A dynamic cycle of O-linked N-acetylglucosamine (O-GlcNAc) addition and removal acts on nuclear pore proteins, transcription factors, and kinases to modulate cellular signaling cascades. This nutrient sensing hexosamine signaling pathway is conserved from nematodes to man. A single nucleotide polymorphism in the human O-GlcNAcase gene is linked to type 2 diabetes, suggesting that perturbation of this pathway results in disease. In collaboration the Hanover lab (NIDDK), we showed that the C. elegans genome encodes the two evolutionarily conserved enzymes that mediate O-GlcNAc cycling, with the genes called ogt-1 and oga-1. We previously characterized a knockout alleles of ogt-1 and oga-1 genes. Using a combination of genomic expression arrays and chromatin immunoprecipitation (ChIP) we are looking for genes that respond to nutrient flux differently in the mutants with the hope of identifying pathways of importance. The expression analysis has revealed widespread de-regulation of gene expression in the mutants, identify affected pathways including longevity and aging. We have tested these pathways in the mutants and find alteration in function that are consistent with the gene expression patterns we observe. From the ChIP studies, we have identified a discrete number of genes associated with O-GlcNAcylated proteins. These associations are pronounced at the promoters of the genes and show some overlap with ChIP signals using RNA PolII antibodies. We are currently investigating the function role, if any, of these restricted O-GlcNAc chromatin marks. These marks have the potential to link nutritional flux in the cell directly to gene regulation, offering a novel insight into the role of O-GlcNAc cycling in animal physiology and development. 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. During the past year, we have demonstrated 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 glucoseinsulin homeostasis. In collaboration with the Hamza Lab (University of Maryland), we continue to use the C. elegans system to explore genes required for proper heme sensing and homeostasis. Further analysis of 288 heme responsive genes (hrgs) by RNAi mediated knockdown in a heme sensing strain has revealed three new genes required for proper heme homeostasis. Our study provides insights into metazoan regulation of organismal heme homeostasis. The identification of parasite-specific hrg homologs may permit the selective design and screening of drugs that specifically target heme uptake pathways in parasites without affecting the host. Thus, this work has therapeutic implications for the treatment of human iron deficiency, one of the top ten mortality factors world-wide. In collaboration with the Kostrouch and Kostrouchova Labs (Charles University, Prague) we have continued are long-term studies of the many nuclear hormone receptors (nhrs) in C. elegans. The current year project focused on a set of related nhrs that were found to have interesting responses to nutrient flux. This information adds to our general understanding of nhr function and provides insights into the biological pressures in nematodes that have led to a huge expansion of this class of ligand-regulated transcription factors. We also continue are long-term study of another gene, nhr-23, that is involved in regulating molting. Expression array analysis of nhr-23 RNAi knockdowns reveal an interesting set of potential target genes.
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