Abstract
One mode of γ-globin gene silencing involves a GATA-1·FOG-1·Mi2β repressor complex that binds to the -566 GATA site relative to the (A)γ-globin gene cap site. However, the mechanism of how this repressor complex is assembled at the -566 GATA site is unknown. In this study, we demonstrate that the O-linked N-acetylglucosamine (O-GlcNAc) processing enzymes, O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA), interact with the (A)γ-globin promoter at the -566 GATA repressor site; however, mutation of the GATA site to GAGA significantly reduces OGT and OGA promoter interactions in β-globin locus yeast artificial chromosome (β-YAC) bone marrow cells. When WT β-YAC bone marrow cells are treated with the OGA inhibitor Thiamet-G, the occupancy of OGT, OGA, and Mi2β at the (A)γ-globin promoter is increased. In addition, OGT and Mi2β recruitment is increased at the (A)γ-globin promoter when γ-globin becomes repressed in postconception day E18 human β-YAC transgenic mouse fetal liver. Furthermore, we show that Mi2β is modified with O-GlcNAc, and both OGT and OGA interact with Mi2β, GATA-1, and FOG-1. Taken together, our data suggest that O-GlcNAcylation is a novel mechanism of γ-globin gene regulation mediated by modulating the assembly of the GATA-1·FOG-1·Mi2β repressor complex at the -566 GATA motif within the promoter.
Highlights
NIGMS under Grant P20GM104936 and NIDDK Grant R01DK100595
We asked the question whether O-GlcNAcylation plays a role in organizing the A␥-globin promoter. We demonstrate that both OGT and OGA interact with the A␥-globin promoter in both immortalized -YAC bone marrow cells (BMCs) and in -YAC transgenic mouse fetal liver
Using WT and Ϫ566 mutant chemical inducer of dimerization (CID)-dependent -YAC BMCs, we demonstrated that both OGT and OGA interact with the A␥-globin promoter (Fig. 1) at the Ϫ566 GATA site occupied by the GATA-11⁄7FOG-11⁄7Mi2 repressor complex when ␥-globin is silenced
Summary
NIGMS under Grant P20GM104936 and NIDDK Grant R01DK100595 Krüppel-like factor 1 (KLF1), required for -globin gene activation, stimulates BCL11A expression, which in turn represses ␥-globin expression [4, 5, 8] Both BCL11A and KLF1 are positively regulated by binding of Mi2 (chromodomain helicase DNAbinding protein 4), a component of the NuRD complex [9]. We previously demonstrated another modality of fetal globin repression using human -globin locus yeast artificial chromo-. The rate of O-GlcNAc cycling (the addition and removal of O-GlcNAc) affects cell cycle progression, mitotic signaling and spindle formation, and cellular respiration [16, 18,19,20,21,22] Both OGT and OGA are essential for cellular function. Disruption of O-GlcNAcylation contributes to the development of diseases including cancer, diabetes, and Alzheimer disease [18, 19, 25,26,27]
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