NN-acetylglucosamine (GlcNAc) units that are β-O-linked to threonine and serine residues (O-GlcNAc) of proteins is a protein modification found within all multicellular eukaryotes1). Well over a thousand proteins within the nucleus and cytoplasm are known to be modified with O-GlcNAc, yet the core functions of this protein modification remain ill defined. Levels of O-GlcNAc are regulated by the action just two enzymes, which allow this modification to be dynamically regulated on target proteins. The glycosyltransferase from GT41 known as O-GlcNAc transferase (OGT) uses uridine diphosphate GlcNAc (UDP-GlcNAc) as a donor sugar to install the O-GlcNAc modification. The glycoside hydrolase from GH84 known as O-GlcNAcase (OGA) acts to remove O-GlcNAc from proteins. These two enzymes are critically important for the developmental of mammals, but only OGT is needed in more simply metazoans such as Drosophila melanogaster. These observations suggest that installation of O-GlcNAc by OGT plays ancient and conserved roles in the developmental programs of metazoans.Within Drosophila the gene super sex combs (sxc) was originally identified as encoding a polycomb group (PcG) protein by virtue of its importance in appropriately silencing the developmentally crucial set of homeotic (Hox) genes. Remarkably, sxc was shown to encode the fly homologue of mammalian OGT2,3). PcG proteins serve to silence gene expression by binding to genomic sequences termed Polycomb group Response Elements (PREs), which are typically found proximal to known targets of PcG proteins, including the Hox genes. Binding of PcG proteins to PREs occurs through the formation of well-defined protein complexes including the Pho repressive complex (PhoRC) which serves to initiate silencing at target loci4). Significantly, various protein modifications of PcG proteins within PhoRC and related silencing complexes influence their stability and ability to binding their interaction partners4). Given the high conservation of OGT within metazoans we were intrigued by the phenotypically clear role of OGT within flies in regulating gene expression2,3).Central to understanding regulation of gene expression by Figure 1. A combined metabolic feeding–chemoselective ligation strategy enables labeling of chromatin-associated proteins from Drosophila S2 cellsa Antibody-free method to enable mapping of b Nuclear proteins from S2 cells in the presence and c Nuclear proteins from S2 cells treated for 16 h with GalNAz PcG proteins has been the ability to map these proteins, along with their post-translationally modified forms, to the genome using chromatin immunoprecipitation followed by sequencing O-GlcNAcylated proteins to the genome. DNA is crosslinked to O-GlcNAcylated proteins that are then ligated to biotin using the chemoselective ligation. Enriched and fragmented DNA is then sequenced. absence (vehicle) of metabolic labeling with Ac4GalNAz (GalNAz), Ac4GlcNAz, and Ac4ManNAz (16 h) were biotinylated by chemoselective ligation. Biotinylated proteins were detected using streptavidin (strvn) blot analysis (upper panel) and Pho antibody (middle panel). or vehicle (DMSO) were biotinylated by chemoselective ligation and detected after streptavidin (strvn) immunoprecipitation by immunoblot. Arrows in the upper panel indicate the size of protein being probed. Figure reproduced from reference7).66In vivo genome wide dynamics of O-GlcNAcylated chromatin-associated proteins brainDavid J. VocadloSimon Fraser University
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