Dynamic O-GlcNAc cycling at promoters of Caenorhabditis elegans genes regulating longevity, stress, and immunity

Abstract

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.

Fig. 1.

O-GlcNAc marks promoters throughout the C. elegans genome. An antibody specific for O-GlcNAc (RL2) was used for ChIP-on-chip analysis of the C. elegans genome. (A) The peak intensities across all six linkage groups: oga-1(ok1207), red; N2 (wild type), green; ogt-1(ok430), blue. Note that the O-GlcNAc-accumulating mutant oga-1(ok1207) (red) has the highest peak intensities, and the O-GlcNAc-deficient mutant ogt-1(ok430) (blue) displayed weak binding and served as a negative control. (B) A threshold was applied to peak intensities to obtain intervals for oga-1(ok1207) (red) and N2 (green). The number of peaks for each linkage group is shown. (C) Examples of the O-GlcNAc peaks at promoters are shown for two separate genes and a micro RNA. ftt-2 shows a large peak at its promoter. O-GlcNAc marks the first and third promoter of daf-16. The mir-54 cluster is also marked by O-GlcNAc. Green arrows represent direction of transcription.

Fig. 2.

O-GlcNAc cycling occurs asymmetrically around the TSS. (A) The average chip value for all peaks within ±2 kb of TSS-associated the genes represented on the Affymetrix expression arrays is shown for each of the strains as indicated by the colored lines sorted using the peak values for oga-1(ok1207). Note that the peak values are highest in oga-1(ok1207) mutants that are unable to remove O-GlcNAc and are at background levels in ogt-1(ok430) mutants that are unable to add O-GlcNAc. (B) The position of the O-GlcNAc peaks within ±2 kb of the annotated TSS for each of the strains as indicated. Note that at this threshold, no peaks were detected in ogt-1(ok430). (C) The position of RNA Pol II peaks relative to the transcription start using antibody ab5095 for each indicated strain as described in Materials and Methods. When averaged together, the peaks for all stains exhibit similar behavior as each do individually (Avg, black line).

Fig. 3.

O-GlcNAc cycling mutant exhibit deregulation of gene expression. Volcano plots of gene expression, as detected by microarray analysis are shown. (A and B) oga-1(ok1207). (C and D) ogt-1(ok430). Larval stages L1 (A and C) and L4 (B and D) were used for analysis. Data are plotted as geometric fold change (x axis) vs. P value (y axis). Genes were considered significantly deregulated if the geometric fold change was greater than 1.5 (bold black line) with a P value of <0.05.

Fig. 4.

O-GlcNAc cycling mutants affect aging and the stress response. The absence of O-GlcNAc modifications in ogt-1(ok430) resulted in shorter lifespan in (A) wild-type and (B) daf-2(e1370) animals. In contrast, an overabundance of O-GlcNAc modification in oga-1(ok1207) mutants had a negligible effect on wild-type lifespan (A), but extended lifespan of daf-2(e1370) animals in a daf-16-independent manner (B and C). Data are from individual representative trials; all results are presented in Dataset S5. Number of animals scored: (A) control, n = 88; ok1207, n = 83; ok430, n = 86; ok430;ok1207, n = 72; (B) control, n = 101; ok1207, n = 101; ok430, n = 95; ok430; ok1207, n = 102; (C) daf-2 daf-16 RNAi, control n = 54; ok1207, n = 59; daf-16(mu86);daf-2, control n = 88; ok1207, n = 76. Experiment in A was performed at 25 °C; similar results were obtained at 20 °C; B and C were performed at 20 °C. Animals in A and B were grown on OP50 food source; animals in C were grown on HT115 bacteria. (D) Neither mutant significantly affected daf-2 fertility at either the semipermissive (22.5 °C) or the restrictive (25 °C) temperature. Error bars represent SD for three independent experiments; P > 0.3. (E) The absence of the O-GlcNAc modification in ogt-1(ok430) decreased resistance to UV stress, whereas the abundance in oga-1(ok1207) increased stress resistance. Young adult hermaphrodites were exposed to 23 mJ of UV radiation and were followed for 3 days to determine the fraction surviving. (F) A large pool of DAF-16:GFP accumulates in the nucleus in ogt-1(ok430) L1 hermaphrodites (Left) without stress induction. In contrast, wild-type animals have very little DAF-16::GFP in the nucleus (Inset). Upon heat challenge at 37 °C for 10 min, nuclear DAF-16::GFP was detected in 100% of N2, ogt-1(ok430), and oga-1(ok1207) animals (Table S4). Quantitation of the fraction of animals with nuclear DAF-16::GFP in the absence of stress (Right).