Undetectable histone O-GlcNAcylation in mammalian cells

Abstract

O-GlcNAcylation is a posttranslational modification catalyzed by the O-Linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and reversed by O-GlcNAcase (OGA). Numerous transcriptional regulators, including chromatin modifying enzymes, transcription factors, and co-factors, are targeted by O-GlcNAcylation, indicating that this modification is central for chromatin-associated processes. Recently, OGT-mediated O-GlcNAcylation was reported to be a novel histone modification, suggesting a potential role in directly coordinating chromatin structure and function. In contrast, using multiple biochemical approaches, we report here that histone O-GlcNAcylation is undetectable in mammalian cells. Conversely, O-GlcNAcylation of the transcription regulators Host Cell Factor-1 (HCF-1) and Ten-Eleven Translocation protein 2 (TET2) could be readily observed. Our study raises questions on the occurrence and abundance of O-GlcNAcylation as a histone modification in mammalian cells and reveals technical complications regarding the detection of genuine protein O-GlcNAcylation. Therefore, the identification of the specific contexts in which histone O-GlcNAcylation might occur is still to be established.

Nutrient regulation of gene expression by O-GlcNAcylation of chromatin

“…Although a recent paper called into question histone O-GlcNAcylation [4], the presence of the sugar on each subunit of the nucleosome has been reported independently by many laboratories and some sites have been mapped (reviewed in [2]). Some of the site-specific functions have been documented (Figure 1).…”

“…As mentioned above, discrepancies and lack of reproducibility regarding Histone O-GlcNAcylation have been observed recently [4]. These issues could arise because investigations have used different models and different techniques to assess histone O-GlcNAcylation.…”

“…As different studies have suggested, some of if not all histone O-GlcNAcylation could be cell type specific, highly dynamic and cell cycle stage specific and/or restricted to a specific developmental stage [5,6,7,11]. Although the H2B S112O-GlcNAc site-specific antibody is commercially available, some concerns have been raised regarding its specificity [2,4]. Re-analysis of five randomly picked loci in the top 20 hits of the H2B S112-O-GlcNAc ChIP-seq data from Fujiki et al, using this antibody by Gambetta et al, has pointed out that, except for the locus of GSK3B , the signal over background of theses specific loci is low and might not be of sufficient quality to justify the authors’ conclusions.…”

Drosophila O-GlcNAcase Deletion Globally Perturbs Chromatin O-GlcNAcylation

“…First, OGT can directly O-GlcNAcylate chromatin remodelers like Sin3A and SET1DA (14,17). Although controversial, OGT is argued to directly O-GlcNAc modify histone 2B, thereby altering chromatin structure (20,21). Beyond affecting gene expression through directly or indirectly changing chromatin, O-GlcNAc influences transcription by affecting the activity and/or stability of key players including RNA polymerase II (RNA Pol II) and many transcription factors (11,16,(22)(23)(24)(25)(26).…”

Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast

“…We selected examples of orthologous proteins with known O-GlcNAcylation sites and identified O-Man glycosites for more detailed analysis. Although the existence of O-GlcNAc on histones was questioned in a recent study (28), O-GlcNAcylation is believed to be one of several posttranslational modifications (PTMs) that constitute the histone code and regulate histone interactions with DNA and effector proteins, and this PTM has been found on histones H2A, H2B, and H4 (29). It has further been demonstrated that O-GlcNAcylation of human histone H2B in response to glucose levels modulates the transcriptional response by promoting monoubiquitination of lysine residue 120 (30).…”