New Roles for the Heterochronic Transcription Factor LIN-29 in Cuticle Maintenance and Lipid Metabolism at the Larval-to-Adult Transition in Caenorhabditis elegans.

Temporal regulation of gene expression is a crucial aspect of metazoan development. In the roundworm Caenorhabditis elegans, the heterochronic pathway controls multiple developmental events in a time-specific manner. The most downstream effector of this pathway, the zinc-finger transcription factor LIN-29, acts in the last larval stage (L4) to regulate elements of the larval-to-adult switch. Here, we explore new LIN-29 targets and their implications for this developmental transition. We used RNA-sequencing to identify genes differentially expressed between animals misexpressing LIN-29 at an early time point and control animals. Among 230 LIN-29-activated genes, we found that genes encoding cuticle collagens were overrepresented. Interestingly, expression of lin-29 and some of these collagens was increased in adults with cuticle damage, suggesting a previously unknown function for LIN-29 in adult cuticle maintenance. On the other hand, genes involved in fat metabolism were enriched among 350 LIN-29-downregulated targets. We used mass spectrometry to assay lipid content in animals overexpressing LIN-29 and observed reduced fatty acid levels. Many LIN-29-repressed genes are normally expressed in the intestine, suggesting cell-nonautonomous regulation. We identified several LIN-29 upregulated genes encoding signaling molecules that may act as mediators in the regulation of intestinally expressed genes encoding fat metabolic enzymes and vitellogenins. Overall, our results support the model of LIN-29 as a major regulator of adult cuticle synthesis and integrity, and as the trigger for metabolic changes that take place at the important transition from rapid growth during larval life to slower growth and offspring production during adulthood.

Nutrient-Driven O-GlcNAcylation at Promoters Impacts Genome-Wide RNA Pol II Distribution.

Nutrient-driven O-GlcNAcylation has been linked to epigenetic regulation of gene expression in metazoans. In C. elegans, O-GlcNAc marks the promoters of over 800 developmental, metabolic, and stress-related genes; these O-GlcNAc marked genes show a strong 5', promoter-proximal bias in the distribution of RNA Polymerase II (Pol II). In response to starvation or feeding, the steady state distribution of O-GlcNAc at promoters remain nearly constant presumably due to dynamic cycling mediated by the transferase OGT-1 and the O-GlcNAcase OGA-1. However, in viable mutants lacking either of these enzymes of O-GlcNAc metabolism, the nutrient-responsive GlcNAcylation of promoters is dramatically altered. Blocked O-GlcNAc cycling leads to a striking nutrient-dependent accumulation of O-GlcNAc on RNA Pol II. O-GlcNAc cycling mutants also show an exaggerated, nutrient-responsive redistribution of promoter-proximal RNA Pol II isoforms and extensive transcriptional deregulation. Our findings suggest a complex interplay between the O-GlcNAc modification at promoters, the kinase-dependent "CTD-code," and co-factors regulating RNA Pol II dynamics. Nutrient-responsive O-GlcNAc cycling may buffer the transcriptional apparatus from dramatic swings in nutrient availability by modulating promoter activity to meet metabolic and developmental needs.

The nematode homologue of Mediator complex subunit 28, F28F8.5, is a critical regulator of C. elegans development.

The evolutionarily conserved Mediator complex is a critical player in regulating transcription. Comprised of approximately two dozen proteins, the Mediator integrates diverse regulatory signals through direct protein-protein interactions that, in turn, modulate the influence of Mediator on RNA Polymerase II activity. One Mediator subunit, MED28, is known to interact with cytoplasmic structural proteins, providing a potential direct link between cytoplasmic dynamics and the control of gene transcription. Although identified in many animals and plants, MED28 is not present in yeast; no bona fide MED28 has been described previously in Caenorhabditis elegans. Here, we identify bioinformatically F28F8.5, an uncharacterized predicted protein, as the nematode homologue of MED28. As in other Metazoa, F28F8.5 has dual nuclear and cytoplasmic localization and plays critical roles in the regulation of development. F28F8.5 is a vital gene and its null mutants have severely malformed gonads and do not reproduce. F28F8.5 interacts on the protein level with the Mediator subunits MDT-6 and MDT-30. Our results indicate that F28F8.5 is an orthologue of MED28 and suggest that the potential to link cytoplasmic and nuclear events is conserved between MED28 vertebrate and nematode orthologues.

A Genetic Analysis of the Caenorhabditis elegans Detoxification Response.

Oxidative damage contributes to human diseases of aging including diabetes, cancer, and cardiovascular disorders. Reactive oxygen species resulting from xenobiotic and endogenous metabolites are sensed by a poorly understood process, triggering a cascade of regulatory factors and leading to the activation of the transcription factor Nrf2 (Nuclear factor-erythroid-related factor 2, SKN-1 in Caenorhabditis elegans). Nrf2/SKN-1 activation promotes the induction of the phase II detoxification system that serves to limit oxidative stress. We have extended a previous C. elegans genetic approach to explore the mechanisms by which a phase II enzyme is induced by endogenous and exogenous oxidants. The xrep (xenobiotics response pathway) mutants were isolated as defective in their ability to properly regulate the induction of a glutathione S-transferase (GST) reporter. The xrep-1 gene was previously identified as wdr-23, which encodes a C. elegans homolog of the mammalian ß-propeller repeat-containing protein WDR-23 Here, we identify and confirm the mutations in xrep-2, xrep-3, and xrep-4 The xrep-2 gene is alh-6, an ortholog of a human gene mutated in familial hyperprolinemia. The xrep-3 mutation is a gain-of-function allele of skn-1 The xrep-4 gene is F46F11.6, which encodes a F-box-containing protein. We demonstrate that xrep-4 alters the stability of WDR-23 (xrep-1), a key regulator of SKN-1 (xrep-3). Epistatic relationships among the xrep mutants and their interacting partners allow us to propose an ordered genetic pathway by which endogenous and exogenous stressors induce the phase II detoxification response.

Caenorhabditis elegans DAF-2 as a Model for Human Insulin Receptoropathies.

Human exome sequencing has dramatically increased the rate of identification of disease-associated polymorphisms. However, examining the functional consequences of those variants has created an analytic bottleneck. Insulin-like signaling in Caenorhabditis elegans has long provided a model to assess consequences of human insulin signaling mutations, but this has not been evaluated in the context of current genetic tools. We have exploited strains derived from the Million Mutation Project (MMP) and gene editing to explore further the evolutionary relationships and conservation between the human and C. elegans insulin receptors. Of 40 MMP alleles analyzed in the C. elegans insulin-like receptor gene DAF-2, 35 exhibited insulin-like signaling indistinguishable from wild-type animals, indicating tolerated mutations. Five MMP alleles proved to be novel dauer-enhancing mutations, including one new allele in the previously uncharacterized C-terminus of DAF-2 CRISPR-Cas9 genome editing was used to confirm the phenotypic consequence of six of these DAF-2 mutations and to replicate an allelic series of known human disease mutations in a highly conserved tyrosine kinase active site residue, demonstrating the utility of C. elegans for directly modeling human disease. Our results illustrate the challenges associated with prediction of the phenotypic consequences of amino acid substitutions, the value of assaying mutant isoform function in vivo, and how recently developed tools and resources afford the opportunity to expand our understanding even of highly conserved regulatory modules such as insulin signaling. This approach may prove generally useful for modeling phenotypic consequences of candidate human pathogenic mutations in conserved signaling and developmental pathways.

X Chromosome Crossover Formation and Genome Stability in Caenorhabditis elegans Are Independently Regulated by xnd-1.

The germ line efficiently combats numerous genotoxic insults to ensure the high fidelity propagation of unaltered genomic information across generations. Yet, germ cells in most metazoans also intentionally create double-strand breaks (DSBs) to promote DNA exchange between parental chromosomes, a process known as crossing over. Homologous recombination is employed in the repair of both genotoxic lesions and programmed DSBs, and many of the core DNA repair proteins function in both processes. In addition, DNA repair efficiency and crossover (CO) distribution are both influenced by local and global differences in chromatin structure, yet the interplay between chromatin structure, genome integrity, and meiotic fidelity is still poorly understood. We have used the xnd-1 mutant of Caenorhabditis elegans to explore the relationship between genome integrity and crossover formation. Known for its role in ensuring X chromosome CO formation and germ line development, we show that xnd-1 also regulates genome stability. xnd-1 mutants exhibited a mortal germ line, high embryonic lethality, high incidence of males, and sensitivity to ionizing radiation. We discovered that a hypomorphic allele of mys-1 suppressed these genome instability phenotypes of xnd-1, but did not suppress the CO defects, suggesting it serves as a separation-of-function allele. mys-1 encodes a histone acetyltransferase, whose homolog Tip60 acetylates H2AK5, a histone mark associated with transcriptional activation that is increased in xnd-1 mutant germ lines, raising the possibility that thresholds of H2AK5ac may differentially influence distinct germ line repair events. We also show that xnd-1 regulated him-5 transcriptionally, independently of mys-1, and that ectopic expression of him-5 suppressed the CO defects of xnd-1 Our work provides xnd-1 as a model in which to study the link between chromatin factors, gene expression, and genome stability.

Regulation of UNC-130/FOXD-mediated mesodermal patterning in C. elegans.

Spatial polarity cues in animals are used repeatedly during development for many processes, including cell fate determination, cell migration, and axon guidance. In Caenorhabditis elegans, the body wall muscle extends the length of the animal in four distinct quadrants and generates an UNC-129/TGF-ß-related signal that is much higher in the dorsal two muscle quadrants compared to their ventral counterparts. This pattern of unc-129 expression requires the activity of the proposed transcriptional repressor UNC-130/FOXD whose body wall muscle activity is restricted to the ventral two body wall muscle quadrants. To understand how these dorsal-ventral differences in UNC-130 activity are established and maintained, we have analyzed the regulation of unc-130 expression and the distribution of UNC-130 protein. We have identified widespread, cis-acting elements in the unc-130 promoter that function to positively regulate ventral body wall muscle expression and negatively regulate dorsal body wall muscle expression. We have defined the temporal distribution of UNC-130 protein in body wall muscle cells during embryogenesis, demonstrated that this pattern is required to establish the dorsal-ventral polarity of UNC-129/TGF-ß, and shown that UNC-130 is not required post-embryonically to maintain the asymmetry of body wall muscle unc-129 expression. Finally, we have tested the impact of the depletion of a variety of transcription factors, repressors, and signaling molecules to identify additional regulators of body wall muscle UNC-130 polarity. Our results confirm and extend earlier studies to clarify the mechanisms by which UNC-130 is controlled and affects the pattern of unc-129 expression in body wall muscle. These results further our understanding of the transcriptional logic behind the generation of polarity cues involving this poorly understood subclass of Forkhead factors.

The Paired-box protein PAX-3 regulates the choice between lateral and ventral epidermal cell fates in C. elegans.

The development of the single cell layer skin or hypodermis of Caenorhabditis elegans is an excellent model for understanding cell fate specification and differentiation. Early in C. elegans embryogenesis, six rows of hypodermal cells adopt dorsal, lateral or ventral fates that go on to display distinct behaviors during larval life. Several transcription factors are known that function in specifying these major hypodermal cell fates, but our knowledge of the specification of these cell types is sparse, particularly in the case of the ventral hypodermal cells, which become Vulval Precursor Cells and form the vulval opening in response to extracellular signals. Previously, the gene pvl-4 was identified in a screen for mutants with defects in vulval development. We found by whole genome sequencing that pvl-4 is the Paired-box gene pax-3, which encodes the sole PAX-3 transcription factor homolog in C. elegans. pax-3 mutants show embryonic and larval lethality, and body morphology abnormalities indicative of hypodermal cell defects. We report that pax-3 is expressed in ventral P cells and their descendants during embryogenesis and early larval stages, and that in pax-3 reduction-of-function animals the ventral P cells undergo a cell fate transformation and express several markers of the lateral seam cell fate. Furthermore, forced expression of pax-3 in the lateral hypodermal cells causes them to lose expression of seam cell markers. We propose that pax-3 functions in the ventral hypodermal cells to prevent these cells from adopting the lateral seam cell fate. pax-3 represents the first gene required for specification solely of the ventral hypodermal fate in C. elegans providing insights into cell type diversification.

Perilipin-related protein regulates lipid metabolism in C. elegans.

Perilipins are lipid droplet surface proteins that contribute to fat metabolism by controlling the access of lipids to lipolytic enzymes. Perilipins have been identified in organisms as diverse as metazoa, fungi, and amoebas but strikingly not in nematodes. Here we identify the protein encoded by the W01A8.1 gene in Caenorhabditis elegans as the closest homologue and likely orthologue of metazoan perilipin. We demonstrate that nematode W01A8.1 is a cytoplasmic protein residing on lipid droplets similarly as human perilipins 1 and 2. Downregulation or elimination of W01A8.1 affects the appearance of lipid droplets resulting in the formation of large lipid droplets localized around the dividing nucleus during the early zygotic divisions. Visualization of lipid containing structures by CARS microscopy in vivo showed that lipid-containing structures become gradually enlarged during oogenesis and relocate during the first zygotic division around the dividing nucleus. In mutant embryos, the lipid containing structures show defective intracellular distribution in subsequent embryonic divisions and become gradually smaller during further development. In contrast to embryos, lipid-containing structures in enterocytes and in epidermal cells of adult animals are smaller in mutants than in wild type animals. Our results demonstrate the existence of a perilipin-related regulation of fat metabolism in nematodes and provide new possibilities for functional studies of lipid metabolism.

Identification of Wnt Pathway Target Genes Regulating the Division and Differentiation of Larval Seam Cells and Vulval Precursor Cells in Caenorhabditis elegans.

The evolutionarily conserved Wnt/ß-catenin signaling pathway plays a fundamental role during metazoan development, regulating numerous processes including cell fate specification, cell migration, and stem cell renewal. Wnt ligand binding leads to stabilization of the transcriptional effector ß-catenin and upregulation of target gene expression to mediate a cellular response. During larval development of the nematode Caenorhabditis elegans, Wnt/ß-catenin pathways act in fate specification of two hypodermal cell types, the ventral vulval precursor cells (VPCs) and the lateral seam cells. Because little is known about targets of the Wnt signaling pathways acting during larval VPC and seam cell differentiation, we sought to identify genes regulated by Wnt signaling in these two hypodermal cell types. We conditionally activated Wnt signaling in larval animals and performed cell type-specific "mRNA tagging" to enrich for VPC and seam cell-specific mRNAs, and then used microarray analysis to examine gene expression compared to control animals. Two hundred thirty-nine genes activated in response to Wnt signaling were identified, and we characterized 50 genes further. The majority of these genes are expressed in seam and/or vulval lineages during normal development, and reduction of function for nine genes caused defects in the proper division, fate specification, fate execution, or differentiation of seam cells and vulval cells. Therefore, the combination of these techniques was successful at identifying potential cell type-specific Wnt pathway target genes from a small number of cells and at increasing our knowledge of the specification and behavior of these C. elegans larval hypodermal cells.

Promotion of bone morphogenetic protein signaling by tetraspanins and glycosphingolipids.

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor ß (TGFß) superfamily of secreted molecules. BMPs play essential roles in multiple developmental and homeostatic processes in metazoans. Malfunction of the BMP pathway can cause a variety of diseases in humans, including cancer, skeletal disorders and cardiovascular diseases. Identification of factors that ensure proper spatiotemporal control of BMP signaling is critical for understanding how this pathway is regulated. We have used a unique and sensitive genetic screen to identify the plasma membrane-localized tetraspanin TSP-21 as a key new factor in the C. elegans BMP-like "Sma/Mab" signaling pathway that controls body size and postembryonic M lineage development. We showed that TSP-21 acts in the signal-receiving cells and genetically functions at the ligand-receptor level. We further showed that TSP-21 can associate with itself and with two additional tetraspanins, TSP-12 and TSP-14, which also promote Sma/Mab signaling. TSP-12 and TSP-14 can also associate with SMA-6, the type I receptor of the Sma/Mab pathway. Finally, we found that glycosphingolipids, major components of the tetraspanin-enriched microdomains, are required for Sma/Mab signaling. Our findings suggest that the tetraspanin-enriched membrane microdomains are important for proper BMP signaling. As tetraspanins have emerged as diagnostic and prognostic markers for tumor progression, and TSP-21, TSP-12 and TSP-14 are all conserved in humans, we speculate that abnormal BMP signaling due to altered expression or function of certain tetraspanins may be a contributing factor to cancer development.

Disruption of O-GlcNAc Cycling in C. elegans Perturbs Nucleotide Sugar Pools and Complex Glycans.

The carbohydrate modification of serine and threonine residues with O-linked beta- N-acetylglucosamine (O-GlcNAc) is ubiquitous and governs cellular processes ranging from cell signaling to apoptosis. The O-GlcNAc modification along with other carbohydrate modifications, including N-linked and O-linked glycans, glycolipids, and sugar polymers, all require the use of the nucleotide sugar UDP-GlcNAc, the end product of the hexosamine biosynthetic pathway (HBP). In this paper, we describe the biochemical consequences resulting from perturbation of the O-GlcNAc pathway in C. elegans lacking O-GlcNAc transferase and O-GlcNAcase activities. In ogt-1 null animals, steady-state levels of UDP-GlcNAc/UDP-GalNAc and UDP-glucose were substantially elevated. Transcripts of genes encoding for key members in the HBP (gfat-2, gna-2, C36A4.4) and trehalose metabolism (tre-1, tre-2, tps-2) were elevated in ogt-1 null animals. While there is no evidence to suggest changes in the profile of N-linked glycans in the ogt-1 and oga-1 mutants, glycans insensitive to PNGase digestion (including O-linked glycans, glycolipids, and glycopolymers) were altered in these strains. Our data support that changes in O-GlcNAcylation alters nucleotide sugar production, overall glycan composition, and transcription of genes encoding glycan processing enzymes. These data along with our previous findings that disruption in O-GlcNAc cycling alters macronutrient storage underscores the noteworthy influence this posttranslational modification plays in nutrient sensing.

Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans.

Homology-directed repair (HDR) of double-strand DNA breaks is a promising method for genome editing, but is thought to be less efficient than error-prone nonhomologous end joining in most cell types. We have investigated HDR of double-strand breaks induced by CRISPR-associated protein 9 (Cas9) in Caenorhabditis elegans. We find that HDR is very robust in the C. elegans germline. Linear repair templates with short (∼30-60 bases) homology arms support the integration of base and gene-sized edits with high efficiency, bypassing the need for selection. Based on these findings, we developed a systematic method to mutate, tag, or delete any gene in the C. elegans genome without the use of co-integrated markers or long homology arms. We generated 23 unique edits at 11 genes, including premature stops, whole-gene deletions, and protein fusions to antigenic peptides and GFP. Whole-genome sequencing of five edited strains revealed the presence of passenger variants, but no mutations at predicted off-target sites. The method is scalable for multi-gene editing projects and could be applied to other animals with an accessible germline.

Use of an activated beta-catenin to identify Wnt pathway target genes in caenorhabditis elegans, including a subset of collagen genes expressed in late larval development.

The Wnt signaling pathway plays a fundamental role during metazoan development, where it regulates diverse processes, including cell fate specification, cell migration, and stem cell renewal. Activation of the beta-catenin-dependent/canonical Wnt pathway up-regulates expression of Wnt target genes to mediate a cellular response. In the nematode Caenorhabditis elegans, a canonical Wnt signaling pathway regulates several processes during larval development; however, few target genes of this pathway have been identified. To address this deficit, we used a novel approach of conditionally activated Wnt signaling during a defined stage of larval life by overexpressing an activated beta-catenin protein, then used microarray analysis to identify genes showing altered expression compared with control animals. We identified 166 differentially expressed genes, of which 104 were up-regulated. A subset of the up-regulated genes was shown to have altered expression in mutants with decreased or increased Wnt signaling; we consider these genes to be bona fide C. elegans Wnt pathway targets. Among these was a group of six genes, including the cuticular collagen genes, bli-1 col-38, col-49, and col-71. These genes show a peak of expression in the mid L4 stage during normal development, suggesting a role in adult cuticle formation. Consistent with this finding, reduction of function for several of the genes causes phenotypes suggestive of defects in cuticle function or integrity. Therefore, this work has identified a large number of putative Wnt pathway target genes during larval life, including a small subset of Wnt-regulated collagen genes that may function in synthesis of the adult cuticle.

GEI-8, a homologue of vertebrate nuclear receptor corepressor NCoR/SMRT, regulates gonad development and neuronal functions in Caenorhabditis elegans.

NCoR and SMRT are two paralogous vertebrate proteins that function as corepressors with unliganded nuclear receptors. Although C. elegans has a large number of nuclear receptors, orthologues of the corepressors NCoR and SMRT have not unambiguously been identified in Drosophila or C. elegans. Here, we identify GEI-8 as the closest homologue of NCoR and SMRT in C. elegans and demonstrate that GEI-8 is expressed as at least two isoforms throughout development in multiple tissues, including neurons, muscle and intestinal cells. We demonstrate that a homozygous deletion within the gei-8 coding region, which is predicted to encode a truncated protein lacking the predicted NR domain, results in severe mutant phenotypes with developmental defects, slow movement and growth, arrested gonadogenesis and defects in cholinergic neurotransmission. Whole genome expression analysis by microarrays identified sets of de-regulated genes consistent with both the observed mutant phenotypes and a role of GEI-8 in regulating transcription. Interestingly, the upregulated transcripts included a predicted mitochondrial sulfide:quinine reductase encoded by Y9C9A.16. This locus also contains non-coding, 21-U RNAs of the piRNA class. Inhibition of the expression of the region coding for 21-U RNAs leads to irregular gonadogenesis in the homozygous gei-8 mutants, but not in an otherwise wild-type background, suggesting that GEI-8 may function in concert with the 21-U RNAs to regulate gonadogenesis. Our results confirm that GEI-8 is the orthologue of the vertebrate NCoR/SMRT corepressors and demonstrate important roles for this putative transcriptional corepressor in development and neuronal function.

O-GlcNAc cycling mutants modulate proteotoxicity in Caenorhabditis elegans models of human neurodegenerative diseases.

O-GlcNAcylation is an abundant posttranslational modification in the brain implicated in human neurodegenerative diseases. We have exploited viable null alleles of the enzymes of O-GlcNAc cycling to examine the role of O-GlcNAcylation in well-characterized Caenorhabditis elegans models of neurodegenerative proteotoxicity. O-GlcNAc cycling dramatically modulated the severity of the phenotype in transgenic models of tauopathy, amyloid ß-peptide, and polyglutamine expansion. Intriguingly, loss of function of O-GlcNAc transferase alleviated, whereas loss of O-GlcNAcase enhanced, the phenotype of multiple neurodegenerative disease models. The O-GlcNAc cycling mutants act in part by altering DAF-16-dependent transcription and modulating the protein degradation machinery. These findings suggest that O-GlcNAc levels may directly influence neurodegenerative disease progression, thus making the enzymes of O-GlcNAc cycling attractive targets for neurodegenerative disease therapies.

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.

NHR-23 dependent collagen and hedgehog-related genes required for molting.

NHR-23, a conserved member of the nuclear receptor family of transcription factors, is required for normal development in Caenorhabditis elegans where it plays a critical role in growth and molting. In a search for NHR-23 dependent genes, we performed whole genome comparative expression microarrays on both control and nhr-23 inhibited synchronized larvae. Genes that decreased in response to nhr-23 RNAi included several collagen genes. Unexpectedly, several hedgehog-related genes were also down-regulated after nhr-23 RNAi. A homozygous nhr-23 deletion allele was used to confirm the RNAi knockdown phenotypes and the changes in gene expression. Our results indicate that NHR-23 is a critical co-regulator of functionally linked genes involved in growth and molting and reveal evolutionary parallels among the ecdysozoa.

O-linked-N-acetylglucosamine cycling and insulin signaling are required for the glucose stress response in Caenorhabditis elegans.

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.

O-GlcNAc cycling: emerging roles in development and epigenetics.

The nutrient-sensing hexosamine signaling pathway modulates the levels of O-linked N-acetylglucosamine (O-GlcNAc) on key targets impacting cellular signaling, protein turnover and gene expression. O-GlcNAc cycling may be deregulated in neurodegenerative disease, cancer, and diabetes. Studies in model organisms demonstrate that the O-GlcNAc transferase (OGT/Sxc) is essential for Polycomb group (PcG) repression of the homeotic genes, clusters of genes responsible for the adult body plan. Surprisingly, from flies to man, the O-GlcNAcase (OGA, MGEA5) gene is embedded within the NK cluster, the most evolutionarily ancient of three homeobox gene clusters regulated by PcG repression. PcG repression also plays a key role in maintaining stem cell identity, recruiting the DNA methyltransferase machinery for imprinting, and in X-chromosome inactivation. Intriguingly, the Ogt gene resides near the Xist locus in vertebrates and is subject to regulation by PcG-dependent X-inactivation. OGT is also an enzymatic component of the human dosage compensation complex. These 'evo-devo' relationships linking O-GlcNAc cycling to higher order chromatin structure provide insights into how nutrient availability may influence the epigenetic regulation of gene expression. O-GlcNAc cycling at promoters and PcG repression represent concrete mechanisms by which nutritional information may be transmitted across generations in the intra-uterine environment. Thus, the nutrient-sensing hexosamine signaling pathway may be a key contributor to the metabolic deregulation resulting from prenatal exposure to famine, or the 'vicious cycle' observed in children of mothers with type-2 diabetes and metabolic disease.