Abstract

The monosaccharide, β-N-acetylglucosamine (GlcNAc), can be added to the hydroxyl group of either serines or threonines to generate an O-linked β-N-acetylglucosamine (O-GlcNAc) residue (Love, D. C., and Hanover, J. A. (2005) Sci. STKE 2005 312, 1-14; Hart, G. W., Housley, M. P., and Slawson, C. (2007) Nature 446, 1017-1022). This post-translational protein modification, termed O-GlcNAcylation, is reversible, analogous to phosphorylation, and has been implicated in many cellular processes. Here, we present evidence that in human cells all four core histones of the nucleosome are substrates for this glycosylation in the relative abundance H3, H4/H2B, and H2A. Increasing the intracellular level of UDP-GlcNAc, the nucleotide sugar donor substrate for O-GlcNAcylation enhanced histone O-GlcNAcylation and partially suppressed phosphorylation of histone H3 at serine 10 (H3S10ph). Expression of recombinant H3.3 harboring an S10A mutation abrogated histone H3 O-GlcNAcylation relative to its wild-type version, consistent with H3S10 being a site of histone O-GlcNAcylation (H3S10glc). Moreover, O-GlcNAcylated histones were lost from H3S10ph immunoprecipitates, whereas immunoprecipitation of either H3K4me3 or H3K9me3 (active or inactive histone marks, respectively) resulted in co-immunoprecipitation of O-GlcNAcylated histones. We also examined histone O-GlcNAcylation during cell cycle progression. Histone O-GlcNAcylation is high in G(1) cells, declines throughout the S phase, increases again during late S/early G(2), and persists through late G(2) and mitosis. Thus, O-GlcNAcylation is a novel histone post-translational modification regulating chromatin conformation during transcription and cell cycle progression.

Highlights

  • Because phosphorylation and O-GlcNAcylation may compete with each other for the same amino acid residue

  • Histone O-GlcNAcylation was high in G1, globally decreased during S phase but re-emerged in late S/early G2, and persisted during mitosis. These findings suggest that regulation of histones by O-GlcNAcylation plays a regulatory role in chromosome metabolism during the cell cycle, with histone H3S10 being a major site of O-GlcNAcylation and global histone O-GlcNAcylation being depleted during DNA replication

  • This work presents primary evidence that serine and threonine residues in histone proteins are modified by the posttranslational addition of an O-linked monosaccharide, ␤-Nacetylglucosamine

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Summary

Introduction

Because phosphorylation and O-GlcNAcylation may compete with each other for the same amino acid residue. Increased O-GlcNAcylation of all forms of histone H3 detected on one-dimensional TAU gels using antibody RL2 correlated with decreased phosphorylation of H3 forms on serine 10 (H3S10ph). Increased histone H3 O-GlcNAcylation upon glucosamine treatment correlated with a decreased level of H3K9Ac but an increased level of H3K9me3, which correspond to post-translational modification characteristic of active and repressed chromatin respectively (Fig. 4A).

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