We present data here indicating that a protein-binding motif, ZF2, contributes to the repression of the HS-40-regulated human -promoter activity in erythroid cell lines and in transgenic mice
To examine the contributions of the putative GATA-1 and RREB1 binding sites within the ZF2 motif to the -globin promoter activity, we introduced three different types of mutations into the ZF2 motif of the promoter
Summary
Plasmids—The construct pBS-HS40--hGH described previously was used in the current study but with replacement of the backbone with that of pBluescript II KS(Ϫ) (Stratagene) [21]. This new pBS-HS40--hGH plasmid was used as the parental plasmid to introduce different mutations into the -globin promoter with the use of the QuikChange site-directed mutagenesis kit from Stratagene. MEL, HeLa, and 293T cells were cultured in the same conditions but in Dulbecco’s modified Eagle’s medium (Invitrogen). The MEL cells were seeded with 5 ml of antibiotic-free Dulbecco’s modified Eagle’s medium for 24 h and induced with 2% DMSO for 96 h before the hGH assay. The 293T and HeLa cells were seeded in 6-well plates with 5 ml of antibiotic-free Dulbecco’s modified Eagle’s medium for 48 h before the hGH assay.
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