A versatile sugar transferase makes the cut.

The nutrient sensor O-GlcNAc transferase modifies proteins with the O-GlcNAc moiety. In this issue, Capotosti et al. (2011) reveal that O-GlcNAc transferase not only glycosylates the cell-cycle regulator host cell factor 1 but activates it through proteolytic cleavage, providing a surprising link between metabolism and epigenetic regulation of the cell cycle.

Cytosolic O-GlcNAcylation and PNG1 maintain Drosophila gut homeostasis by regulating proliferation and apoptosis.

Tissue homeostasis requires a delicate balance between stem cell self-renewal, proliferation, and differentiation. Essential to this process is glycosylation, with both intra-and extra-cellular glycosylation being required for stem cell homeostasis. However, it remains unknown how intracellular glycosylation, O-GlcNAcylation, interfaces with cellular components of the extracellular glycosylation machinery, like the cytosolic N-glycanase NGLY1. In this study, we utilize the Drosophila gut and uncover a pathway in which O-GlcNAcylation cooperates with the NGLY1 homologue PNG1 to regulate proliferation in intestinal stem cells (ISCs) and apoptosis in differentiated enterocytes. Further, the CncC antioxidant signaling pathway and ENGase, an enzyme involved in the processing of free oligosaccharides in the cytosol, interact with O-GlcNAc and PNG1 through regulation of protein aggregates to contribute to gut maintenance. These findings reveal a complex coordinated regulation between O-GlcNAcylation and the cytosolic glycanase PNG1 critical to balancing proliferation and apoptosis to maintain gut homeostasis.

O-GlcNAc transferase plays a non-catalytic role in C. elegans male fertility.

Animal behavior is influenced by the competing drives to maintain energy and to reproduce. The balance between these evolutionary pressures and how nutrient signaling pathways intersect with mating remains unclear. The nutrient sensor O-GlcNAc transferase, which post-translationally modifies intracellular proteins with a single monosaccharide, is responsive to cellular nutrient status and regulates diverse biological processes. Though essential in most metazoans, O-GlcNAc transferase (ogt-1) is dispensable in Caenorhabditis elegans, allowing genetic analysis of its physiological roles. Compared to control, ogt-1 males had a four-fold reduction in mean offspring, with nearly two thirds producing zero progeny. Interestingly, we found that ogt-1 males transferred sperm less often, and virgin males had reduced sperm count. ogt-1 males were also less likely to engage in mate-searching and mate-response behaviors. Surprisingly, we found normal fertility for males with hypodermal expression of ogt-1 and for ogt-1 strains with catalytic-dead mutations. This suggests OGT-1 serves a non-catalytic function in the hypodermis impacting male fertility and mating behavior. This study builds upon research on the nutrient sensor O-GlcNAc transferase and demonstrates a role it plays in the interplay between the evolutionary drives for reproduction and survival.

Ancestry-specific high-risk gene variant profiling unmasks diabetes-associated genes.

How ancestry-associated genetic variance affects disparities in the risk for polygenic diseases and influences the identification of disease-associated genes warrant a deeper understanding. We hypothesized that the discovery of genes associated with polygenic diseases may be limited by overreliance on single-nucleotide polymorphism (SNP)-based genomic investigation, since most significant variants identified in genome-wide SNP association studies map to introns and intergenic regions of the genome. To overcome such potential limitation, we developed a gene-constrained and function-based analytical method centered on high-risk variants (hrV) that encode frameshifts, stopgains, or splice site disruption. We analyzed the total number of hrV per gene in populations of different ancestry, representing a total of 185 934 subjects. Using this analysis, we developed a quantitative index of hrV (hrVI) across 20 428 genes within each population. We then applied hrVI analysis to the discovery of genes associated with type 2 diabetes mellitus (T2DM), a polygenic disease with ancestry-related disparity. HrVI profiling and gene-to-gene comparisons of ancestry-specific hrV between the case (20 781 subjects) and control (24 440 subjects) populations in the T2DM national repository identified 57 genes associated with T2DM, 40 of which were discoverable only by ancestry-specific analysis. These results illustrate how function-based and ancestry-specific analysis of genetic variations can accelerate the identification of genes associated with polygenic diseases. Besides T2DM, such analysis may facilitate our understanding of the genetic basis for other polygenic diseases that are also greatly influenced by environmental and behavioral factors, such as obesity, hypertension, and Alzheimer's disease.

Tools, tactics and objectives to interrogate cellular roles of O-GlcNAc in disease.

The vast array of cell types of multicellular organisms must individually fine-tune their internal metabolism. One important metabolic and stress regulatory mechanism is the dynamic attachment/removal of glucose-derived sugar N-acetylglucosamine on proteins (O-GlcNAcylation). The number of proteins modified by O-GlcNAc is bewildering, with at least 7,000 sites in human cells. The outstanding challenge is determining how key O-GlcNAc sites regulate a target pathway amidst thousands of potential global sites. Innovative solutions are required to address this challenge in cell models and disease therapy. This Perspective shares critical suggestions for the O-GlcNAc field gleaned from the international O-GlcNAc community. Further, we summarize critical tools and tactics to enable newcomers to O-GlcNAc biology to drive innovation at the interface of metabolism and disease. The growing pace of O-GlcNAc research makes this a timely juncture to involve a wide array of scientists and new toolmakers to selectively approach the regulatory roles of O-GlcNAc in disease.

Genetic and functional modulation by agonist MRS5698 and allosteric enhancer LUF6000 at the native A3 adenosine receptor in HL-60 cells.

The A3 adenosine receptor (AR) is an important inflammatory and immunological target. However, the underlying mechanisms are not fully understood. Here, we report the gene regulation in HL-60 cells treated acutely with highly selective A3AR agonist MRS5698, positive allosteric modulator (PAM) LUF6000, or both. Both pro- and anti-inflammatory genes, such as IL-1a, IL-1ß, and NFκBIZ, are significantly upregulated. During our observations, LUF6000 alone produced a lesser effect, while the MRS5698 + LUF6000 group demonstrated generally greater effects than MRS5698 alone, consistent with allosteric enhancement. The number of genes up- and down-regulated are similar. Pathway analysis highlighted the critical involvement of signaling molecules, including IL-6 and IL-17. Important upstream regulators include IL-1a, IL-1ß, TNF-α, NF-κB, etc. PPAR, which modulates eicosanoid metabolism, was highly downregulated by the A3AR agonist. Considering previous pharmacological results and mathematical modeling, LUF6000's small enhancement of genetic upregulation suggested that MRS5698 is a nearly full agonist, which we demonstrated in both cAMP and calcium assays. The smaller effect of LUF6000 on MRS5698 in comparison to its effect on Cl-IB-MECA was shown in both HL-60 cells endogenously expressing the human (h) A3AR and in recombinant hA3AR-expressing CHO cells, consistent with its HL-60 cell genetic regulation patterns. In summary, by using both selective agonists and PAM, we identified genes that are closely relevant to immunity and inflammation to be regulated by A3AR in differentiated HL-60 cells, a cell model of neutrophil function. In addition, we demonstrated the previously uncharacterized allosteric signaling-enhancing effect of LUF6000 in cells endogenously expressing the hA3AR.

Bittersweet memories: linking metabolism to epigenetics through O-GlcNAcylation.

O-GlcNAcylation, which is a nutrient-sensitive sugar modification, participates in the epigenetic regulation of gene expression. The enzymes involved in O-linked ß-D-N-acetylglucosamine (O-GlcNAc) cycling - O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) - target key transcriptional and epigenetic regulators including RNA polymerase II, histones, histone deacetylase complexes and members of the Polycomb and Trithorax groups. Thus, O-GlcNAc cycling may serve as a homeostatic mechanism linking nutrient availability to higher-order chromatin organization. In response to nutrient availability, O-GlcNAcylation is poised to influence X chromosome inactivation and genetic imprinting, as well as embryonic development. The wide range of physiological functions regulated by O-GlcNAc cycling suggests an unexplored nexus between epigenetic regulation in disease and nutrient availability.

Nutrient-responsive O-GlcNAcylation dynamically modulates the secretion of glycan-binding protein galectin 3.

Endomembrane glycosylation and cytoplasmic O-GlcNAcylation each play essential roles in nutrient sensing, and characteristic changes in glycan patterns have been described in disease states such as diabetes and cancer. These changes in glycosylation have important functional roles and can drive disease progression. However, little is known about the molecular mechanisms underlying how these signals are integrated and transduced into biological effects. Galectins are proteins that bind glycans and that are secreted by a poorly characterized nonclassical secretory mechanism. Once outside the cell, galectins bind to the terminal galactose residues of cell surface glycans and modulate numerous extracellular functions, such as clathrin-independent endocytosis (CIE). Originating in the cytoplasm, galectins are predicted substrates for O-GlcNAc addition and removal; and as we have shown, galectin 3 is a substrate for O-GlcNAc transferase. In this study, we also show that galectin 3 secretion is sensitive to changes in O-GlcNAc levels. We determined using immunoprecipitation and Western blotting that there is a significant difference in O-GlcNAcylation status between cytoplasmic and secreted galectin 3. We observed dramatic alterations in galectin 3 secretion in response to nutrient conditions, which were dependent on dynamic O-GlcNAcylation. Importantly, we showed that these O-GlcNAc-driven alterations in galectin 3 secretion also facilitated changes in CIE. These results indicate that dynamic O-GlcNAcylation of galectin 3 plays a role in modulating its secretion and can tune its function in transducing nutrient-sensing information coded in cell surface glycosylation into biological effects.

The O-GlcNAc transferase OGT is a conserved and essential regulator of the cellular and organismal response to hypertonic stress.

The conserved O-GlcNAc transferase OGT O-GlcNAcylates serine and threonine residues of intracellular proteins to regulate their function. OGT is required for viability in mammalian cells, but its specific roles in cellular physiology are poorly understood. Here we describe a conserved requirement for OGT in an essential aspect of cell physiology: the hypertonic stress response. Through a forward genetic screen in Caenorhabditis elegans, we discovered OGT is acutely required for osmoprotective protein expression and adaptation to hypertonic stress. Gene expression analysis shows that ogt-1 functions through a post-transcriptional mechanism. Human OGT partially rescues the C. elegans phenotypes, suggesting that the osmoregulatory functions of OGT are ancient. Intriguingly, expression of O-GlcNAcylation-deficient forms of human or worm OGT rescue the hypertonic stress response phenotype. However, expression of an OGT protein lacking the tetracopeptide repeat (TPR) domain does not rescue. Our findings are among the first to demonstrate a specific physiological role for OGT at the organismal level and demonstrate that OGT engages in important molecular functions outside of its well described roles in post-translational O-GlcNAcylation of intracellular proteins.

O-GlcNAcylation protein disruption by Thiamet G promotes changes on the GBM U87-MG cells secretome molecular signature.

Glioblastoma (GBM) is a grade IV glioma highly aggressive and refractory to the therapeutic approaches currently in use. O-GlcNAcylation plays a key role for tumor aggressiveness and progression in different types of cancer; however, experimental evidence of its involvement in GBM are still lacking. Here, we show that O-GlcNAcylation plays a critical role in maintaining the composition of the GBM secretome, whereas inhibition of OGA activity disrupts the intercellular signaling via microvesicles. Using a label-free quantitative proteomics methodology, we identified 51 proteins in the GBM secretome whose abundance was significantly altered by activity inhibition of O-GlcNAcase (iOGA). Among these proteins, we observed that proteins related to proteasome activity and to regulation of immune response in the tumor microenvironment were consistently downregulated in GBM cells upon iOGA. While the proteins IGFBP3, IL-6 and HSPA5 were downregulated in GBM iOGA cells, the protein SQSTM1/p62 was exclusively found in GBM cells under iOGA. These findings were in line with literature evidence on the role of p62/IL-6 signaling axis in suppressing tumor aggressiveness and our experimental evidence showing a decrease in radioresistance potential of these cells. Taken together, our findings provide evidence that OGA activity may regulate the p62 and IL-6 abundance in the GBM secretome. We propose that the assessment of tumor status from the main proteins present in its secretome may contribute to the advancement of diagnostic, prognostic and even therapeutic tools to approach this relevant malignancy.

O-GlcNAcylation regulates dopamine neuron function, survival and degeneration in Parkinson disease.

The dopamine system in the midbrain is essential for volitional movement, action selection, and reward-related learning. Despite its versatile roles, it contains only a small set of neurons in the brainstem. These dopamine neurons are especially susceptible to Parkinson's disease and prematurely degenerate in the course of disease progression, while the discovery of new therapeutic interventions has been disappointingly unsuccessful. Here, we show that O-GlcNAcylation, an essential post-translational modification in various types of cells, is critical for the physiological function and survival of dopamine neurons. Bidirectional modulation of O-GlcNAcylation importantly regulates dopamine neurons at the molecular, synaptic, cellular, and behavioural levels. Remarkably, genetic and pharmacological upregulation of O-GlcNAcylation mitigates neurodegeneration, synaptic impairments, and motor deficits in an animal model of Parkinson's disease. These findings provide insights into the functional importance of O-GlcNAcylation in the dopamine system, which may be utilized to protect dopamine neurons against Parkinson's disease pathology.

O-GlcNAc cycling mediates energy balance by regulating caloric memory.

Caloric need has long been thought a major driver of appetite. However, it is unclear whether caloric need regulates appetite in environments offered by many societies today where there is no shortage of food. Here we observed that wildtype mice with free access to food did not match calorie intake to calorie expenditure. While the size of a meal affected subsequent intake, there was no compensation for earlier under- or over-consumption. To test how spontaneous eating is subject to caloric control, we manipulated O-linked ß-N-acetylglucosamine (O-GlcNAc), an energy signal inside cells dependent on nutrient access and metabolic hormones. Genetic and pharmacological manipulation in mice increasing or decreasing O-GlcNAcylation regulated daily intake by controlling meal size. Meal size was affected at least in part due to faster eating speed. Without affecting meal frequency, O-GlcNAc disrupted the effect of caloric consumption on future intake. Across days, energy balance was improved upon increased O-GlcNAc levels and impaired upon removal of O-GlcNAcylation. Rather than affecting a perceived need for calories, O-GlcNAc regulates how a meal affects future intake, suggesting that O-GlcNAc mediates a caloric memory and subsequently energy balance.

A genetic model to study O-GlcNAc cycling in immortalized mouse embryonic fibroblasts.

O-GlcNAcylation is an abundant posttranslational protein modification in which the monosaccharide O-GlcNAc is added to Ser/Thr residues by O-GlcNAc transferase and removed by O-GlcNAcase. Analyses of O-GlcNAc-mediated signaling and metabolic phenomena are complicated by factors including unsatisfactory inhibitors and loss-of-function cell lines lacking identical genetic backgrounds. In this work, we generated immortalized WT, Oga knockout, and Ogt floxed allele (Ogt floxed) mouse embryonic fibroblast (MEF) cell lines with similar genetic backgrounds. These lines will facilitate experiments and serve as a platform to study O-GlcNAc cycling in mammals. As a test paradigm, we used the immortalized MEF lines to investigate how changes in O-GlcNAcylation affected pathological phosphorylation of the tau protein. The activity of glycogen synthase kinase 3ß (GSK3ß), a kinase that phosphorylates tau, decreases when expressed in Oga knockout MEFs compared with WT cells. Phosphorylation at Thr231 in recombinant, tauopathy-associated tau with a proline-to-leucine mutation at position 301 (P301L) was altered when expressed in MEFs with altered O-GlcNAc cycling. In aggregate, our data support that O-GlcNAc cycling indirectly affects tau phosphorylation at Thr231, but tau phosphorylation was highly variable, even in genetically stable, immortalized MEF cells. The variable nature of tau phosphorylation observed here supports the need to use cells akin to those generated here with genetically defined lesions and similar backgrounds to study complex biological processes.

Nutrient-driven O-linked N-acetylglucosamine (O-GlcNAc) cycling impacts neurodevelopmental timing and metabolism.

Nutrient-driven O-GlcNAcylation is strikingly abundant in the brain and has been linked to development and neurodegenerative disease. We selectively targeted the O-GlcNAcase (Oga) gene in the mouse brain to define the role of O-GlcNAc cycling in the central nervous system. Brain knockout animals exhibited dramatically increased brain O-GlcNAc levels and pleiotropic phenotypes, including early-onset obesity, growth defects, and metabolic dysregulation. Anatomical defects in the Oga knockout included delayed brain differentiation and neurogenesis as well as abnormal proliferation accompanying a developmental delay. The molecular basis for these defects included transcriptional changes accompanying differentiating embryonic stem cells. In Oga KO mouse ES cells, we observed pronounced changes in expression of pluripotency markers, including Sox2, Nanog, and Otx2. These findings link the O-GlcNAc modification to mammalian neurogenesis and highlight the role of this nutrient-sensing pathway in developmental plasticity and metabolic homeostasis.

Nutrient-driven O-GlcNAc in proteostasis and neurodegeneration.

Proteostasis is essential in the mammalian brain where post-mitotic cells must function for decades to maintain synaptic contacts and memory. The brain is dependent on glucose and other metabolites for proper function and is spared from metabolic deficits even during starvation. In this review, we outline how the nutrient-sensitive nucleocytoplasmic post-translational modification O-linked N-acetylglucosamine (O-GlcNAc) regulates protein homeostasis. The O-GlcNAc modification is highly abundant in the mammalian brain and has been linked to proteopathies, including neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's. C. elegans, Drosophila, and mouse models harboring O-GlcNAc transferase- and O-GlcNAcase-knockout alleles have helped define the role O-GlcNAc plays in development as well as age-associated neurodegenerative disease. These enzymes add and remove the single monosaccharide from protein serine and threonine residues, respectively. Blocking O-GlcNAc cycling is detrimental to mammalian brain development and interferes with neurogenesis, neural migration, and proteostasis. Findings in C. elegans and Drosophila model systems indicate that the dynamic turnover of O-GlcNAc is critical for maintaining levels of key transcriptional regulators responsible for neurodevelopment cell fate decisions. In addition, pathways of autophagy and proteasomal degradation depend on a transcriptional network that is also reliant on O-GlcNAc cycling. Like the quality control system in the endoplasmic reticulum which uses a 'mannose timer' to monitor protein folding, we propose that cytoplasmic proteostasis relies on an 'O-GlcNAc timer' to help regulate the lifetime and fate of nuclear and cytoplasmic proteins. O-GlcNAc-dependent developmental alterations impact metabolism and growth of the developing mouse embryo and persist into adulthood. Brain-selective knockout mouse models will be an important tool for understanding the role of O-GlcNAc in the physiology of the brain and its susceptibility to neurodegenerative injury.

Coronary calcification in adults with Turner syndrome.

PurposeAdults with Turner syndrome (TS) have an increased predisposition to ischemic heart disease. The quantitative relationship between coronary atherosclerosis and TS has yet to be established.MethodsA total of 128 females (62 with TS) participated in this prospective study. Coronary computed tomography angiography was performed to measure coronary calcified plaque burden, and prevalent noncalcified plaque burden. Regression analysis was used to study the effects of TS and traditional cardiovascular disease risk factors on coronary plaque burden.ResultsAdults with TS were 63% more likely to have coronary calcifications than controls (odds ratio 1.63, 95% confidence interval: 1.02, 2.61, P = 0.04), with an age cutoff of 51.7 years for a probability of >50% for the presence of coronary calcifications, when compared to 55.7 years in female controls. The average age of TS patients with calcified plaques was significantly lower than that of controls with calcified plaques (51.5 ± 8.9 years vs. 60.5 ± 7.0 years, P < 0.001). Age increased the likelihood of coronary calcifications by 13% per year (odds ratio 1.13, confidence interval 95%: 1.07-1.19, P < 0.001).ConclusionThis study demonstrates a higher prevalence and earlier onset of calcified coronary plaques in TS. These findings have important implications for cardiovascular risk assessment and the management of patients with TS.

O-GlcNAc in cancer: An Oncometabolism-fueled vicious cycle.

Cancer cells exhibit unregulated growth, altered metabolism, enhanced metastatic potential and altered cell surface glycans. Fueled by oncometabolism and elevated uptake of glucose and glutamine, the hexosamine biosynthetic pathway (HBP) sustains glycosylation in the endomembrane system. In addition, the elevated pools of UDP-GlcNAc drives the O-GlcNAc modification of key targets in the cytoplasm, nucleus and mitochondrion. These targets include transcription factors, kinases, key cytoplasmic enzymes of intermediary metabolism, and electron transport chain complexes. O-GlcNAcylation can thereby alter epigenetics, transcription, signaling, proteostasis, and bioenergetics, key 'hallmarks of cancer'. In this review, we summarize accumulating evidence that many cancer hallmarks are linked to dysregulation of O-GlcNAc cycling on cancer-relevant targets. We argue that onconutrient and oncometabolite-fueled elevation increases HBP flux and triggers O-GlcNAcylation of key regulatory enzymes in glycolysis, Kreb's cycle, pentose-phosphate pathway, and the HBP itself. The resulting rerouting of glucose metabolites leads to elevated O-GlcNAcylation of oncogenes and tumor suppressors further escalating elevation in HBP flux creating a 'vicious cycle'. Downstream, elevated O-GlcNAcylation alters DNA repair and cellular stress pathways which influence oncogenesis. The elevated steady-state levels of O-GlcNAcylated targets found in many cancers may also provide these cells with a selective advantage for sustained growth, enhanced metastatic potential, and immune evasion in the tumor microenvironment.

Drosophila O-GlcNAcase Deletion Globally Perturbs Chromatin O-GlcNAcylation.

Gene expression during Drosophila development is subject to regulation by the Polycomb (Pc), Trithorax (Trx), and Compass chromatin modifier complexes. O-GlcNAc transferase (OGT/SXC) is essential for Pc repression suggesting that the O-GlcNAcylation of proteins plays a key role in regulating development. OGT transfers O-GlcNAc onto serine and threonine residues in intrinsically disordered domains of key transcriptional regulators; O-GlcNAcase (OGA) removes the modification. To pinpoint genomic regions that are regulated by O-GlcNAc levels, we performed ChIP-chip and microarray analysis after OGT or OGA RNAi knockdown in S2 cells. After OGA RNAi, we observed a genome-wide increase in the intensity of most O-GlcNAc-occupied regions including genes linked to cell cycle, ubiquitin, and steroid response. In contrast, O-GlcNAc levels were strikingly insensitive to OGA RNAi at sites of polycomb repression such as the Hox and NK homeobox gene clusters. Microarray analysis suggested that altered O-GlcNAc cycling perturbed the expression of genes associated with morphogenesis and cell cycle regulation. We then produced a viable null allele of oga (oga(del.1)) in Drosophila allowing visualization of altered O-GlcNAc cycling on polytene chromosomes. We found that trithorax (TRX), absent small or homeotic discs 1 (ASH1), and Compass member SET1 histone methyltransferases were O-GlcNAc-modified in oga(del.1) mutants. The oga(del.1) mutants displayed altered expression of a distinct set of cell cycle-related genes. Our results show that the loss of OGA in Drosophila globally impacts the epigenetic machinery allowing O-GlcNAc accumulation on RNA polymerase II and numerous chromatin factors including TRX, ASH1, and SET1.

O-GlcNAc cycling and the regulation of nucleocytoplasmic dynamics.

The dynamic carbohydrate post-translational modification (PTM) O-linked ß-N-acetyl glucosamine (O-GlcNAc) is found on thousands of proteins throughout the nucleus and cytoplasm, and rivals phosphorylation in terms of the number of substrates and pathways influenced. O-GlcNAc is highly conserved and essential in most organisms, with disruption of O-GlcNAc cycling linked to diseases ranging from cancer to neurodegeneration. Nuclear pore proteins were the first identified O-GlcNAc-modified substrates, generating intense and ongoing interest in understanding the role of O-GlcNAc cycling in nuclear pore complex structure and function. Recent advances in detecting and altering O-GlcNAcylation levels have provided insights into many mechanisms by which O-GlcNAcylation influences the nucleocytoplasmic localization and stability of protein targets. The emerging view is that the multifunctional enzymes of O-GlcNAc cycling are critical nutrient-sensing components of a complex network of signaling cascades involving multiple PTMs. Furthermore, O-GlcNAc plays a role in maintaining the structural integrity of the nuclear pore and regulating its function as the gatekeeper of nucleocytoplasmic trafficking.

Chemical tools to explore nutrient-driven O-GlcNAc cycling.

Posttranslational modifications (PTM) including glycosylation, phosphorylation, acetylation, methylation and ubiquitination dynamically alter the proteome. The evolutionarily conserved enzymes O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and O-GlcNAcase are responsible for the addition and removal, respectively, of the nutrient-sensitive PTM of protein serine and threonine residues with O-GlcNAc. Indeed, the O-GlcNAc modification acts at every step in the "central dogma" of molecular biology and alters signaling pathways leading to amplified or blunted biological responses. The cellular roles of OGT and the dynamic PTM O-GlcNAc have been clarified with recently developed chemical tools including high-throughput assays, structural and mechanistic studies and potent enzyme inhibitors. These evolving chemical tools complement genetic and biochemical approaches for exposing the underlying biological information conferred by O-GlcNAc cycling.