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.
Author summary: The ability to sense and adapt to changes in the environment is an essential feature of cellular life. Changes in environmental salt and water concentrations can rapidly cause cell volume swelling or shrinkage and, if left unchecked, will lead to cell and organismal death. All organisms have developed similar physiological strategies for maintaining cell volume. However, the molecular mechanisms that control these physiological outputs are not well understood in animals. Using unbiased genetic screening in C. elegans, we discovered that a highly conserved enzyme called O-GlcNAc transferase (OGT) is essential for regulating physiological responses to increased environmental solute levels. A human form of OGT can functionally substitute for worm OGT, showing that this role is conserved across evolution. Surprisingly, the only known enzymatic activity of OGT was not required for this role, suggesting this enzyme has important undescribed molecular functions. Our studies reveal a new animal-specific role for OGT in the response to osmotic stress and show that C. elegans is an important model for defining the conserved molecular mechanisms that respond to alterations in cell volume.
Cells must adapt to perturbations in extracellular osmolarity to maintain cell volume, membrane tension, and turgor pressure [[
Cells adapt to hypertonic stress primarily through the cytosolic accumulation of small uncharged molecules called organic osmolytes [[
One chemical class of osmolytes is carbohydrate polyols such as sorbitol and glycerol. During hypertonic stress, mammalian kidney epithelial cells upregulate the enzyme aldose reductase to synthesize sorbitol from glucose [[
The O-GlcNAc transferase OGT is the sole protein that adds the single ring sugar, O-GlcNAc, to serine and threonine residues of hundreds of intracellular proteins to modify their function, stability, and localization. The O-GlcNAcase OGA is the sole enzyme that removes O-GlcNAc from proteins. OGT and OGA together regulate cellular O-GlcNAc homeostasis, which is important to a variety of cellular processes including metabolism, stress responses, and proteostasis [[
All metazoans express a single ogt gene, which is absent from yeast [[
Knockout of OGT in most metazoans is lethal at either the single cell or developmental level. The notable exception to this is C. elegans, where ogt-1 null mutants are viable under standard cultivation conditions. Here, we show that C. elegans ogt-1 mutants are non-viable under a specific physiological condition, hypertonic stress. Through an unbiased screen, we identified ogt-1 as being required for expression of the osmosensitive gpdh-1p:GFP reporter. We found that under hypertonic stress conditions, ogt-1 is required for accumulation of GPDH-1-GFP protein, but not gpdh-1 mRNA. Additionally, ogt-1 mutants are unable to develop following exposure to mild hypertonic environments. Finally, we demonstrate that expression of human OGT can partially rescue the C. elegans hypertonic stress phenotype. The ability of either human or C. elegans OGT to rescue is independent of O-GlcNAcylation catalytic activity but dependent on the OGT TPR domain. These results demonstrate for the first time a specific role for OGT in the essential process of osmoregulation and suggest that this function is conserved across >700 million years of evolution.
In C. elegans, hypertonic stress rapidly and specifically upregulates expression of the osmolyte biosynthesis gene gpdh-1, which we visualized with a gpdh-1p:GFP transcriptional reporter [[
Graph: Fig 1 gpdh-1 transcriptional and translational reporters are upregulated by hypertonic stress.(A) Wide-field fluorescence microscopy of day 2 adult animals expressing drIs4 (col-12p:dsRed; gpdh-1p:GFP) exposed to 50 or 250 mM NaCl NGM plates for 18 hours. Images depict merged GFP and RFP channels. Scale bar = 100 microns. (B) COPAS Biosort quantification of GFP and RFP signal in day 2 adult animals expressing drIs4 exposed to 50 or 250 mM NaCl NGM plates for 18 hours. Each point represents the quantified signal from a single animal. N ≥ 276 for each group. (C) Population mean of the normalized GFP/RFP ratio from data in 1B. Data are expressed as mean ± S.D. with individual points shown. ****—p<0.0001 (Mann-Whitney test). (D) Wide-field fluorescence microscopy of day 2 adult animals expressing a kbIs6 (gpdh-1p:GPDH-1-GFP) translational fusion protein exposed to 50 or 250 mM NaCl NGM plates for 18 hours. Scale bar = 100 microns. (E) COPAS Biosort quantification of GFP and TOF signal in day 2 adult animals expressing the kbIs6 translational fusion protein exposed to 50 or 250 mM NaCl NGM plates for 18 hours. N ≥ 276 for each group. (F) Population mean of the normalized GFP/TOF ratio from data in 1E. Data are expressed as mean ± S.D. ****—p<0.0001 (Mann-Whitney test).
Taking advantage of the binary nature of GFP activation by hypertonic stress in the drIs4 strain, we designed an unbiased F2 forward genetic screen for mutants that fail to activate GFP expression during hypertonic stress, but exhibit no effects on RFP (No induction of osmolyte biosynthesis gene expression or Nio mutants; Fig 2A). From this screen of ~120,000 haploid genomes, we identified two recessive alleles, dr15 and dr20, that genetically fail to complement each other. Whole genome sequencing and bioinformatics revealed that each allele contained a distinct nonsense mutation in the gene encoding the O-GlcNAc transferase ogt-1 (S1, S2 and S3 Tables, Fig 2B). Two independently isolated ogt-1 deletion alleles, ok430 and ok1474, as well as wild type worms exposed to ogt-1(RNAi) also exhibited a Nio phenotype, and ok430 and ok1474 failed to complement the dr15 and dr20 alleles. (Fig 2B, 2C, 2D, S1A, S1B and S2 Tables). CRISPR reversion of the dr20 Q600STOP mutation back to wild type was sufficient to rescue the ogt-1 Nio phenotype, indicating that other ENU induced mutations in the background do not contribute to the Nio phenotype (Fig 2E). Additionally, transgenic overexpression of ogt-1 in the ogt-1(dr20) mutant led to supra-physiological rescue (S1C Fig). Finally, we found that knock down of ogt-1 during post-developmental stages with ogt-1(RNAi) was sufficient to cause a Nio phenotype, suggesting that OGT-1 is not required for the establishment of developmental structures necessary for responding to hypertonic stress (Fig 2F). ogt-1 is not required for the activation of other stress inducible reporters since inhibition of ogt-1 resulted in small but significant increase in a heat shock inducible GFP reporter and had no effect on an endoplasmic reticulum stress inducible GFP reporter (S2 Fig). A reporter for the expression of the antimicrobial peptide nlp-29 (frIs7) is induced by several stressors including hypertonic stress [[
Graph: Fig 2 The conserved O-GlcNAc transferase OGT-1 is required for the upregulation of the gpdh-1 transcriptional reporter by hypertonic stress.(A) ENU-based forward genetic screening strategy and mutant identification workflow. (B) C. elegans OGT-1 and Homo sapiens OGT protein domain diagrams detailing the positions of the two LOF ogt-1 alleles identified in the screen (dr15 and dr20), two independently isolated ogt-1 deletion mutations (ok430 and ok1474), and two mutations that disrupt catalytic activity of the enzyme (H612A and K957M). The precise breakpoints of ok1474 have not been determined. (C) Wide-field fluorescence microscopy of day 2 adult drIs4 and ogt-1;drIs4 mutant animals exposed to 50 or 250 mM NaCl NGM plates for 18 hours. Images depict merged GFP and RFP channels. Scale bar = 100 microns. (D) COPAS Biosort quantification of GFP and RFP signal in day 2 adult animals expressing drIs4 or ogt-1;drIs4 exposed to 50 or 250 mM NaCl NGM plates for 18 hours. Data are represented as the relative fold induction of normalized GFP/RFP ratio on 250 mM NaCl NGM plates versus 50 mM NaCl NGM plates, with WT fold induction set to 1. Each point represents the quantified signal from a single animal. Data are expressed as mean ± S.D. ****—p<0.0001 (Kruskal-Wallis test with post hoc Dunn's test). N ≥ 62 for each group. (E) COPAS Biosort quantification of GFP and RFP signal in day 2 adult animals expressing drIs4 or drIs4;ogt-1(dr20) exposed to 50 or 250 mM NaCl NGM plates for 18 hours. ogt-1(dr20 dr36) is a strain in which the dr20 mutation is converted back to WT using CRISPR/Cas9 genome editing. Data are represented as relative fold induction of normalized GFP/RFP ratio on 250 mM NaCl NGM plates versus 50 mM NaCl NGM plates, with WT fold induction set to 1. Each point represents the quantified signal from a single animal. Data are expressed as mean ± S.D. ****—p<0.0001 (Kruskal-Wallis test with post hoc Dunn's test). N ≥ 170 for each group. Inset: Wide-field fluorescence microscopy of day 2 adult animals expressing drIs4 in the WT or indicated ogt-1 mutant background exposed to 250 mM NaCl NGM plates for 18 hours. Images depict merged GFP and RFP channels. Scale bar = 100 microns. (F) COPAS Biosort quantification of GFP and RFP signal in day 2 adult animals expressing drIs4 exposed to 50 or 250 mM NaCl NGM plates for 18 hours. Animals were placed on empty vector(RNAi) (ev(RNAi)) or ogt-1(RNAi) plates at the indicated stage. Data are represented as normalized fold induction of normalized GFP/RFP ratio on 250 mM NaCl RNAi plates relative to on 50 mM NaCl RNAi plates, with ev(RNAi) set to 1 for each RNAi timepoint. Each point represents the quantified signal from a single animal. Data are expressed as mean ± S.D. ****—p<0.0001 (Mann-Whitney test). N ≥ 144 for each group.
Since ogt-1 is required for induction of the gpdh-1p:GFP transgenic reporter by hypertonic stress, we hypothesized that endogenous osmosensitive mRNAs would not be upregulated in an ogt-1 mutant. To test this, we used qPCR to measure the expression levels of several previously described mRNAs that are induced by osmotic stress [[
Graph: Fig 3 OGT-1 functions post-transcriptionally to regulate osmosensitive GPDH-1-GFP protein expression.(A) qPCR of gpdh-1, hmit-1.1, and nlp-29 mRNA from WT and ogt-1(dr20) day 2 adult animals expressing drIs4 exposed to 50 or 250 mM NaCl NGM plates for 24 hours. Data are represented as fold induction of RNA on 250 mM NaCl relative to 50 mM NaCl. Data are expressed as mean ± S.D. **—p<0.01, n.s. = nonsignificant (Student's two-tailed t-test). N ≥ 3 biological replicates of 35 animals for each group. (B) qPCR of GFP mRNA from WT and ogt-1(dr20) day 2 animals expressing drIs4 exposed to 50 or 250 mM NaCl NGM plates for 24 hours. Data are represented as fold induction of RNA on 250 mM NaCl relative to 50 mM NaCl. Data are expressed as mean ± S.D. *—p<0.05 (Student's two-tailed t-test). N ≥ 3 biological replicates of 35 animals for each group. (C) COPAS Biosort quantification of GFP and TOF signal in day 2 adult animals expressing the kbIs6 GPDH-1 translational fusion exposed to 50 or 250 mM NaCl NGM plates for 18 hours. The ogt-1(dr34) allele carries the same homozygous Q600STOP mutation as the ogt-1(dr20) allele and was introduced using CRISPR/Cas9. Data are represented as relative fold induction of normalized GFP/TOF ratio on 250 mM NaCl NGM plates versus 50 mM NaCl NGM plates. Each point represents the quantified signal from a single animal. Data are expressed as mean ± S.D. ****—p<0.0001 (Mann-Whitney test). N ≥ 84 for each group. Inset: Wide-field fluorescence microscopy of day 2 adult animals expressing the kbIs6 translational fusion protein exposed to 250 mM NaCl NGM plates for 18 hours. Scale bar = 100 microns. (D) qPCR of gpdh-1 mRNA from day 2 adult animals expressing the kbIs6 translational fusion exposed to 50 or 250 mM NaCl NGM plates for 24 hours. Strains include WT and ogt-1(dr34). The ogt-1(dr34) allele is the dr20 point mutation introduced using CRISPR/Cas9. Data are represented as fold induction of RNA on 250 mM NaCl relative to 50 mM NaCl. Data are expressed as mean ± S.D. n.s. = nonsignificant (Student's two-tailed t-test). N = 3 biological replicates of 35 animals for each group. (E) Immunoblot of GFP and β-actin in lysates from day 2 adult animals exposed to 50 mM or 250 mM NaCl for 18 hours. The animals express a CRISPR/Cas9 edited knock-in of GFP into the endogenous gpdh-1 gene (gpdh-1(dr81)). ogt-1 carries the dr83 allele, which is the same homozygous Q600STOP mutation as the ogt-1(dr20) allele and was introduced using CRISPR/Cas9. Top: Normalized quantification of immunoblots. *—p<0.05 (One-way ANOVA with post hoc Dunnett's test). Bottom: Representative immunoblot. N = 3 biological replicates.
To further examine if OGT-1 affects the coupling between hypertonic stress induced mRNA and protein expression, we measured GPDH-1-GFP protein levels in an ogt-1 mutant (dr34; CRISPR/Cas9 knock-in of the dr20(Q600STOP) mutation) expressing a GPDH-1 translational reporter (GPDH-1:GFP). As we observed for the gpdh-1p:GFP transcriptional reporter, ogt-1(dr34) mutants failed to induce the GPDH-1:GFP protein in response to hypertonic stress (Fig 3C, S3B Fig). However, the mRNA from this translational reporter was still induced to wild type levels (Fig 3D). mRNA induction of the translational reporter did not exceed wild type levels like we saw for the transcriptional reporter for unknown reasons. Importantly the requirement for ogt-1 in the hypertonic stress response is not transgene dependent because ogt-1 is also required for the hypertonic induction of a CRISPR/Cas9 engineered endogenously expressed GPDH-1:GFP fusion protein, which we confirmed to be functional based on its ability to exhibit acute adaptation to hypertonic stress (Fig 3E and S4D Fig). Like in the transcriptional and translational gpdh-1 reporters, gpdh-1:gfp mRNA levels in the gpdh-1:gfp CRISPR allele were induced to WT levels or higher (S3C and S3D Fig). In conclusion, these results suggest that ogt-1 functions downstream of osmosensitive mRNA upregulation, but upstream of osmosensitive GPDH-1-GFP protein expression.
C. elegans upregulates osmosensitive genes, including gpdh-1, to survive and adapt to hypertonic challenges. Survival and adaptation can be measured in several ways. Survival measures the ability of animals grown under standard laboratory isotonic conditions to survive a 24 hour exposure to an indicated level of hypertonic stress. Acute adaptation measures the ability of animals to activate adaptive responses that permit survival under normally lethal hypertonic conditions using a pre-conditioning stimulus. Chronic adaptation measures the ability of animals develop under non-lethal hypertonic conditions.
We found that loss of ogt-1 had no effect on acute survival during hypertonic stress (S4A Fig) [[
Graph: Fig 4 ogt-1 is required for physiological and genetic adaptation to hypertonic stress.(A) Percent of moving unadapted and adapted day 3 adult animals exposed to 600 mM NaCl NGM plates for 24 hours. Strains expressing drIs4 are on the left of the dashed orange line and those not expressing drIs4 are on the right. ok1558 is an out-of-frame deletion allele that generates a premature stop codon in exon 2 of gpdh-1 and is therefore a likely null allele. Data are expressed as mean ± S.D. ****—p<0.0001 (One-way ANOVA with post hoc Dunnett's test). N = 5 replicates of 20 animals for each strain. (B) COPAS Biosort quantification of GFP and RFP signal in day 2 adult animals expressing drIs4 exposed to 50 mM NaCl NGM plates. Data are represented as the fold induction of normalized GFP/RFP ratio on 50 mM NaCl NGM plates, with osm-8(dr9) set to 1. osm-8(dr9) was isolated in a previous genetic screen for new osm-8 alleles but encodes the same mutation as the n1518 reference allele. Each point represents the quantified signal from a single animal. Data are expressed as mean ± S.D. ****—p<0.0001 (Mann-Whitney test). N ≥ 109 for each group. Inset: Wide-field fluorescence microscopy of day 2 adult animals expressing drIs4 exposed to 50 mM NaCl NGM plates. Images depict merged GFP and RFP channels. Scale bar = 100 microns. (C) Percent of moving (OSR, osmotic stress resistant) day 1 animals after exposure to 500 mM NaCl or 700 mM NaCl for 10 minutes. Data are represented as mean ± S.D. ***—p<0.001, ****—p<0.0001 (Student's two-tailed t-test). N = 5 replicates of 10 animals for each strain.
In addition to physiological exposures, adaptation to hypertonic stress can also be induced genetically via loss of function mutations in several hypodermis expressed secreted extracellular matrix (ECM) proteins [[
In C. elegans, a CRISPR generated OGT-1-GFP allele is functional and is ubiquitously expressed throughout somatic cells in the nucleus, consistent with previous observations (S4D and S5D Figs) [[
Graph: Fig 5 Non-canonical activity of ogt-1 primarily in the hypodermis regulates gpdh-1 induction by hypertonic stress through a functionally conserved mechanism.(A) Wide-field fluorescence microscopy of day 2 adult animals expressing drIs4 exposed to 50 or 250 mM NaCl NGM plates for 18 hours. Strains express an ogt-1 cDNA from the indicated tissue-specific promoter. Images depict the GFP channel only for clarity. The RFP signal was unaffected in these rescue strains (not shown). Scale bar = 100 microns. (B) COPAS Biosort quantification of GFP and RFP signal in day 2 adult animals expressing drIs4 exposed to 50 or 250 mM NaCl NGM plates for 18 hours. Data are represented as the 'Degree of Rescue' on 250 mM NaCl NGM plates relative to on 50 mM NaCl NGM plates (see 'Methods' for description of this calculation). Each point represents the quantified signal from a single animal. Data are expressed as mean ± S.D. ***—p<0.001, ****—p<0.0001, n.s. = nonsignificant (Kruskal-Wallis test with post hoc Dunn's test). N ≥ 110 for each group. (C) Wide-field fluorescence microscopy of day 2 adult animals expressing drIs4 exposed to 50 or 250 mM NaCl NGM plates for 18 hours. For the WT and catalytically inactive human rescue strains, we expressed a human cDNA corresponding to isoform 1 of OGT using an extrachromosomal array. Images depict the GFP channel for clarity. The RFP signal was unaffected in these rescue strains (not shown). Scale bar = 100 microns. (D) COPAS Biosort quantification of GFP and RFP signal in day 2 adult animals expressing drIs4 exposed to 50 or 250 mM NaCl NGM plates for 18 hours. Data are represented as the 'Degree of Rescue' on 250 mM NaCl NGM plates relative to on 50 mM NaCl NGM plates (see 'Methods' for description of this calculation). Each point represents the quantified signal from a single animal. Data are expressed as mean ± S.D. ****—p<0.0001, ***—p <0.001, **—p<0.01 (Kruskal-Wallis test with post hoc Dunn's test). N ≥ 40 for each group. (E) Wide-field fluorescence microscopy of day 2 adult animals expressing drIs4 exposed to 50 or 250 mM NaCl NGM plates for 18 hours. Images depict the GFP channel only for clarity. The RFP signal was unaffected in these rescue strains (not shown). Scale bar = 100 microns. (F) COPAS Biosort quantification of GFP and RFP signal in day 2 adult animals expressing drIs4 exposed to 50 or 250 mM NaCl NGM plates for 18 hours. Data are represented as the fold induction of normalized GFP/RFP ratio on 50 and 250 mM NaCl NGM plates relative to on 50 mM NaCl NGM plates. Each point represents the quantified signal from a single animal. Data are expressed as mean ± S.D. ****—p<0.0001, n.s = nonsignificant (Kruskal-Wallis test with post hoc Dunn's test). N ≥ 81 for each group. (G) Percent of moving unadapted and adapted day 3 adult animals expressing drIs4 exposed to 600 mM NaCl NGM plates for 24 hours. Data are expressed as mean ± S.D. ****—p<0.0001 (One-way ANOVA with post hoc Tukey's test). N = 5 replicates of 20 animals for each strain.
Given that C. elegans OGT-1 is highly conserved with human OGT (Fig 2B), we asked if human OGT could functionally replace C. elegans OGT-1 in the hypertonic stress response. Overexpression of a human OGT cDNA from the native C. elegans ogt-1 promoter exhibited weak but statistically significant rescue of gpdh-1p:GFP induction by hypertonic stress in an ogt-1 LOF mutant (Fig 5C and 5D and S5B Fig). Unexpectedly, catalytically inhibited human OGT (OGT H498A) rescued gpdh-1p:GFP induction by hypertonic stress in an ogt-1(dr20) LOF mutant to the same extent as wild type human OGT (Fig 5C and 5D and S5B Fig) [[
In addition to the catalytic domain of OGT, another functionally important domain in OGT is the tetratricopeptide repeat (TPR) domain. This domain mediates protein-protein interactions thought to be important for the binding of O-GlcNAcylation substrates [[
Through an unbiased forward genetic screen for mutants that disrupt osmosensitive expression of a gpdh-1:GFP reporter in C. elegans, we identified multiple alleles of the O-GlcNAc transferase OGT-1. ogt-1 likely functions as a key signaling component of the hypertonic stress response, since post-developmental knockdown of ogt-1 is sufficient to cause the Nio phenotype. ogt-1-dependent signaling in the hypertonic stress response appears to occur primarily in the hypodermis, a known osmosensitive tissue in C. elegans [[
Graph: Fig 6 A non-catalytic function of ogt-1 is required to couple hypertonic stress induced transcription and translation to enable physiological adaptation to hypertonic stress.In WT, animals exposed to hypertonic stress induce the transcription of osmosensitive mRNAs, such as gpdh-1. These mRNAs are rapidly translated into protein by the ribosome, facilitating adaptation to hyperosmotic stress. Loss of ogt-1 does not interfere with hypertonic stress induced transcription. Rather, loss of ogt-1 decreases hypertonic stress induced protein levels. ogt-1 may facilitate stress-induced translation via several potential mechanisms, including regulation of mRNA cleavage and 3'UTR usage, mRNA export, initiation factor interactions, or ribosomal elongation of the transcript. Importantly, the tetratricopeptide repeat (TPR) domain but not the O-GlcNAcylation function of OGT-1 is required in the hypertonic stress response.
C. elegans is the primary genetic model system for studies of ogt-1 because it is the only organism in which loss of ogt-1 is viable [[
Our studies reveal a critical and previously unappreciated condition-specific role of OGT-1 in adaptation to hypertonic stress. This phenotype is completely penetrant and one of the strongest ogt-1 phenotypes described to date. Although their ability to survive acute hypertonic stress is unaffected, ogt-1 mutants are unable to adapt and develop following extremely mild shifts in extracellular osmolarity (250 mM NaCl). Such conditions have minimal effects on the ability of wild type animals to adapt and develop [[
In mammals, OGT is essential for cell division, a physiological process that involves tight regulation of cell volume [[
Knockout of OGT in mammalian cells leads to a rapid loss in cellular viability [[
Cell volume regulation during environmental stress requires upregulation of osmoprotective proteins, including those that regulate osmolyte accumulation. In almost all cases, these genes are upregulated at the transcriptional level [[
The regulation of stress responsive gene expression by OGT is not a new paradigm. Previous data has shown that it plays both a transcriptional and post-transcriptional role in stress response gene expression. For example, OGT-1 O-GlcNAcylates the oxidative stress responsive transcription factor SKN-1 to facilitate upregulation of antioxidant gene transcription [[
Since the discovery of OGT, C. elegans has been an important tool for characterizing the role of OGT in cell signaling because it is the only organism in which genetic loss of OGT generates viable cells and organisms [[
In conclusion, our unbiased genetic screening approaches in C. elegans have revealed a previously unappreciated requirement for non-canonical OGT signaling in a critical and conserved aspect of cell physiology. The primary function of OGT has long been assumed to be due to its catalytic O-GlcNAcylation activity. However, as we and others have shown, OGT also has critical and conserved non-catalytic functions that warrant further study [[
Strains were cultured on standard NGM media with E.coli OP50 bacteria at 20°C unless otherwise noted. The following strains were used; N2 Bristol WT, OG119 drIs4 [gpdh-1p:GFP; col-12p:dsRed2], VP223 kbIs6 [gpdh-1p:gpdh-1-GFP], OG971 ogt-1(dr15);drIs4, OG969 ogt-1(dr20);drIs4, OG1034 ogt-1(ok430);drIs4, OG1035 ogt-1(ok1474);drIs4, OG1066 ogt-1(dr20 dr36);drIs4, OG1064 ogt-1(dr34);unc-119(ed3);kbIs6, OG1115 gpdh-1(dr81) [gpdh1:GFP], OG1123 gpdh-1(dr81);ogt-1(dr84), RB1373 gpdh-1(ok1558), OG1048 osm-8(dr9);unc-4(e120);drIs4, OG1049 osm-8(dr9);unc-4(e120);ogt-1(dr20):drIs4, OG1111 ogt-1(dr20);drIs4;drEx468 [ogt-1p:ogt-1cDNA:ogt-13'utr; rol-6(su1006)], OG1119 ogt-1(dr20);drIs4;drEx469 [dpy-7p:ogt-1cDNA:ogt-13'utr; rol-6(su1006)], OG1120 ogt-1(dr20);drIs4;drEx470 [nhx-2p:ogt-1cDNA:ogt-13'utr; rol-6(su1006)], OG1121 ogt-1(dr20);drIs4;drEx471 [myo-2p:ogt-1cDNA:ogt-13'utr; rol-6(su1006)], OG1122 ogt-1(dr20);drIs4;drEx472 [rab-3p:ogt-1cDNA:ogt-13'utr; rol-6(su1006)], OG1125 ogt-1(dr20);drIs4;drEx473 [ogt-1p:human OGT isoform 1cDNA:ogt-13'utr; rol-6(su1006)], OG1126 ogt-1(dr20);drIs4;drEx474 [ogt-1p:human OGT isoform 1 H498AcDNA:ogt-13'utr; rol-6(su1006)], OG1046 ogt-1(dr20);drIs4;drEx465 [ogt-1p:ogt-1 genomic], TJ375 gpIs1 [hsp16.2p:GFP], SJ4005 zcIs4 [hsp4:GFP] V, OG1081 ogt-1(dr50);zcIs4, MT3643 osm-11(n1604), OG1083 ogt-1(dr52);osm-11(n1604), OG1135 ogt-1(dr86);drIs4, OG1140 ogt-1(dr90);drIs4, OG1124 ogt-1(dr84) [ogt-1:GFP], OG1139 ogt-1(dr84 dr89), OG1141 ogt-1(dr84 dr91), OG1156 ogt-1(dr93);drIs4, OG1157 ogt-1(dr84 dr94), IG274 frIs7 [nlp-29p:GFP + col-12p:dsRed]. To create mutant combinations, we used either standard genetic crossing approaches or CRISPR/Cas9 genetic engineering (see below for CRISPR methods). The homozygous genotype of every strain was confirmed either by DNA sequencing of the mutant lesion, restriction digest, or a loss of function phenotype.
L4 stage drIs4 animals (P
Each nio mutant was backcrossed to drIs4 males three times. F
nio/+ males were crossed with hermaphrodites homozygous for the mutation being complementation tested. The F
DNA was isolated from starved OP50 NGM plates with WT(drIs4) or mutant animals using the Qiagen Gentra Puregene Tissue Kit (Cat No 158667). The supplementary protocol for "Purification of archive-quality DNA from nematode suspensions using the Gentra Puregene Tissue Kit" available from Qiagen was used to isolate DNA. DNA samples were sequenced by BGI Americas (Cambridge, MA) with 20X coverage and paired-end reads using the Illumina HiSeq X Ten System.
A Galaxy workflow was used to analyze the FASTQ forward and reverse reads obtained from BGI. The forward and reverse FASTQ reads from the animal of interest, C. elegans reference genome Fasta file (ce11m.fa), and SnpEff download gene annotation file (SnpEff4.3 WBcel235.86) were input into the Galaxy workflow. The forward and reverse FASTQ reads were mapped to the reference genome Fasta files with the Burrows-Wheeler Aligner (BWA) for Illumina. The resultant Sequence Alignment Map (SAM) dataset was filtered using bitwise flag and converted to the Binary Alignment Map (BAM) format [[
Gravid adult animals on RNAi plates (NGM + 1mM IPTG + 25ug/ml carbenicillin) were hypochlorite treated. Synchronized L1s from the hypochlorite treatment were allowed to develop on RNAi plates until day one adult. Day 1 adults were seeded onto either 50 mM or 250 mM RNAi or OP50 NaCl plates. For the developmental timed RNAi experiment (Fig 2F), hypochlorite synchronized L1 animals were seeded onto empty vector(RNAi) (ev(RNAi)) or ogt-1(RNAi). At the indicated stages, animals were manually transferred from ev(RNAi) to ogt-1(RNAi). For the adult-specific RNAi, day 1 adult animals were transferred from ev(RNAi) to ogt-1(RNAi) plates containing either 50 mM NaCl or 250 mM NaCl. The identity of all RNAi clones was confirmed by sequencing.
Day one adults from a synchronized egg lay or hypochlorite preparation were seeded on 50 or 250 mM NaCl OP50 or the indicated RNAi NGM plates. After 18 hours, the GFP and RFP fluorescence intensity, time of flight (TOF), and extinction (EXT) of each animal was acquired with the COPAS Biosort. Events in which the RFP intensity of adult animals (TOF 400–1200) was <20 (dead worms or other objects) were excluded from the analysis. The GFP fluorescence intensity of each animal was normalized to its RFP fluorescence intensity or TOF. To determine the fold induction of GFP for each animal, each GFP/RFP or GFP/TOF was divided by the average GFP/RFP or GFP/TOF of that strain exposed to 50 mM NaCl. The relative fold induction was determined by setting the fold induction of drIs4 exposed to 250 mM NaCl to 1. For the data in Fig 5, the 'Degree of Rescue' was calculated as (A
The drIs4 strain was made by injecting wild type animals with gpdh-1p:GFP (20ng/μL) and col-12p:dsRed2 (100ng/μL) to generate the extrachromosomal array drEx73, which was integrated using UV bombardment, followed by isolation of animals exhibiting 100% RFP fluorescence. The resulting strain was outcrossed five times to wild type to generate the homozygous integrated transgene drIs4. kbIs6 was generated from a Gene Gun bombardment of unc-119(ed3) animals with a gpdh-1p:gpdh-1:GFP plasmid and an unc-119(+) rescue plasmid (pMM051). The resulting strain was outcrossed five times to generate kbIs6. drIs4 is integrated on LGIV. The integration site for kbIs6 is unmapped.
All the primers used to generate the rescue constructs can be found in S5 Table. The genomic ogt-1 rescue construct (used in the drEx465 extrachromosomal array) was made by amplifying ogt-1 with 2 kb of sequence upstream of the start codon and 1 kb of sequence downstream of the stop codon. All other rescue constructs (used in extrachromosomal arrays drEx468 –drEx474) were made using Gibson Assembly. The ogt-1 promoter, ogt-1 cDNA, and ogt-1 3'UTR were cloned into the pPD61.125 vector through a four component Gibson Assembly reaction. This vector was used as the backbone for all other promoter and human OGT rescue constructs. All rescue constructs were confirmed by Sanger sequencing. Extrachromosomal array lines were made by injecting day one adult animals with the rescue construct (20 ng/μL) and rol-6(su1006) (100 ng/μL).
CRISPR allele generation and TPR deletion was preformed using the single-stranded oliodeoxynucelodite donors (ssODN) method [[
Day one animals were plated on 50 mM or 250 mM NaCl OP50 NGM plates for 24 hours. Unless noted otherwise, after 24 hours, 35 animals were picked into 50 μL Trizol for mRNA isolation. RNA isolation followed a combined Trizol/RNeasy column purification method as previously described [[
Cell lysates were prepared from hypochlorite synchronized day 1 adult animals exposed to 50 mM or 250 mM NaCl plates for 18 hours. 3–5 non-starved 10 cm plates were concentrated into a 100 μL mixture. NuPage LDS Sample Buffer (4X) and NuPAGE Sample Reducing Agent (10X) were added and the sample was frozen and thawed three times at -80°C and 37°C. Prior to get loading, the sample was heated to 100°C for 10 minutes and cleared by centrifugation at 4°C, 12,000 x g for 15 minutes. The cleared supernatant was run on a 4–12% or 8% Bis-Tris Mini Plus gel and transferred to a nitrocellulose membrane using iBlot 2 NC Regular Stacks and the iBlot 2 Dry Blotting System. The membranes were placed on iBind cards and the iBind western device was used for the antibody incubation and blocking. The Flex Fluorescent Detection (FD) Solution Kit or the iBind Solution Kit was used to dilute the antibodies and block the membrane. The antibodies used are listed in S7 Table. The following antibody dilutions were used: 1:1000 α-GFP, 1:2000 α-ß-Actin, 1:2000 α-mouse HRP, and 1:4000 Goat α-Mouse IgG (H+L) Cross-Absorbed Secondary DyLight 800. A C-DiGit Licor Blot Scanner (LI-COR Biosciences, Lincoln, NE) or an Odyssey CLx imaging System (LI-COR Biosciences, Lincoln, NE) were used to image membranes incubated with a chemiluminescent or fluorescent secondary antibody, respectively.
Worms were anesthetized (10mM levamisole) and mounted on either agar plates for low magnification stereo fluorescence microscopy or silicone greased slide chambers for high magnification wide-field microscopy. Images were collected on either a Leica MZ16FA fluorescence stereo dissecting scope with a DFC345 FX camera or a Leica DMI4000B inverted compound microscope with a Leica DFC 340x digital camera using the Leica Advanced Fluorescence software (Leica Microsystems, Wetzlar, Germany). Unless noted, images within an experiment were collected using the same exposure and zoom settings. Unless noted, images depict merged GFP and RFP channels of age matched day 1 adult animals exposed to 50 or 250 mM NaCl for 18 hours.
Embryos from a hypochlorite preparation were freeze-cracked on a superfrost slide, fixed with 4% paraformaldehyde, blocked with bovine serum albumin (BSA), incubated with a 1:400 dilution of α-O-GlcNAc monoclonal antibody (RL2) overnight, and incubated with 1:400 dilution of 1:400 goat α-mouse IgG, IgM (H+L) Secondary Antibody, Alexa Fluor 488 for 4–6 hours [[
Day one adult animals were transferred to five 50 mM NaCl OP50 NGM plates and five 200 mM NaCl OP50 plates. ~25 animals were transferred to each plate (i.e. ~125 animals total per condition per genotype). After 24 hours, 20 animals from each 50 mM or 200 mM plate were transferred to 600 mM NaCl OP50 NGM plates. Animals were scored for movement after 24 hours on the 600 mM NaCl OP50 NGM plates. The experimenter was blinded to genotype. To be counted as moving, the animal had to move greater than half a body length. Animals that were not moving were lightly tapped on the nose to confirm that they were paralyzed or dead.
5 L4 animals were transferred to 50 or 250 mM NaCl OP50 NGM plates. Plates were monitored over several days. For the brood and development assays, a single L4 animal was transferred to a 50 or 250 mM NaCl OP50 NGM plate. Embryos counts and transfer of the mother to a new plate were done daily until the mother stopped laying eggs. Progeny from each animal were allowed to develop and the number of L4s was counted. Percent of developed embryos was calculated by dividing the number of L4s on a plate by the number of embryos originally laid on that plate.
Survival and Osr assays were performed as previously described [[
Comparisons of means were analyzed with either a two-tailed Students t-test (2 groups) or ANOVA (3 or more groups) using the Dunnett's or Tukey's post-test analysis as indicated in GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, CA). COPAS biosort data is nonparametric and was therefore analyzed using a Mann-Whitney test (2 groups) or Kruskal-Wallis test (3 or more groups) using the Dunn's post-test analysis in GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, CA). p-values of <0.05 were considered significant. Data are expressed as mean ± S.D. with individual points shown. Underlying numerical data for all data are found in S9–S45 Tables.
S1 Fig. ogt-1 is required for upregulation of the transcriptional gpdh-1:GFP reporter (drIs4) by hypertonic stress.
(A) Immunoblot of GFP and β-actin in lysates from WT and ogt-1 mutant animals expressing drIs4 exposed to 50 or 250 mM NaCl NGM plates for 18 hours. (B) COPAS Biosort quantification of GFP and RFP signal in day 2 adult WT animals expressing drIs4 exposed to 50 or 250 mM NaCl NGM plates for 18 hours. Animals were grown on ev(RNAi) or ogt-1(RNAi) plates for multiple generations. Data are represented as fold induction of normalized GFP/RFP ratio on 250 mM NaCl RNAi plates relative to on 50 mM NaCl RNAi plates. Each point represents the quantified signal from a single animal. Data are expressed as mean ± S.D. ****—p<0.0001 (Mann-Whitney test). N ≥ 334 for each group. Inset: Wide-field fluorescence microscopy of day 2 adult animals expressing drIs4 exposed to 250 mM NaCl NGM plates for 18 hours. Animals were grown on ev(RNAi) or ogt-1(RNAi) plates for multiple generations. Images depict merged GFP and RFP channels. Scale bar = 100 microns. (C) COPAS Biosort quantification of GFP and RFP signal in day 2 adult animals expressing drIs4 exposed to 50 or 250 mM NaCl NGM plates for 18 hours in the indicated genetic background. drEx465 is an extrachromosomal array expressing a 10.3 Kb ogt-1 genomic DNA fragment containing ~2Kb upstream and ~1Kb downstream of the ogt-1 coding sequence. Data are represented as fold induction of normalized GFP/RFP ratio on 250 mM NaCl NGM plates relative to on 50 mM NaCl NGM plates. Each point represents the quantified signal from a single animal. Data are expressed as mean ± S.D. ****—p<0.0001, *—p<0.05 (Kruskal-Wallis test with post hoc Dunn's test). N ≥ 37 for each group.
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S2 Fig. ogt-1 is not required for upregulation of transcriptional reporters by heat shock or ER stress.
(A) Wide-field fluorescence microscopy of day 2 adult animals expressing hsp-16.2p:GFP (gpIs1) grown on ev(RNAi), ogt-1(RNAi), or hsf-1(RNAi) plates and exposed to control or heat shock conditions (35°C for 3 hours, 18 hour recovery at 20°C). Images depict the GFP channel, since there is not a normalizing RFP reporter in these strains. Scale bar = 100 microns. (B) COPAS Biosort quantification of GFP and TOF signal from animals in (A). Data are represented as fold induction of normalized GFP/TOF ratio of animals exposed to heat shock conditions relative to animals exposed to control conditions. Each point represents the quantified signal from a single animal. Data are expressed as mean ± S.D. ****—p<0.0001 (Kruskal-Wallis test with post hoc Dunn's test). N ≥ 158 for each group. (C) Wide-field fluorescence microscopy of day 2 adult animals expressing hsp-4p:GFP (zcIs4) exposed to DTT plates for 18 hours. The ogt-1(dr50) allele carries the same homozygous Q600STOP mutation as the ogt-1(dr20) allele and was introduced using CRISPR/Cas9. Images depict the GFP channel. Scale bar = 100 microns. (D) COPAS Biosort quantification of GFP and TOF signal from animals in (C). Data are represented as fold induction of normalized GFP/TOF ratio of animals exposed to DTT plates relative to animals exposed to control plates. Each point represents the quantified signal from a single animal. Data are expressed as mean ± S.D. ****—p<0.0001, n.s = nonsignificant (Kruskal-Wallis test with post hoc Dunn's test). N ≥ 48 for each group. (E) COPAS Biosort quantification of GFP and RFP signal from day 2 animals expressing gpdh-1p:GFP (drIs4) or nlp-29:GFP (frIs7) exposed to 250 mM NaCl for 18 and 24 hours respectively. Data are represented as fold induction of normalized GFP/RFP ratio of animals exposed to 250 mM NaCl plates relative to animals exposed to 50 mM NaCl plates. Each point represents the quantified signal from a single animal. Data are expressed as mean ± S.D. ****—p<0.0001 (Kruskal-Wallis test with post hoc Dunn's test). N ≥ 173 for each group.
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S3 Fig. ogt-1 is not required for upregulation of gpdh-1 mRNA by hypertonic stress but is required for upregulation of GPDH-1-GFP protein.
(A) qPCR of gpdh-1 mRNA in day 2 adult animals expressing drIs4 exposed to 50 or 250 mM NaCl NGM plates for 3 hours. Strains include WT and ogt-1(dr20). Data are represented as fold induction of RNA on 250 mM NaCl relative to 50 mM NaCl. Data are expressed as mean ± S.D. ****—p<0.0001 (Student's two-tailed t-test). N = 3 biological replicates of 35 animals for each group. (B) Immunoblot of GFP and β-actin in lysates from WT and ogt-1(dr34) animals expressing the kbIs6 translational fusion exposed to 50 or 250 mM NaCl NGM plates for 18 hours. The ogt-1(dr34) allele carries the same homozygous Q600STOP mutation as the ogt-1(dr20) allele and was introduced using CRISPR/Cas9. Numbers under the GFP bands represent GFP signal normalized to β-actin signal for each sample, with the WT 250 mM NaCl sample set to 1. (C) qPCR of gpdh-1 mRNA from day 2 adult animals exposed to 50 or 250 mM NaCl NGM plates for 24 hours. The animals express a CRISPR/Cas9 edited knock-in of GFP into the endogenous gpdh-1 gene (gpdh-1(dr81)). ogt-1 carries the dr83 allele, which is the same homozygous Q600STOP mutation as the ogt-1(dr20) allele and was introduced using CRISPR/Cas9. Data are represented as fold induction of RNA on 250 mM NaCl relative to 50 mM NaCl. Data are expressed as mean ± S.D. *—p<0.05 (Student's two-tailed t-test). N ≥ 3 biological replicates of 35 animals for each group. (D) qPCR of gfp mRNA from day 2 animals) exposed to 50 or 250 mM NaCl NGM plates for 24 hours. The strains are the same as in (C). Data are represented as fold induction of RNA on 250 mM NaCl relative to 50 mM NaCl. Data are expressed as mean ± S.D. n.s. = nonsignificant (Student's two-tailed t-test). N ≥ 3 biological replicates of 35 animals for each group.
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S4 Fig. ogt-1 is not required for acute hypertonic stress survival but is required for chronic physiological and genetic adaptation to hypertonic stress.
(A) Percent survival of day 2 adult animals expressing drIs4 exposed to 100–600 mM NaCl NGM plates for 24 hours. Strains include WT, ogt-1(dr15), and ogt-1(dr20). Data are expressed as mean ± S.D. N = 5 replicates of 20 animals for each salt concentration. (B) Brightfield microscopy images of animals grown on 50 mM or 250 mM NaCl for 5 and 10 days respectively. Strains include WT (drIs4) and ogt-1(dr20);drIs4. Scale bar = 100 microns (C) Percent of progeny that developed into L4s. L4 animals were placed on 50 or 250 mM NaCl and the total number of eggs laid and progeny that developed into L4s were counted each day as described in the 'Methods'. Data are expressed as mean ± S.D. ****—p<0.0001, **—p<0.01 (One-way ANOVA with post hoc Tukey's test). N = 10 independent broods for each strain. (D) Percent of moving unadapted and adapted day 3 adult animals after exposure to 600 mM NaCl NGM plates for 24 hours. ogt-1(dr20 dr36) is a strain in which the dr20 mutation was converted back to WT using CRISPR/Cas9 genome editing. The gpdh-1(dr81) allele is a CRISPR/Cas9 edited C-terminal knock-in of GFP into the endogenous gpdh-1. The ogt-1(dr84) allele is a CRISPR/Cas9 edited C-terminal knock-in of GFP into the endogenous ogt-1. Data are expressed as mean ± S.D. ****—p<0.0001, n.s. = nonsignificant (One-way ANOVA with post hoc Dunnett's test). N = 5 replicates of 20 animals for each strain. (E) COPAS Biosort quantification of GFP and RFP signal in day 2 adult drIs4 animals drIs4 exposed to 50 mM NaCl NGM plates. Data are represented as the fold induction of normalized GFP/RFP ratio on 50 mM NaCl NGM plates, with osm-11(n1604) set to 1. The ogt-1(dr52) allele carries the same homozygous Q600STOP mutation as the ogt-1(dr20) allele, which was introduced using CRISPR/Cas9. Each point represents the quantified signal from a single animal. Data are expressed as mean ± S.D. ****—p<0.0001 (Mann-Whitney test). N ≥ 163 for each group. Inset: Wide-field fluorescence microscopy of day 2 adult animals expressing drIs4 exposed to 50 mM NaCl NGM plates. Images depict merged GFP and RFP channels. Scale bar = 100 microns.
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S5 Fig. Rescue of the ogt-1 Nio phenotype by tissue specific ogt-1 expression and effect of catalytically impaired ogt-1 mutations on O-GlcNAcylation and OGT-1-GFP protein levels and localization.
(A) COPAS Biosort quantification of GFP and RFP signal in day 2 adult animals expressing drIs4 exposed to 50 or 250 mM NaCl NGM plates for 18 hours. Data are represented as the fold induction of normalized GFP/RFP ratio on 250 mM NaCl NGM plates relative to on 50 mM NaCl NGM plates. Each point represents the quantified signal from a single animal. Data are expressed as mean ± S.D. ***—p<0.001, ****—p<0.0001, n.s. = nonsignificant (Kruskal-Wallis test with post hoc Dunn's test). N ≥ 37 for each group. These data were used to calculate the 'Degree of Rescue' in Fig 5B. (B) COPAS Biosort quantification of GFP and RFP signal in day 2 adult animals expressing drIs4 exposed to 50 or 250 mM NaCl NGM plates for 18 hours. Data are represented as the fold induction of normalized GFP/RFP ratio on 50 and 250 mM NaCl NGM plates relative to on 50 mM NaCl NGM plates. Each point represents the quantified signal from a single animal. Data are expressed as mean ± S.D. ****—p<0.0001, ***—p <0.001, **—p<0.01 (Kruskal-Wallis test with post hoc Dunn's test). N ≥ 40 for each group. These data were used to calculate the 'Degree of Rescue' in Fig 5D. (C) Wide-field fluorescence microscopy of fixed and stained embryos. RL2 was used to stain for nuclear pore O-GlcNAc modifications and Hoechst 33258 was used to visualize the DNA. Images are exposure matched. White arrowheads indicate RL2 staining in OGT-1
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S6 Fig. The tetratricopeptide repeat (TPR) domain of OGT-1 is required for O-GlcNAcylation, gpdh-1p:GFP induction, and hypertonic adaptation but does not alter OGT-1-GFP levels or localization.
(A) Wide-field fluorescence microscopy of fixed and stained embryos. RL2 was used to stain for nuclear pore O-GlcNAc modifications and Hoechst 33258 was used to visualize the DNA. Images are exposure matched. Scale bar = 10 microns. (B) Wide-field fluorescence microscopy of day 1 adult animals expressing endogenously CRISPR/Cas9 GFP tagged OGT-1 exposed to 50 mM NaCl NGM plates. Scale bar = 100 microns. Images are exposure matched. Inset: Zoomed in images of the boxed area. Scale bar = 10 microns. (C) Wide-field fluorescence microscopy of day 2 adult animals expressing drIs4 exposed to 50 or 250 mM NaCl NGM plates for 18 hours. Images depict the GFP channel only for clarity. The RFP signal was unaffected in these rescue strains (not shown). Scale bar = 100 microns. (D) COPAS Biosort quantification of GFP and RFP signal in day 2 adult animals expressing drIs4 exposed to 50 or 250 mM NaCl NGM plates for 18 hours. Data are represented as the fold induction of normalized GFP/RFP ratio on 50 and 250 mM NaCl NGM plates relative to on 50 mM NaCl NGM plates. Each point represents the quantified signal from a single animal. Data are expressed as mean ± S.D. ****—p<0.0001, ***—p<0.001, n.s. = nonsignificant (Kruskal-Wallis test with post hoc Dunn's test). N ≥ 92 for each group. (E) Percent of moving unadapted and adapted day 3 adult animals expressing drIs4 exposed to 600 mM NaCl NGM plates for 24 hours. Data are expressed as mean ± S.D. ****—p<0.0001, n.s. = nonsignificant (One-way ANOVA with post hoc Tukey's test). N = 5 replicates of 20 animals for each strain.
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S7 Fig. Inhibition of proteasomal or autophagic pathways does not rescue gpdh-1p:GFP expression during hypertonic stress in ogt-1(dr20) mutants.
(A) COPAS Biosort quantification of GFP and RFP signal in day 2 adult animals expressing drIs4 exposed to 50 or 250 mM NaCl NGM plates for 18 hours. Animals were placed on empty vector(RNAi) (ev(RNAi)) or rpn-8(RNAi) plates as L1s. Data are represented as normalized fold induction of normalized GFP/RFP ratio on 250 mM NaCl RNAi plates relative to on 50 mM NaCl RNAi plates. Each point represents the quantified signal from a single animal. Data are expressed as mean ± S.D. ****—p<0.0001 (Kruskal-Wallis test with post hoc Dunn's test). N ≥ 14 for each group. (B) COPAS Biosort quantification of GFP and RFP signal in day 2 adult animals expressing drIs4 exposed to 50 or 250 mM NaCl NGM plates for 18 hours. Animals were placed on empty vector(RNAi) (ev(RNAi)) or lgg-1(RNAi) plates as L1s. Data are represented as normalized fold induction of normalized GFP/RFP ratio on 250 mM NaCl RNAi plates relative to on 50 mM NaCl RNAi plates. Each point represents the quantified signal from a single animal. Data are expressed as mean ± S.D. ****—p<0.0001 (Kruskal-Wallis test with post hoc Dunn's test). N ≥ 47 for each group.
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S1 Table. ogt-1(dr15) and ogt-1(dr20) genetics are consistent with recessive and single gene alleles.
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S2 Table. All ogt-1 alleles fail to complement for the Nio phenotype.
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S3 Table. Backcrossing does not substantially reduce the number of SNPs and INDELS in the ogt-1(dr20) strain.
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S4 Table. Strain used in this study.
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S5 Table. DNA oligos used in this study.
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S6 Table. Bacterial strains used in this study.
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S7 Table. Chemicals, antibodies, peptides, and recombinant proteins.
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S8 Table. Critical commercial assays.
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S9 Table. Underlying numerical data for Fig 1B.
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S10 Table. Underlying numerical data for Fig 1C.
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S11 Table. Underlying numerical data for Fig 1E.
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S12 Table. Underlying numerical data for Fig 1F.
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S13 Table. Underlying numerical data for Fig 2D.
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S14 Table. Underlying numerical data for Fig 2E.
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S15 Table. Underlying numerical data for Fig 2F.
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S16 Table. Underlying numerical data for Fig 3A.
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S17 Table. Underlying numerical data for Fig 3B.
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S18 Table. Underlying numerical data for Fig 3C.
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S19 Table. Underlying numerical data for Fig 3D.
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S20 Table. Underlying numerical data for Fig 3E.
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S21 Table. Underlying numerical data for Fig 4A.
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S22 Table. Underlying numerical data for Fig 4B.
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S23 Table. Underlying numerical data for Fig 4C.
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S24 Table. Underlying numerical data for Fig 5B.
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S25 Table. Underlying numerical data for Fig 5D.
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S26 Table. Underlying numerical data for Fig 5F.
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S27 Table. Underlying numerical data for Fig 5G.
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S28 Table. Underlying numerical data for S1B Fig.
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S29 Table. Underlying numerical data for S1C Fig.
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S30 Table. Underlying numerical data for S2B Fig.
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S31 Table. Underlying numerical data for S2D Fig.
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S32 Table. Underlying numerical data for S2E Fig.
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S33 Table. Underlying numerical data for S3A Fig.
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S34 Table. Underlying numerical data for S3C Fig.
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S35 Table. Underlying numerical data for S3D Fig.
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S36 Table. Underlying numerical data for S4A Fig.
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S37 Table. Underlying numerical data for S4C Fig.
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S38 Table. Underlying numerical data for S4D Fig.
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S39 Table. Underlying numerical data for S4E Fig.
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S40 Table. Underlying numerical data for S5A Fig.
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S41 Table. Underlying numerical data for S5B Fig.
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S42 Table. Underlying numerical data for S6D Fig.
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S43 Table. Underlying numerical data for S6E Fig.
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S44 Table. Underlying numerical data for S7A Fig.
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S45 Table. Underlying numerical data for S7B Fig.
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Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We thank the labs of Arjumand Ghazi, Judy Yanowitz, and Aditi Gurkar (University of Pittsburgh) for many helpful suggestions, the lab of Gary Ruvkun (Harvard University) for the Galaxy whole genome sequencing workflow, the lab of Oliver Hobert (Columbia University) for providing additional ogt-1 strains, and David Raizen (University of Pennsylvania) for critical reading of the manuscript.
By Sarel J. Urso; Marcella Comly; John A. Hanover and Todd Lamitina
Reported by Author; Author; Author; Author