Dynamic O-GlcNAc cycling at promoters of
Caenorhabditis elegans genes regulating longevity,
stress, and immunity
Dona C. Lovea,1, Salil Ghosha,1, Michelle A. Mondouxa,1, Tetsunari Fukushigea, Peng Wanga, Mark A. Wilsonb,
Wendy B. Iserb, Catherine A. Wolkowb, Michael W. Krausea, and John A. Hanovera,2
aNational Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0851; andbNational Institute on Aging,
National Institutes of Health, Baltimore, MD 21224
Edited by Iva Greenwald, Columbia University, New York, NY, and approved March 16, 2010 (received for review October 15, 2009)
Nutrient-driven O-GlcNAcylation of key components of the tran-
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
O-GlcNAc-marked promoters are biased toward genes associated
with PIP3 signaling, hexosamine biosynthesis, and lipid/carbohy-
drate metabolism. These marked genes are linked to insulin-like sig-
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 molec-
ular programimpacting nutrient-responsive pathways in C. elegans,
including stress, pathogen response, and adult lifespan. The ob-
served impact of O-GlcNAc cycling on both signaling and transcrip-
tion in C. elegans has important implications for human diseases of
aging, including diabetes and neurodegeneration.
mellitus, Alzheimer’s disease, and coronary artery disease (1–7).
Genetic approaches to understanding the links between aging,
human disease, and hexosamine signaling have been hampered by
the factthat many ofthegenesimportant for hexosaminesignaling
are essential in mammals, making it difficult to analyze perturba-
tions in the pathway in the context of the whole organism (8–10).
The HSP leads to the dynamic and reversible Ser/Thr-O-
GlcNAc modification ofkey nuclearand cytoplasmic proteins,and
is a major cellular sensor of nutrient flux (1, 3, 5, 6). Levels of the
sugar nucleotide UDP-GlcNAc are highly responsive to intra-
cellular concentrations of glucose, amino acid, and lipids (11). In
transferase (OGT) posttranslationally GlcNAcylates key compo-
nents of intracellular signaling pathways, mitochondrial proteins,
metabolic enzymes, the proteasome, and transcription factors. O-
GlcNAc modification, like other forms of posttranslational mod-
ification, serves to regulate the function of these target proteins.
The O-GlcNAcase (OGA) catalyzes cycling by promoting O-
GlcNAc removal (1, 3, 5, 6). In addition to this enzymatic activity,
the OGA also contains a putative chromatin-interacting histone
acetyltransferase-like (HAT) domain (12).
A growing body of evidence suggests that the enzymes of O-
GlcNAc cycling may play a role in maintaining chromatin structure
and modulating transcription (13–16). Unlike vertebrate and Dro-
sophila model systems (6, 8–10, 13, 14, 17), null mutations in the
lterations in the hexosamine signaling pathway (HSP) have
been linked to diseases of human aging, including diabetes
elegans (18, 19), allowing a global analysis of the consequences of
altered O-GlcNAc cycling on chromatin structure and gene expres-
sion in a living organism. Here, using whole-genome chromatin
immunoprecipitation (ChIP)-on-chip tiling arrays, and transcrip-
associated with defects in O-GlcNAc addition and removal. The
of a subset of genes and microRNAs and that loss of O-GlcNAc
cycling results in a dramatic deregulation of gene expression.
Genome-Wide Analysis of Chromatin-Associated O-GlcNAc Proteins.
ChIP-on-chip analysis was performed to determine the location of
O-GlcNAc throughout the C. elegans genome. Synchronous L1
larvae were used as a source of chromatin for these experiments.
Three O-GlcNAc specific antibodies, RL2 (20), HGAC85 (21),
and CTD 110.6(22), were tested. Based on specific signal intensity
in preliminary tiling experiments, RL2 was chosen for all sub-
(N2), ogt-1 mutants, and oga-1 mutants was subjected to whole-
important negative control; it encodes a catalytically null allele,
and the strain completely lacks the O-GlcNAc modification. The
oga-1 mutant strain also is a catalytically null allele and exhibits
been characterized previously (18, 19). The normalized peak
intensities across eachofthe Caenorhabditiseleganslinkagegroups
is shown in Fig. 1A. Thresholding of wild-type and oga-1(ok1207)
was performed by subtractingnormalized signal intensities of ogt-1
(ok430) from those of wild type and oga-1(ok1207). Peaks with
intensities of 2.2 or greater were called as O-GlcNAc-specific
peaks. After thresholding, wild-type and the oga-1 mutant strain
retained 1166 O-GlcNAc-specific peaks. The number and dis-
tribution of O-GlcNAc-marked peaks (active regions) per chro-
mosome is indicated in Fig. 1B. These peaks were associated with
828 genes (Dataset S1). A nearly identical set of O-GlcNAc-
marked chromatin peaks was identified in both the oga-1 mutant
Author contributions: D.C.L., S.G., M.A.M., T.F., P.W., M.A.W., W.B.I., C.A.W., M.W.K., and
J.A.H. designed research; D.C.L., S.G., M.A.M., T.F., P.W., M.A.W., W.B.I., and C.A.W. per-
formed research; D.C.L., S.G., M.A.M., T.F., P.W., M.A.W., C.A.W., M.W.K., and J.A.H.
analyzed data; and D.C.L., M.A.M., C.A.W., M.W.K., and J.A.H. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
Data deposition: The data reported in this paper have been deposited in the Gene Ex-
pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (SuperSeries no.
1D.C.L., S.G., and M.A.M. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
| April 20, 2010
| vol. 107
| no. 16
few exceptions, were always higher in oga-1(ok1207) compared
with wild type (Fig. 1 A and C). This is apparent when the average
peak value is plotted for the 718 peaks within ±2 kb of known
transcription starts; oga-1(1207) peak values are almost invariably
higher than the other strains (Fig. 2A). This finding presumably
reflects active cycling of O-GlcNAc in the wild-type strain, due to
the action of O-GlcNAcase. In the absence of O-GlcNAcase,
of the marked loci in the oga-1 mutant.
O-GlcNAc Marks Are Associated with Promoters. Closerinspectionof
the O-GlcNAc-modified regions on chromatin showed that they
were almost invariably associated with the promoters of genes. A
(Fig. 1C). Note the enhanced 5′ peak of O-GlcNAc in the oga-1
(ok1207) mutant (red line) compared with wild type (green line),
whereas ogt-1(ok430) (blue line) shows minimal O-GlcNAc asso-
ciation. These differences in the O-GlcNAc peaks are consistent
with active cycling at the promoter. Many genes containing mul-
tiple promoters exhibit O-GlcNAc peaks at selective sites. For
first and third promoters are strongly marked by O-GlcNAc; the
middle promoter is not marked by the modification (Fig. 1C). In
addition to protein-coding genes, O-GlcNAc marks were asso-
ciated with ≈24 known microRNAs (Dataset S2), including the
mir-54 cluster (Fig. 1C). Of the marked genes that are part of a
the first gene. One striking example is the operon containing p38
MAP-kinase genes (pmk-1–3), where the first gene (F42G8.5) is
heavily marked by O-GlcNAc (Fig. S1B).
To determine whether O-GlcNAc marks were correlated with
transcriptional activity, we compared the O-GlcNAc ChIP-on-
chip data set with an RNA Pol II ChIP-on-chip carried out under
identical conditions. We have used two anti-RNA Pol II CTD
antibodies: 5095 (Abcam) and 8WG16 (MMS-126R; Covance).
The 5095 is marketed as specific for Ser-2-phosphorylation of the
heptad repeat (YSPTSPS), a modification that is associated with
the elongating form of the polymerase. However, 5095 was
recently shown to also recognize unphosphorylated Pol II in C.
elegans (23). The CTD heptad repeat-specific antibody (8WG16)
is phosphorylation independent (24) and has been used pre-
viously for C. elegans ChIPs (23, 25). ChIP signals throughout the
marked genes were much stronger with 5095 compared with
8WG16 (Fig. S1A). We found that O-GlcNAc marks a subset of
the genes (∼40%) that were transcriptionally active as deter-
mined by 5095 Pol II ChIP. Two specific examples are shown in
Fig. S1 A and B. Averaged across the genome, O-GlcNAc marks
were almost exclusively associated with the 5′ end of genes, with
a profile very similar, but not identical, to the distribution of
RNA polymerase II (cf. Fig. 2 B and C). The asymmetric O-
GlcNAc peak distribution is centered 100 bp upstream of the
transcriptional start site (TSS), dropping off dramatically after
crossing the TSS, whereas the RNA Pol II peak is centered near
the TSS. The distribution of the O-GlcNAc peaks relative to the
TSS was similar in N2 and oga-1(ok1207) thresholded samples
(Fig. 2B, green and red lines), and was absent in the negative
control strain ogt-1(ok430) (Fig. 2A, blue line). The distribution
of elongating Pol II, relative to the TSS, was essentially identical
in all of the strains (Fig. 2C), similar to previous reports (23, 25).
O-GlcNAc Marks Promoters of Genes Implicated in Growth, Aging, and
the Stress Response. To determine what kinds of proteins were
encoded by genes that actively accumulate O-GlcNAc marks, we
genome.(A)Thepeakintensitiesacross all sixlinkagegroups:oga-1(ok1207), red;N2(wildtype), green; ogt-1(ok430), blue.Notethat 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 thresholdwas applied to peak intensities to obtain intervals for oga-1(ok1207)(red)andN2 (green). The number of peaks foreach linkage
groupisshown.(C) Examples oftheO-GlcNAcpeaksat promoters are shownfor twoseparate genesanda micro RNA. ftt-2showsa large peakat itspromoter.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.
theAffymetrixexpressionarraysisshown foreach ofthestrains asindicatedby
the colored lines sorted using the peak values for oga-1(ok1207). Note that the
add O-GlcNAc. (B) The position of the O-GlcNAc peaks within ±2 kb of the
peaks were detected in ogt-1(ok430). (C) The position of RNA Pol II peaks rel-
stains exhibit similar behavior as each do individually (Avg, black line).
| www.pnas.org/cgi/doi/10.1073/pnas.0911857107Love et al.
using both traditional GO terms and the DAVID bioinformatic
tools (26, 27). The O-GlcNAc marked promoters are strikingly
biased toward genes involved in the biochemical pathways of
PIP3 signaling (enrichment score 2.4), glyoxylate and glycerone-
phosphate metabolism (enrichment score 1.2), and amine-
sites (28, 29), and amine-metabolism is central to the formation of
UDP-GlcNAc via the hexosamine biosynthetic pathway (11).
Further, DAVID functional annotation clustering analysis of the
O-GlcNAc marked genes suggested that the biological pathways
enriched in this data set involved reproductive behavior (enrich-
ment score 8.5), aging (enrichment score 3.9), morphogenesis
(enrichment score 3.2), neuron differentiation (enrichment score
3.0), mitotic spindle localization (enrichment score 2.7), and gly-
colysis (enrichment score 1.9; Table S1). Among the genes identi-
3 proteins, which are known to sequester transcription factors like
DAF-16/FOXO in the cytoplasm (see below). Previous work has
implicated O-GlcNAc in many of these processes (1, 5).
Disruption of O-GlcNAc Cycling Leads to Changes in Transcription. In
addition to the global analysis of O-GlcNAc marks on chromatin,
we examined changes in the transcriptome associated with the loss
of O-GlcNAc cycling. From the same synchronous L1 larvae used
for ChIP-on-chip, as well as synchronized L4 larvae, we prepared
triplicate RNA samples for Affymetrix whole-genome expression
arrays. The gene expression data from ogt-1 and oga-1 mutant
strains were compared with wild type to determine how defects in
O-GlcNAc cycling affected the transcriptome. A complete list of
deregulated genes and the associated GO term analysis for both
developmental stages is presented in Dataset S3.
In the oga-1(ok1207) L1 sample, 509 genes were deregulated
greater than 1.5-fold relative to wild type (Fig. 3A). Of these, 291
genes (57%) were down-regulated and 218 were up-regulated.
predicted to regulate lifespan and aging, the stress response,
innate immunity, and carbohydrate/lipid metabolism (Table S2).
In the L4 larvae, knockout of oga-1 led to deregulation of ≈165
genes [69 down-regulated (41%); 96 up-regulated; Fig. 3B].
Though fewer genes were deregulated in the L4 stage compared
with L1, bioinformatics analysis suggested that these genes were
enriched for similar pathways, including aging (enrichment score
3.7),carbohydrate metabolism(enrichment score2.7),membrane
transport (enrichment score 2.5), chromatin remodeling (enrich-
and in L4 larvae, ≈100 representative genes altered in the oga-1
(ok1207) were confirmed by a quantitative reverse transcriptase
PCR (qRT-PCR; Dataset S4).
A similar transcriptional analysis was performed using the ogt-1
(ok430) knockout strain. At the L1 stage, 688 genes were up- or
down-regulated relative to wild type; 389 (56%) were down-
regulated and 299 were up-regulated (Fig. 3C). These genes were
highly enriched in pathways involved in innate immunity (C-type
lectins, major facilitator superfamily, peptidoglycan catabolism),
1(ok430) showed deregulation of 1,641 genes compared with wild
type; 659 (40%) were down-regulated and 982 were up-regulated
(Fig. 3D). These deregulated genes were highly enriched for loci
encoding insulin and secreted peptides (enrichment score 11.7),
regulators of amino acid metabolism (enrichment score 9.6),
in reproduction, stress, detoxification, and aging (Table S3). Both
in L1 larvae and in L4 larvae, many of these changes were con-
firmed by qRT-PCR (Dataset S4). These expression data sets
strongly suggest that at two developmental stages, the loss of O-
involved in the response to stress, adult lifespan, carbohydrate
breakdown, pathogen resistance, and transcriptional regulation.
Disruption of O-GlcNAc Cycling Affects the Aging Pathway and the
Stress Response. ThegenomicandtranscriptionalanalysesoftheO-
GlcNAc cycling mutants predicted a role for O-GlcNAc cycling in
several processes, including the stress response and aging. We first
examined the lifespan of the O-GlcNAc cycling mutants in both a
DAF-2isthe C.elegansinsulin-likereceptor,anddaf-2 mutants are
40% respectively; Fig. 4 A and B). The oga-1 mutation significantly
extended lifespan in the daf-2 mutant background (∼12% exten-
sion), but not in the wild-type background (Fig. 4 A and B).
The lifespan extension observed in a daf-2 mutant is depend-
ent on the downstream transcription factor DAF-16/FOXO (30),
so next we tested whether the lifespan extension observed with
the oga-1 mutant was dependent on DAF-16. Surprisingly, we
found that the lifespan extension of a daf-2;oga-1 mutant is not
dependent on DAF-16. oga-1(ok1207) increased daf-2 lifespan in
either a daf-16(mu86) mutant background (28% extension) or in
response to daf-16 RNAi (19% extension, P < 0.0001; Fig. 4C).
Thus, oga-1(1207) increased lifespan in a manner dependent
upon daf-2, but independent of daf-16.
insulin-like receptor also regulates fertility: daf-2(e1370) mutations
reduce fertility in a DAF-16 and temperature-dependent manner
(31) (Fig. 4D). However, although mutations in oga-1 and ogt-1
modulate dauer formation and lifespan in a daf-2(e1370) mutant,
there was no statistical difference in brood size among the three
strains (Fig. 4D). These findings suggest that O-GlcNAc cycling is
important for control of dauer formation and lifespan regulation,
but not hermaphrodite self-fertilization and reproduction.
cano plots of gene expression, as detected by microarray analysis are shown. (A
and D) were usedfor analysis. Data are plotted as geometric fold change (x axis)
vs. P value (y axis). Genes were considered significantly deregulated if the geo-
metricfoldchange was greater than 1.5 (bold black line) with aP value of<0.05.
O-GlcNAc cycling mutant exhibit deregulation of gene expression. Vol-
Love et al. PNAS
| April 20, 2010
| vol. 107
| no. 16
Next, we tested whether O-GlcNAc cycling was involved in
transcriptome data. We found that ogt-1(ok430) was hypersensitive
(ok1207) strain was slightly more resistant to UV stress compared
with wild type. The observed altered sensitivity to UV stress is con-
for O-GlcNAc in mediating the stress response (32–34).
The FOXO homolog DAF-16 is a key mediator of the stress
responseand longevityinC.elegans,andisknown totranslocateto
the nucleus in response to stress, including heat stress and starva-
was normally cytoplasmic intheoga-1(ok1207) strain (Fig. 4F) and
entered the nucleus upon heat stress. However, in ogt-1(ok430)
animals, we found nuclear-localized DAF-16::GFP in the absence
(Fig. 4F), suggesting chronic activation of DAF-16. Such chronic
activation of DAF-16 does not appear to be protective, because
these animals are more sensitive to acute stress (Fig. 4E). The
constitutive translocation detected in ogt-1(ok430), in the absence
ofacutestress,is consistent witha deregulationofinsulin signaling
and its downstream effectors in the ogt-1 knockout.
O-GlcNAc Cycling Occurs at Promoters of Nutrient-Responsive Genes
and microRNAs. Kelly and Hart (36) first demonstrated that gly-
coprotein-binding lectins are localized to Drosophila polytene
chromosomes. Using whole-genome tiling array technology, we
have identified over 800 genes as being linked to sites of active
O-GlcNAc cycling in C. elegans. A comparison of genes marked
by either O-GlcNAc or by Pol II (5095) shows a substantial
overlap (∼40%). Thus O-GlcNAc marks provide a clear and
convenient means of identifying promoters in C. elegans, many of
which are actively transcribing, even in growth-arrested L1s.
The chromatin-associated O-GlcNAc marks share several prop-
erties with the chromatin marks of the histone variant HTZ-1, the
of the transcriptional start and tend to mark the first, but not sub-
sequent, starts sites for genes within operons. Both also show a
strong, but not absolute, correlation with transcriptional activity as
defined by RNA Pol II ChIP (pan-Pol II CTD antibody 8WG16)
GlcNAc data. For example, HTZ-1 is underincorporated on the X
chromosome, whereas the X chromosome has the most abundant
reflective of overlapping, yet distinct, biological processes.
easily correlated to direct transcriptional changes at those loci as
revealed by expression array analysis. This could be due to several
factors. First, the influence of O-GlcNAc on signaling cascades
could lead to transcriptional changes independent of marked loci.
Second, many transcription factors are modified by O-GlcNAc (e.
g., daf-16), and changes in these key regulators could influence the
transcription of many downstream target genes. Additionally, the
O-GlcNAc-marked miRNAs highlight the possible role of post-
transcriptional regulation of message stability as a confounding
factor in correlating the steady-state mRNA analysis provided by
GlcNAcylated, chromatin-associated proteins and transcriptional
networks will have to be much better understood before we can
elucidate the molecular mechanisms linking O-GlcNAc marks to
transcriptional effects on individual genes.
Despite the imperfect correlation between our O-GlcNAc
ChIP data and expression array results gene by gene, a more
global approach was informative. Bioinformatic examination of
the O-GlcNAc marked genes revealed a significant enrichment
for genes involved in hexosamine synthesis, phosphoinositol
signaling, lipid and carbohydrate metabolism, and chromatin
remodeling. These observed enrichments are consistent with a
number of the proposed biological functions of the O-GlcNAc
modification (11, 18, 19, 28, 37–40). The predictive ability of the
O-GlcNAc ChIP-on-chip data set suggests that these data can be
mined to identify new O-GlcNAc-responsive pathways.
O-GlcNAc Marks, Transcriptional Repression, and Polycomb. Previous
studies have hinted at a role for O-GlcNAc in transcriptional
repression (29, 41, 42). Two recent papers have demonstrated that
Drosophila ogt is allelic with super sex combs (sxc), a gene involved in
repression of homeotic genes (13, 14). Flies deficient in catalytically
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) con-
trol, 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 hermaph-
rodites (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).
O-GlcNAc cycling mutants affect aging and the stress
| www.pnas.org/cgi/doi/10.1073/pnas.0911857107Love et al.
that have a variety of homeotic transformations (13, 14, 43). Poly-
GlcNAc modified and was suggested to be the mediator of OGT-
marked genes in the fly is associated with polycomb response ele-
ments (PREs), which are correlated with homeotic repression and
genes in C. elegans also have a connection with transcriptional re-
pression, polycomb, and homeotic gene expression (44–50).
Although Drosophila O-GlcNAc is essential for PcG repression
and normal development, O-GlcNAc cycling does not appear to
be essential for normal C. elegans development. However, many
homeotic transformations in C. elegans manifest as cell lineage
defects that are often subtle and not obvious upon casual exami-
nation (51). Further investigation will be required to determine
what role O-GlcNAc cycling may play in C. elegans HOX gene
expression. The robustness of the C. elegans developmental pro-
gram may be due to the complex interplay between Wnt signaling
and HOX gene expression (52).
O-GlcNAc Cycling Modulates the C. elegans Insulin-Like Signaling
Pathway Impacting Longevity and the Stress Response. We have
provided several lines of evidence pointing to deregulation of
cellular signaling and transcriptional pathways in the mutants of
lifespan and sensitivity to stress. The ogt-1 null mutants also show
an aberrant, constitutive nuclear accumulation of the critical
transcription factor DAF-16. Among the genes most deregulated
stress and longevity pathways (reviewed in ref. 53). These findings
are consistent with our previous work implicating O-GlcNAc
cycling in the dauer diapause (18, 19). The modulation of O-
GlcNAc in the insulin-signaling pathway can be thought of as a
fine-tuning mechanism, not an on/off switch.
daf-16 independent. Combined with the ogt-1 lifespan results, these
findings suggest that O-GlcNAc cycling influences lifespan via both
the insulin-like signaling pathway (daf-2 dependent; daf-16 depen-
dent) and additional signaling pathways (daf-16 independent).
Two of the three daf-16 promoters are heavily marked by O-
GlcNAc in the tiling array, and the mammalian DAF-16 homolog
55, 56), and O-GlcNAc cycling could represent a modulator that
distinguishes between these, as it regulates the DAF-16-dependent
lifespan extension of daf-2 mutants but not the DAF-16-dependent
fertility suppression of daf-2 mutants. These findings point to con-
siderable complexity in the interaction between O-GlcNAc cycling
and the well-studied insulin-like signaling pathway in C. elegans.
(Fig. 4F); however, given that ogt-1 mutants are sensitive to acute
stress (Fig. 4E) and display longevity defects (Fig. 4 A–C), the
nuclear localized DAF-16 in the ogt-1 background is further evi-
may include the insulin-signaling pathway and the p38 MAPK
pathways (6, 57, 58). OGT-1 and PMK-1 physically interact in C.
elegans (59), and the absence of OGT-1 could cause the dereg-
ulation of pmk-1, leading to dramatic changes in longevity and the
stress response that are unrelated to daf-16-dependent tran-
scription. Thus the nuclear localization of daf-16 could reflect a
constitutive, unregulated activation of several stress-induced path-
OGT-1 and is known to activate O-GlcNAcylation of some sub-
strates during starvation (60). Our data are also consistent with
results from mammalian cells demonstrating that nuclear local-
ization of the DAF-16 homolog FOXO1 is not sufficient to stim-
in smk-1 (a presumptive phosphatase) affect stress, longevity, and
innate immunity downstream of DAF-16 nuclear localization (62).
O-GlcNAc Cycling and Diseases of Aging. C. elegans is a widely used
model for aging, implicating the insulin-like signaling pathway in
determination of adult lifespan (63). A growing body of evidence
suggeststhat alteredO-GlcNAccycling maybeassociated withthe
diseases of aging, including obesity, type II diabetes mellitus, car-
diovascular disease, cancer, and neurodegenerative disease (3, 6).
Our data derived from a global analysis of O-GlcNAc promoters
are consistent with these proposed associations. The alterations in
dauer and longevity observed for the O-GlcNAc cycling mutant
alleles suggest that the insulin-like signaling pathway is normally
addition, the multiple pathways leading to induction of the stress
cycling mutants. Thus, the genetic and molecular analysis pre-
sented here reinforces the growing body of evidence that O-
GlcNAc cycling is critical for maintaining the delicate balance
and MAPK pathways during senescence. Because O-GlcNAc has
wearepursuing thehypothesis that O-GlcNAccould alsofunction
in metabolic reprogramming in the intrauterine environment.
Materials and Methods
Strains. The following alleles were used in these studies: wild-type N2 Bristol
(discovered by Sydney Brenner in 1964), ogt-1(ok430) (18), ogt-1(ok1474),
ogt-1(tm1046) (National Bioresource Project of Japan), oga-1(ok1207) (19),
the temperature-sensitive insulin like receptor mutant daf-2(e1370), daf-16
(mu86), and TJ356 [daf-16::GFP (zIs356)]. Unless otherwise noted, all strains
were obtained from the Caenorhabditis Genetics Center. For details of allele
conformation and genetic crosses, see SI Materials and Methods.
Chromatin Preparation and ChIP-on-Chip. Synchronized C. elegans L1-stage
animalpelletswerecollected,immediatelyfrozeninwater,and shipped ondry
ice for processing by Genpathway. Genomic DNA regions of interest were iso-
lated using antibodiesagainstthe followingepitopes: the unmodifiedand Ser-
5-P-modified heptad repeat (YSPTSPS) of the C-terminal domain (CTD) of RNA
heptad repeat of the CTD domain (ab5095, Abcam); O-linked N-acetylglucos-
amine modified peptides (RL2; ab2739; Abcam); HGAC85-derived O-linked N-
acetylglucosamine (21) (MA1-076; Thermo Scientific); and Ser/Thr-O-linked N-
acetylglucosamine (CTD 110.6; MMS-248R; Covance). Standard chromatin
immunoprecipitation methods were used (25). For more detailed methods, see
SI Materials and Methods. Bioinformatic analysis (Genpathway) was used to
identify genes associated with RL2 peaks above threshold and located within
2 kb of the transcriptional start site (genome version WS195). All data were
submitted to the Gene Expression Omnibus (GEO) as SuperSeries GSE18132.
Gene Expression Analysis. Embryoswereobtainedbyhypochlorite-NaOHlysisof
gravid hermaphrodite adults. The embryos were hatched in M9 medium by
continuous overnight shaking at 20 °C. Growth-arrested L1 larvae were lysed
using glass beads with Minibeadbeater 8 (Biospec Products) at 4 °C. L4 animals
and Methods. All data were submitted to GEO as SuperSeries GSE18132.
Longevity and Fertility Assays. Longevity assays were conducted as described
(65). The fertility assays were conducted as described (31). Modifications to
either assay are provided in SI Materials and Methods. RNAi feeding vector
L4440 and daf-16 (RNAi) were obtained from the RNAi feeding library (66).
UV Treatment and DAF-16::GFP Translocation. UV stress performed essentially
as described (67). At least 20 animals were used for each allele tested. Any
changes are detailed in SI Materials and Methods.
ACKNOWLEDGMENTS. This research was supported by the Intramural
Research Program of the NIH, NIDDK, and NIA.
Love et al.PNAS
| April 20, 2010
| vol. 107
| no. 16
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| www.pnas.org/cgi/doi/10.1073/pnas.0911857107Love et al.