Nutrient-driven O-GlcNAcylation of key components of the transcription machinery may epigenetically modulate gene expression in metazoans. The global effects of GlcNAcylation on transcription can be addressed directly in C. elegans because knockouts of the O-GlcNAc cycling enzymes are viable and fertile. Using anti-O-GlcNAc ChIP-on-chip whole-genome tiling arrays on wild-type and mutant strains, we detected over 800 promoters where O-GlcNAc cycling occurs, including microRNA loci and multigene operons. Intriguingly, O-GlcNAc-marked promoters are biased toward genes associated with PIP3 signaling, hexosamine biosynthesis, and lipid/carbohydrate metabolism. These marked genes are linked to insulin-like signaling, metabolism, aging, stress, and pathogen-response pathways in C. elegans . Whole-genome transcriptional profiling of the O-GlcNAc cycling mutants confirmed dramatic deregulation of genes in these key pathways. As predicted, the O-GlcNAc cycling mutants show altered lifespan and UV stress susceptibility phenotypes. We propose that O-GlcNAc cycling at promoters participates in a molecular program impacting nutrient-responsive pathways in C. elegans , including stress, pathogen response, and adult lifespan. The observed impact of O-GlcNAc cycling on both signaling and transcription in C. elegans has important implications for human diseases of aging, including diabetes and neurodegeneration.

As Western diets continue to include an ever-increasing amount of sugar, there has been a rise in obesity and type 2 diabetes. To avoid metabolic diseases, the body must maintain proper metabolism, even on a high-sugar diet. In both humans and , excess sugar (glucose) is stored as glycogen. Here, we find that animals increased stored glycogen as they aged, whereas even young adult animals had increased stored glycogen on a high-sugar diet. Decreasing the amount of glycogen storage by modulating the glycogen synthase, , a key enzyme in glycogen synthesis, can extend lifespan, prolong healthspan, and limit the detrimental effects of a high-sugar diet. Importantly, limiting glycogen storage leads to a metabolic shift whereby glucose is now stored as trehalose. Two additional means to increase trehalose show similar longevity extension. Increased trehalose is entirely dependent on a functional FOXO transcription factor DAF-16 and autophagy to promote lifespan and healthspan extension. Our results reveal that when glucose is stored as glycogen, it is detrimental, whereas, when stored as trehalose, animals live a longer, healthier life if DAF-16 is functional. Taken together, these results demonstrate that trehalose modulation may be an avenue for combatting high-sugar-diet pathology.

In a variety of organisms, including worms, flies, and mammals, glucose homeostasis is maintained by insulin-like signaling in a robust network of opposing and complementary signaling pathways. The hexosamine signaling pathway, terminating in O-linked-N-acetylglucosamine (O-GlcNAc) cycling, is a key sensor of nutrient status and has been genetically linked to the regulation of insulin signaling in Caenorhabditis elegans. Here we demonstrate that O-GlcNAc cycling and insulin signaling are both essential components of the C. elegans response to glucose stress. A number of insulin-dependent processes were found to be sensitive to glucose stress, including fertility, reproductive timing, and dauer formation, yet each of these differed in their threshold of sensitivity to glucose excess. Our findings suggest that O-GlcNAc cycling and insulin signaling are both required for a robust and adaptable response to glucose stress, but these two pathways show complex and interdependent roles in the maintenance of glucose-insulin homeostasis.

Protein ubiquitination is an important regulator of cytokine-activated signal transduction pathways and hematopoietic cell growth. Protein ubiquitination is controlled by the coordinate action of ubiquitin-conjugating enzymes and deubiquitinating enzymes. Recently a novel family of genes encoding growth-regulatory deubiquitinating enzymes (DUB-1 and DUB-2) has been identified. DUBs are immediate-early genes and are induced rapidly and transiently in response to cytokine stimuli. By means of polymerase chain reaction amplification with degenerate primers for the DUB-2 complementary DNA, 3 murine bacterial artificial chromosome (BAC) clones that contain DUB gene sequences were isolated. One BAC contained a novel DUB gene (DUB-2A) with extensive homology to DUB-2. Like DUB-1 and DUB-2, the DUB-2A gene consists of 2 exons. The predicted DUB-2A protein is highly related to other DUBs throughout the primary amino acid sequence, with a hypervariable region at its C-terminus. In vitro, DUB-2A had functional deubiquitinating activity; mutation of its conserved amino acid residues abolished this activ- IntroductionProtein ubiquitination controls many intracellular processes, including cell cycle progression. 1,2 transcriptional activation, 3 and signal transduction 4 (reviewed in Ciechanover 5 and D'Andrea and Pellman 6 ). Like protein phosphorylation, protein ubiquitination is dynamic, involving enzymes that add ubiquitin (ubiquitinconjugating enzymes) and enzymes that remove ubiquitin (deubiquitinating enzymes). Considerable progress has been made in understanding ubiquitin conjugation and its role in regulating protein degradation. Recent studies have demonstrated that regulation also occurs at the level of deubiquitination. Deubiquitinating enzymes are cysteine proteases that specifically cleave ubiquitin from ubiquitin-conjugated protein substrates. Deubiquitinating enzymes have significant sequence diversity and may therefore have a broad range of substrate specificity.There are 2 major families of deubiquitinating enzymes, the ubiquitin-processing proteases (ubp) family 7-9 and the ubiquitin carboxy-terminal hydrolase (uch) family. 10,11 The ubp family and the uch family have also been referred to as the type 1 uch and type 2 uch families. 12 Both ubps and uchs are cysteine proteases containing an active site cysteine, aspartate, and histidine residue. Ubps vary greatly in size and structural complexity, but all contain 6 characteristic conserved homology domains. 13 Uchs, in contrast, include a group of small, closely related proteases that lack the 6 characteristic homology domains of the ubps. 11 Little is known regarding the precise cellular function of ubps and uchs. For instance, despite the broad range and structural diversity of these enzymes, only a few specific candidate substrates have been identified. 14-16 Also, whether these enzymes act exclusively on ubiquitinated substrates or on substrates with ubiquitinlike modifications, such as SUMO-1 17,18 and NEDD8, 19 remains unknown. Recently, a distinct fa...