PNAS PLUS
AMPK blocks starvation-inducible transgenerational
defects in Caenorhabditis elegans
Emilie Demoineta,b, Shaolin Lia, and Richard Roya,1
a
Department of Biology, McGill University, Montreal, QC, Canada H3A 1B1; and bInstitute of Biology Valrose (iBV), CNRS, INSERM, Université Nice Sophia
Antipolis, 06100 Nice, France
Edited by Iva Greenwald, Columbia University, New York, NY, and approved February 14, 2017 (received for review September 30, 2016)
epigenetics
| AMPK | histone methyltransferase | COMPASS | C. elegans
I
n the winter of 1944/1945, the Nazi regime blockaded regions
of Holland and rationed food for all inhabitants without exception, including pregnant women and newborn children. The
infamous “Hongerwinter” came to an end in the spring of 1945,
with the arrival of the allied forces providing supplies and medical
help for the survivors. Although birth weight and size of the
Hongerwinter infants varied with the period of gestation that occurred during the famine, surprisingly few developmental anomalies were immediately observed despite the extreme mal/
undernutrition of the mothers (1–3). However, years afterward,
the same individuals showed an abnormally elevated frequency of
obesity, cardiac disease, and even schizophrenia, despite the lack
of genetic history of these diseases. Further analysis suggested that
DNA methylation patterns in specific regions of the genome were
altered in many of the Hongerwinter children (1, 4).
Although largely correlative, the study of the children born
during and after the Hongerwinter, along with other studies of
famine victims, provide compelling evidence to support a developmental origin of a broad spectrum of disorders. Some of these
disorders may rely on epigenetic changes that alter gene expression
downstream of a single event or prolonged environmental conditions (5, 6). At present it has proven difficult to link the observed
epigenetic changes to any obvious adaptive response to the nutrient
stress, or how such a response may underlie the clinical manifestations that arise in individuals several years after the initial stress.
Moreover, the nature of such changes may potentially extend beyond the described changes in DNA methylation to impinge upon
chromatin marks that could heritably modify gene expression in a
DNA sequence-independent manner.
www.pnas.org/cgi/doi/10.1073/pnas.1616171114
Although Caenorhabditis elegans does not possess the enzymes
involved in cytosine methylation, it does exhibit various forms of
epigenetic inheritance. These epigenetic traits are mediated largely
through chromatin modifications and a heritable population of
small RNAs that collectively dictate whether regions of the genome
are expressed or silenced (7–9). Recently, it was shown that the
composition of a large repertoire of RNAs is sensitive to starvation
during the first larval (L1) diapause, and could reflect some of the
adaptive changes in gene expression that are transmitted in subsequent generations (10). Furthermore, starvation during early
stages of development can induce long-term phenotypic consequences for exposed animals and increase stress resistance in their
descendants (11). These data therefore suggest that in C. elegans
early life-history events can exert a lasting transgenerational impact
associated with an adaptive response to the initial event.
We show here that the AMP-activated protein kinase (AMPK)
is required not only to adjust to the stress associated with starvation during the L1 stage in C. elegans, but also to block epigenetic modifications typical of gene activation during this period of
acute starvation. In its absence, the histone methyltransferasecontaining COMPASS complex (complex proteins associated
with Set1) becomes misregulated, generating chromatin modifications that have detrimental effects on reproductive fitness, not
only in the generation of animals that experienced the starvation,
but in subsequent generations that were never starved.
Results
Following embryogenesis, emergent C. elegans L1-stage larvae remain in a nondeveloping, diapause-like state until they encounter a
nutrient/energy source that will trigger the onset of the postembryonic developmental program (12, 13). Whereas wild-type
Significance
Following periods of famine, a reproducible spectrum of disorders often manifest long after the period of starvation. Curiously,
many of these diseases arise in individuals that have no apparent
genetic history of the disorder, although they do correlate with
specific epigenetic modifications. We used Caenorhabditis elegans as a model to understand how acute periods of starvation
might result in physiological or developmental consequences in a
single generation or over multiple generations following the
initial period of stress. Our data suggest that the AMP-activated
protein kinase, an enzyme that mediates metabolic adjustment
during starvation, is required to block germ-line gene expression
during these conditions. In its absence the inappropriate activation of germ-line transcription results in sterility and transgenerational reproductive defects.
Author contributions: E.D., S.L., and R.R. designed research; E.D. and S.L. performed research; E.D., S.L., and R.R. analyzed data; and E.D. and R.R. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. Email: richard.roy@mcgill.ca.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1616171114/-/DCSupplemental.
PNAS | Published online March 13, 2017 | E2689–E2698
DEVELOPMENTAL
BIOLOGY
Life history events, such as traumatic stress, illness, or starvation, can
influence us through molecular changes that are recorded in a
pattern of characteristic chromatin modifications. These modifications are often associated with adaptive adjustments in gene expression that can persist throughout the lifetime of the organism, or
even span multiple generations. Although these adaptations may
confer some selective advantage, if they are not appropriately regulated they can also be maladaptive in a context-dependent manner.
We show here that during periods of acute starvation in Caenorhabditis
elegans larvae, the master metabolic regulator AMP-activated protein kinase (AMPK) plays a critical role in blocking modifications to
the chromatin landscape. This ensures that gene expression remains
inactive in the germ-line precursors during adverse conditions. In its
absence, critical chromatin modifications occur in the primordial
germ cells (PGCs) of emergent starved L1 larvae that correlate with
compromised reproductive fitness of the generation that experienced the stress, but also in the subsequent generations that never
experienced the initial event. Our findings suggest that AMPK regulates the activity of the chromatin modifying COMPASS complex
(complex proteins associated with Set1) to ensure that chromatin
marks are not established until nutrient/energy contingencies are
satisfied. Our study provides molecular insight that links metabolic
adaptation to transgenerational epigenetic modification in response
to acute periods of starvation.
L1 larvae can survive up to 2 wk in this starved state, loss of activity in
the metabolic regulatory kinase AMPK/aak-1/2, the tumor suppressor ortholog phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/daf-18, or the histone deacetylase 1 ortholog hda-1,
sensitizes animals to this stress, causing them to die prematurely
en masse (Fig. S1A) (13–16). Although it is less clear how the
other two genes affect survival of starved L1 larvae, AMPK
becomes activated in response to starvation and phosphorylates
diverse substrates to facilitate metabolic adjustment (17). These
targets include key metabolic enzymes, whereas AMPK activation
has also been linked to changes in gene expression through
chromatin modification (18). To determine whether AMPK may
establish or maintain changes within the chromatin landscape
downstream of acute starvation, we examined the role of AMPK
during the metabolic adaptation associated with the L1 diapause
in C. elegans.
In many organisms the loss of AMPK is lethal (19, 20); however,
C. elegans mutants that have no AMPK signaling because of the
disruption of its two catalytic subunits (AMPK/aak-1/2) are viable
but exhibit defects during periods of nutrient/energy stress. This is
most notable during the L1 diapause stage and during an alternative
postembryonic developmental stage called dauer, where it has been
shown to affect germ-line stem cell quiescence, dauer maintenance,
and survival (21–23). Mutations in the C. elegans tumor-suppressor
ortholog PTEN/daf-18 cause similar defects in both dauer and
L1 diapause-arrested larvae, although it most probably acts through
a genetically independent pathway (15, 24, 25).
AMPK and PTEN Are Both Required for Primordial Germ Cell
Quiescence During the L1 Diapause and Post-L1 Diapause Fertility,
but Act Through Independent Pathways. When starved AMPK/aak-
1/2 or PTEN/daf-18 mutant L1 larvae that have been arrested in
the L1 diapause are subsequently placed into replete growth
conditions, the L1 larvae (which we will refer to hereafter as postL1 diapause larvae) initiate postembryonic development, although
their progression through the various larval stages is slower and
more variable than wild-type L1 diapause larvae that were starved
for the same duration. Once the surviving post-L1 diapause mutants reach the adult stage they show both somatic and reproductive defects, including a reduced brood size, and many
mutant animals die prematurely as adults (Fig. 1A, Fig. S1 A–E,
and Table S1). The survival of the L1 diapause larvae, and the
severity and frequency of the somatic growth defects that manifest
following recovery of the starved mutant animals are dependent
on the duration of the starvation. For example, after 5 d of starvation most animals die, but after 3 d of starvation half the daf-18
and aak-1/2 animals were unable to recover, and those that did
exhibited a spectrum of somatic and reproductive defects that
included vulval defects, sterility, and premature adult lethality.
This is not unique to the mutants because we also observe the
same types of growth and reproductive abnormalities in wild-type
animals that were starved for substantially longer durations (11 d)
before transferring them to replete conditions (Fig. 1B, Fig. S1 D
and E, and Table S1).
The premature death that occurs in all of the post-L1 diapause
animals appears to be independent of the observed morphological
somatic defects, because 60% of the post-L1 diapause AMPK/aak1/2 or PTEN/daf-18 mutant adults, and 70% of the wild-type animals that were starved 11 d, die prematurely, 6 d after reaching
the L4 stage with no visible somatic defects. Unlike the postL1 diapause AMPK/aak-1/2 mutants, daf-18 adults die earlier than
wild-type in a manner that is independent of the L1 diapause (Fig.
S1E). Therefore, although wild-type animals show a progressive
decline in their reproductive fitness during an extended period of
starvation (11 d), this same decline occurs in the AMPK/aak-1/2
and daf-18 mutants after only a short period of starvation (1–3 d)
(Fig. 1 A and B and Fig. S1 C and D).
E2690 | www.pnas.org/cgi/doi/10.1073/pnas.1616171114
The duration of the starvation at the L1 stage has a cumulative
impact on fertility in wild-type, AMPK/aak-1/2, and PTEN/daf-18
animals, whereas the sterility observed in the triple mutant combination (aak-1; daf-18; aak-2) is even more severely affected: 71 ±
6% of post-L1 diapause triple mutants became sterile after 1 d in
the diapause compared with 4.6 ± 4% and 35 ± 6% for AMPK/
aak-1/2 and PTEN/daf-18 mutants, respectively. No triple mutants
survive after 3 d of starvation at the L1 stage, consistent with
AMPK and PTEN acting through independent pathways to ensure
survival, robust and timely growth, and somatic and germ-line
development following starvation (Fig. S1 A–D). After being
maintained 3 d in the L1 diapause stage, only 30% of the viable
post-L1 diapause aak-1/2 larvae grew to become fertile adults (Fig.
1A). The majority (80%) of these fertile parents display a strikingly reduced brood size, generating fewer than 100 F1 progeny.
We refer to these fertile parents as “reduced” (parents that produce broods with less than 100 F1 progeny) (Fig. 1B and Fig. S1 C
and D). Surprisingly, the remaining 20% of the fertile adults
produced an almost normal brood size and seem almost unaffected. We refer to these animals as “normal” (parents that
produce more than 100 F1 progeny).
Because the primordial germ cells (PGCs) undergo inappropriate supernumerary divisions in both AMPK/aak-1/2– and
PTEN/daf-18–starved L1 mutant larvae (Fig. S1F) (14, 15), we
wondered whether this aberrant proliferation may be responsible
for the compromise in fertility and brood size observed in the postL1 diapause AMPK/aak-1/2 and PTEN/daf-18 mutant adults.
Using previously described genetic suppressors that block the supernumerary PGC divisions in both PTEN/daf-18 and AMPK/aak1/2 mutants (14, 15, 23), we determined whether they might also
restore fertility in the post-L1 diapause mutant adults. Whereas,
age-1 (PI 3-kinase) mutations restore both germ-line quiescence
and fertility of post-L1 diapause PTEN/daf-18 mutants (Fig. S1G),
age-1 did not suppress the sterility of AMPK/aak-1/2 mutants, but
alternatively, enhanced sterility in the triple mutants (87± 6% of
3-d post-L1 diapause age-1; aak-1/2 adults became sterile compared with 66 ± 3% in aak-1/2 adults) (Fig. S1G). Moreover, recent data suggest that blocking the target of rapamycin complex 1
(TOR) pathway by compromising raga-1 or ragc-1 can restore
PGC quiescence in both AMPK/aak-1/2– and PTEN/daf-18–
starved L1 mutant larvae (14). We observed that raga-1(lf) restored the fertility of PTEN/daf-18 adults that had transited
through the L1 diapause, but did not restore the fertility of postL1 diapause AMPK/aak-1/2 mutant adults (Fig. S1G). This finding
suggests that the supernumerary PGC divisions are unlikely to be
the sole basis of the sterility seen in post-L1 diapause AMPK/aak1/2 mutants, although the supernumerary PGC divisions may
contribute to the sterility observed in PTEN/daf-18 mutants.
Germ-Line and Gonad Defects in Post-L1 Diapause AMPK Mutant
Hermaphrodite Adults. To better understand the basis of the
AMPK/aak-1/2 reproductive defects that result from starvation
at the L1 stage, we examined the germ lines of adult hermaphrodites (Fig. 1 C and D and Fig. S2 A–C). The gonads of the
post-L1 diapause AMPK/aak-1/2 adults were highly disorganized, often possessing smaller or empty arms. The size of the
germ-cell nuclei was variable, whereas the typical germ-cell
morphology appeared abnormal in many animals. In wild-type
adult hermaphrodites, germ cells exit a mitotically active zone at
the distal end of the gonad and enter meiotic prophase I as they
move proximally. This region of the gonad called the transition
zone is cytologically distinct based on DAPI staining (26). The
length of the transition zones seen in post-L1 diapause AMPK/
aak-1/2 mutants was extremely variable and sometimes transition
zones were completely absent. The chromosomal morphology
typical of cells in the transition zone based on DAPI staining was
abnormal, suggesting that the progression from mitosis to meiosis was affected. None of these features were reproducibly
Demoinet et al.
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PNAS PLUS
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Fig. 1. Loss of AMPK results in sensitivity to acute starvation and subsequent postrecovery reproductive effects. (A) AMPK is required to ensure adult reproductive fitness after recovering from L1 starvation. Following varying durations of starvation (“Days” refer to days of starvation here and throughout), postL1 diapause larvae were singled to replete plates, allowed to grow to adulthood when their fertility was scored. “–” indicates nonstarved animals. Error bars
represent the confidence interval at 95% (95% CI); n ≥ 80 animals were scored per experimental condition. WT, wild type. (B) Prolonged starvation in the
L1 diapause results in a reduction in the F1 brood size for post-L1 diapause aak-1/2 mutant and wild-type animals. Total number of F1 progeny born from fertile
post-L1 diapause parents were scored following varying durations in the L1 diapause [nonstarved (–), 1, 3, 5, 7, 9, 11, or 13 d]. (Upper) Brood size distribution for
wild-type and aak-1/2 animals after varying durations of L1 diapause. The average brood size is indicated by horizontal bars for each genotype/condition. The
dotted line represents the minimum number of F1 progeny used to define reduced brood size (fewer than 100 F1 progeny). (Lower) The proportion of individuals
with reduced brood size is represented, n ≥ 80 animals. Error bars: 95% CI. (C) L1 starvation disrupts gonad morphology in post-L1 diapause aak-1/2 adult
hermaphrodites. L1 larvae were either allowed to eat immediately (–) or maintained in the L1 diapause for varying durations before they were singled to OP50seeded plates. Whole-animal DAPI staining was performed on wild-type or aak-1/2 adult hermaphrodites to visualize the germ cells. The gonads of (a) nonstarved
wild-type controls or (b) aak-1/2 mutant adult hermaphrodites possess two symmetric, morphologically normal gonadal arms, which are also observed in (c) wildtype adults that were previously subjected to a 3-d period in the L1 diapause indicated by the asterisk (*). In contrast, young adult hermaphrodite aak-1/2 mutants
that developed after a 3-d L1 diapause (*) displayed abnormal gonads that lack or have reduced germ cell numbers (d–f), where the mitosis–meiosis zone is
disorganized. In addition, many germ cells undergo endomitotic cell cycles (arrowheads). Dotted lines delineate the gonad boundary, whereas solid gray lines
outline the cuticle. (Scale bar, 50 μm.) (D) Morphological defects in the gonad are unique to aak-1/2 mutants that were previously subjected to 3 d in the
L1 diapause. Gonadal defects observed in young-adult post-L1 diapause aak-1/2 mutant adults from C were quantified and represented graphically. Oocyte-only
animals lack sperm; sperm-only animals lack oocytes. Oocytes and sperm-contain both; n ≥ 20 animals. (E) Sperm numbers are reduced in post-L1 diapause aak-1/2
mutants. Sperm were counted following DAPI staining in aak-1/2 mutant adults subjected to none (–), or a 3-d duration in the L1 diapause and subsequent
recovery on replete plates until the adult stage; n ≥ 8 gonad arms. Error bars: SE, *P < 0.05 using Student’s t test. (F) Reproductive defects observed in postL1 diapause aak-1/2 mutants are caused predominantly by compromised oocyte integrity with comparatively less contribution from the sperm. Crosses performed
with post-L1 diapause hermaphrodites were partially rescued by mating with nonstarved aak-1/2 mutant males. Reciprocal crosses were performed between postL1 diapause aak-1/2 mutant males (_dia), or hermaphrodites (adia) that were previously maintained 3 d in the L1 diapause. The resulting proportion of animals that
exhibited a reduced brood size from successful mating (as judged by 50% frequency of males in the F1 progeny) was tabulated for comparison. Error bars: 95% CI;
*P < 0.05 Fisher’s exact test.
observed in AMPK/aak-1/2 mutant hermaphrodites that were
not subjected to the L1 diapause. These germ-line abnormalities
likely impinge on gamete integrity because many of the disrupted
gonads possessed oocytes, while several of these appear to have
undergone endomitotic cycles based on DAPI staining (Emo,
76%, n = 20). In addition, most of the post-L1 diapause aak-1/2
Demoinet et al.
adults exhibit gonads with a strong reduction, or a complete
absence of sperm (Fig. 1 D and E and Fig. S2C). This reduced
abundance and efficiency of the sperm, compounded with the
observed defects in oogenesis after starvation, likely contribute
to the observed brood-size defect in post-L1 diapause AMPK/
aak-1/2 mutant hermaphrodite adults.
PNAS | Published online March 13, 2017 | E2691
DEVELOPMENTAL
BIOLOGY
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The observed reproductive defects could result from either
somatic or germ-cell deficiencies that arise during or following the
L1 diapause. To determine if the defects were a result of the integrity of the oocytes, the sperm, or both, we performed reciprocal
crosses and assessed the brood size of the resulting cross progeny
(Fig. 1F). When we mated AMPK/aak-1/2 mutant hermaphrodites
that had never been starved with post-L1 diapause AMPK/aak-1/2
males, the resulting brood sizes were normal and we did not observe any significant increase in sterility, suggesting that the
abundance or the quality of the sperm in post-L1 diapause
AMPK/aak-1/2 adult hermaphrodites is not a major factor in the
observed sterility. However, when the crosses were performed with
post-L1 diapause AMPK/aak-1/2 mutant hermaphrodites and
nonstarved male AMPK/aak-1/2 mutant animals, we noted that
the brood-size defects could only be partially restored. A similar
partial rescue was observed when nonstarved wild-type males were
mated with post-L1 diapause AMPK/aak-1/2 mutant hermaphrodites. This finding suggests that only some of the reproductive
defects (sterility or reduced brood size) of the post-L1 diapause
AMPK/aak-1/2 mutants are derived from the sperm defects described above (Fig. 1E). These results collectively suggest that the
germ line of the hermaphrodite is particularly sensitive to starvation during this period and that when the PGCs are affected, a
defect occurs in the oocytes such that fertility cannot be fully restored through crossing with unaffected males. The memory of the
starvation remains molecularly recorded and ultimately affects the
integrity of the oocyte.
Transgenerational Reproductive Defects Arise in Post-L1 Diapause
AMPK Mutants. This phenomenon is consistent with epigenetic
modifications that alter the chromatin, ultimately affecting cellular
outcomes over the course of one or more cell divisions or even
generations. To test whether a single bout of starvation during the
L1 stage could result in transgenerational epigenetic defects in
subsequent generations of progeny that never experienced the
initial starvation, we carried out three distinct multigenerational
analyses based on different hypotheses (Fig. 2 A and B and Fig.
S2E). Using the first method (method #1 in Fig. 2A), we postulated that post-L1 diapause parents that have a reduced brood size
(fewer than 100 F1 progeny) might have an increased chance of
transmitting these epigenetic changes transgenerationally compared with post-L1 diapause parents that exhibit a normal brood
size (more than 100 F1 progeny). To test this hypothesis, we followed animals through multiple generations by continuously
selecting L4 larvae born from parents that produced a reduced
brood size at each generation. The larvae were then allowed to
develop, produce progeny, and their individual brood sizes were
evaluated and recorded for each generation (Fx).
Alternatively, it is possible that all of the post-L1 diapause
AMPK/aak-1/2 mutant adults are affected by the stress of starvation during the L1 diapause, regardless of whether the parents
exhibit reproductive defects initially. We noted that some (20%)
of the fertile post-L1 diapause AMPK/aak-1/2 mutant adults had
normal broods, but their progeny fell into two categories: those
that generated F2 broods that were reduced in size and those that
produced normal F2 broods. By systematically selecting L4 larvae
from each generation (Materials and Methods), we then tracked
individuals from the reduced and normal categories and constructed multigenerational lineages that were based on whether
they were initially selected from parents that had a reduced brood
size (method #2), or whether they were generated from parents
that had a normal brood size (method #3) (Fig. 2A; additional
information in Materials and Methods).
All of the animals that were tracked through successive generations using method #3 generated progeny with comparatively less
impact on brood size. The minor effects that did manifest in the
early generations were resolved rapidly, but in later generations
(F5–F9) reproductive defects did appear, despite our continuous
E2692 | www.pnas.org/cgi/doi/10.1073/pnas.1616171114
selection of normal parents. This finding suggests that even though
most of these post-L1 diapause parents were either phenotypically
unaffected by the diapause, or that any initially occurring defects
may have been resolved in the first generation, some heritable
record of the initial event persisted and was manifested, albeit at
low penetrance, up to nine generations after the initial event.
Alternatively, when we continuously selected post-L1 diapause
AMPK/aak-1/2 descendants from parents that had reduced brood
sizes using methods #1 or #2, we consistently observed transgenerational brood-size defects (Fig. 2B). Curiously, this transmissible brood-size defect occurred despite the fact that these
successive generations of progeny had never been starved or, in
some cases, whether their parents appeared reproductively compromised or not (method #2). Therefore, regardless of whether
the initially affected aak-1/2 post-L1 diapause animals had a
normal or reduced brood size, both types of parents are very likely
to transmit this reduction in brood size to their descendants (Fig.
2B). This progressive transgenerational reduction in reproductive
fitness is never observed in nonstarved AMPK/aak-1/2 mutant
animals, in 3-d post-L1 diapause PTEN/daf-18 animals, or in 3-d
or 11-d starved wild-type animals (Fig. S2E).
There is a substantial degree of variability in the brood-size
defects between these parental lineages that may represent a
graded threshold effect in response to the epigenetic stimulus
(starvation). However, this variability seems typical of an epigenetic
response where not all of the germ cells are affected by starvation
in an identical way, potentially because of individual differences in
availability to nutrient/energy levels within the PGCs. Nevertheless,
most AMPK/aak-1/2 mutant animals that survive the acute stress
associated with starvation in the L1 diapause will produce progeny
that will eventually be adversely affected by the initial challenge,
even though the affected animals in subsequent generations may
never have experienced the stress directly.
To determine the extent to which each individual animal was
affected we quantified the brood size of 10 individual fertile postL1 diapause AMPK/aak-1/2 mutant parents and that of their
progeny over several generations (Fig. 2C and Fig. S2F). Nine of
these lineages (P0-I to P0-IX) were analyzed using method #1 and
one lineage (P0-X) was analyzed using method #2. In each of the
parental lineages analyzed by method #1 (P0-I to P0-IX) we observed a similar progressive reduction in brood size that varied in
expressivity and in the delay that preceded either the correction of
the reproductive defects or the eventual terminal arrest of the
lineage. This progressive brood size reduction is similar to the
progressive mortal germ-line phenotypes (Mrt) that remain fertile
indefinitely at permissive temperature but become progressively
sterile after growth for multiple generations at higher temperatures
(27). These phenotypes are typically caused by mutations in genes
involved in genome integrity or epigenetic regulation (27–30).
Consistent with this, in four independent lineages (see † in Fig.
2C), we noted that animals became very sickly in late generations,
segregating a high percentage of male progeny (Him) and other
spontaneous mutant phenotypes [Dumpy (Dpy), Roller (Rol),
Long (Lon), Small (Sma)]: in the F6 plates of P0-VIII and the F8
plates of lineage P0-IX, 9% and 14% of the animals segregate a
strong Him phenotype in next generation; 6% and 14% segregate a
Lon phenotype; whereas 3% and 5% give rise to a Dpy phenotype,
respectively, before laying a large number of dead eggs. In the
animals present on the F3 plates of the P0-VI and P0-VII lineages,
up to 33% of the progeny segregate a Sma phenotype, 4–8% give
rise to a Him phenotype, and 80–92% of the remaining viable
progeny eventually arrested between the L1 and L3 stages.
We were able to correct the brood-size defects of post-L1
diapause AMPK/aak-1/2 mutant descendants from later generations in P0-VIII and P0-IX by crossing them with nonstarved wildtype (N2) males (Fig. 2D). However, although AMPK/aak-1/2
mutant males could partially suppress the brood-size defect in the
first generation of affected animals born from post-L1 diapause
Demoinet et al.
PNAS PLUS
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that produce initially broods >100 progeny
and then reduced broods <100 progeny
P0
F1
F2
F3
F4
F5
F7
F9
80
N
%)
Reduced brood size (%
Red
duced brood size
e (%)
B 100
development
Adult
80
60
F1
F2
F3
F4
F5
F6
F7
F8
†
*
100
†
Reduced brood size (%)
N
P0
no starvation (always on foo
od)
no starvvation (always on food)
R=“Reduced”
Brood size >100
L1
development
Adult
N=“Normal”
starvation
L1
development
†
†
40
20
80
*
60
40
20
0
0
F8
F7
WT x F8
3 days
#1
WT x F7
#2
Fig. 2. Post-L1 diapause AMPK mutants display variable transgenerational reproductive defects. (A) Schematic representation of the three selection methods
used for our transgenerational analyses. Method #1: transgenerational analysis performed using fertile parents (P0) that have a reduced brood size of less than
100 F1 progeny. Methods #2 and #3: Multigenerational analyses were carried out first using parents that produce a normal brood size of more than 100 F1
progeny (see Materials and Methods for additional details). Red arrows indicate the selection at each successive generation. (B) Loss of AMPK causes transgenerational reproductive defects following a short duration in the L1 diapause. The percentage of post-L1 diapause animals that generated descendants that
bore smaller broods (≤100 progeny) after either no starvation (−), or 3 d in the diapause. For multigenerational analyses, the F1 progeny generated from reduced
parents using method #1, or parents with a normal brood size using methods #2 and #3 were analyzed over multiple generations (see Fig. S2E for additional
information). Error bars: 95% CI. (C) aak-1/2 mutants exhibit a progressive transgenerational reduction in brood size. Phenotypic variability in brood size is inherent in subsequent generations analyzed from fertile post-L1 diapause aak-1/2 parents. Brood sizes were analyzed over multiple generations in the descendants
of 10 independent post-L1 diapause aak-1/2 mutant parents. Brood-size defects were monitored in lineages P0-I to P0-IX using section method #1, whereas for
comparison, the analysis in P0-X was performed using method #2. Descendants that showed spontaneous mutant phenotypes that did not subsequently breed
true (Him, Lon, Dpy or Sma) are shown by a dagger (†). (D) The multigenerational reproductive defects that occur in post-L1 diapause aak-1/2 mutant animals arise
because of adverse epigenetic changes that occur as a result of the absence of AMPK. aak-1/2 transgenerational brood-size defects are only partially rescued by
the introduction of a wild-type copy of AMPK. Well-fed wild-type males (nonstarved) were crossed with aak-1/2 hermaphrodites that were selected from two
independently maintained transgenerationally compromised lineages (P0-VIII and P0-IX) that produced smaller broods (≤100 progeny) after seven or eight
generations following the initial diapause (F7 and F8, respectively). The brood sizes of cross progeny were determined and represented as the percentage of the
total population of animals born from the same successful cross. Error bars: 95% CI; *P < 0.05 Fisher’s exact test.
AMPK/aak-1/2 mutant parents (Fig. 1F), crossing nonstarved
AMPK mutant males with these later generation (F4 or F5)
AMPK/aak-1/2 mutants did not suppress the brood-size defects
in the resulting cross progeny (Fig. S2G; compare with Fig. 1F).
These observations are consistent with an epigenetic phenomenon that affects germ-line integrity in the post-L1 diapause
AMPK/aak-1/2 mutants that becomes progressively worse with
each successive generation. Furthermore, although this transgenerational defect can be corrected by wild-type gene activity,
the inability of nonstarved AMPK/aak-1/2 mutant males to rescue the transgenerational brood size defects of the postL1 diapause AMPK/aak-1/2 mutant hermaphrodites suggests that
AMPK activity may also be required to resolve these epigenetic
modifications once they are established.
The possibility that the stress of an initial starvation event
might confer increased resistance to a subsequent bout of starvation as an evolutionary trade-off was excluded because F1
animals obtained from fertile post-L1 diapause AMPK/aak-1/2
mutant adults exhibited similar L1 survival rates to animals that
Demoinet et al.
had never been previously starved. The F1 animals that arose
from post-L1 diapause AMPK/aak-1/2 mutant parents with reduced broods did, however, die earlier than nonstarved mutant
controls (Fig. S2H; see asterisks, F1*).
Our data therefore indicate that both PTEN/daf-18 and AMPK
are required for survival and adult fertility in response to acute
starvation during the early L1 stage. Whereas PTEN/daf-18 is
required acutely in a pathway that is independent of AMPK, its
effects are resolved in the first generation following the starvation.
In AMPK/aak-1/2 mutants however, the reproductive consequences that manifest in the post-L1 diapause animals persist
throughout multiple generations after the initial event resulting in
the progressive extinction of the affected lineages.
H3K4me3 Levels Are Abnormally High in the PGCs of Post-L1 Diapause
AMPK Mutants. Changes in chromatin modification have been
linked to epigenetic transmission in several contexts (31). To
determine whether chromatin marks are affected in AMPK/aak1/2 mutants following starvation in the L1 stage and whether
PNAS | Published online March 13, 2017 | E2693
DEVELOPMENTAL
BIOLOGY
starvation
no starvation (always on foo
od)
A
these marks persist in the subsequent affected generations, we
examined the levels of various histone modifications in the PGCs
of post-L1 diapause wild-type and mutant L1 larvae. Consistent
with recent reports, the levels of phosphorylated H2B were reduced in starved AMPK/aak-1/2 mutants (18), whereas the levels
of H3K4me3 were increased in the PGCs of starved AMPK/aak-1/2
mutants (Fig. 3 A and B and Fig. S3 A and B). Emergent L1 larvae
in the subsequent F2, F4, and F6 generation derived from post-L1
diapause AMPK/aak-1/2 mutants also possess increased levels of
H3K4me3 (Fig. 3B).
In many organisms H3K4me3 is catalyzed by a COMPASS-like
complex, the catalytic core of which includes a histone methyltransferase of the Su(var)3-9/enhancer of zeste/trithorax domain
protein (SET1)/mixed lineage leukaemia (MLL) family (32–34).
The SET1/MLL orthologs in C. elegans are SET-2 and SET-16
(35, 36) and they exist in a large multisubunit assembly (34, 37).
Mutations in the COMPASS-like complex exhibit somatic phenotypes that include disruptions in vulva development by attenuating LET-60/RAS signaling, and a reduced brood size (35, 36).
We noted that increases in H3K4me3 levels, such as those seen in
mutants that harbor mutations in rbr-2: a member of a family of
the histone demethylases involved in removing methyl groups
H3K4me3
B 10
WT
T
aa
ak-1/2
C
*
6
*
H3K4me3
2
α-tubulin
-
3d
3d
F2
F4
F6
aak-1/2
*
*
40
20
*
Reduced brood
d size (%)
E
*
Fertile adults (%)
*
4
WT
60
0
3 days
8
0
D
-
F
P0
F1
100
80
60
40
20
*
*
DAPI
SET-2
WT
HTP-3
H3K4me3/ HTP
P-3
DAPI
*
*
a
aak-1/2
A
from H3K4, lead to somatic defects, reduced brood size, and
premature adult death (38). These phenotypes mirror those we
observed in our post-L1 diapause AMPK/aak-1/2 mutants, suggesting that the misregulation of H3K4me3 levels may be linked to
the observed defects (Fig. S3 C and D).
Consistent with the increases in H3K4me3 observed in the
PGCs of the starved AMPK/aak-1/2 mutants, we also observed a
global increase in H3K4me3 in later-stage (L4) post-L1 diapause
animals, suggesting that early modifications that occur in the PGCs
are not reduced to a baseline threshold more typical of wild-type
upon the onset of postembryonic development, but rather remain
abnormally elevated (Fig. 3C). The increased H3K4me3 levels
were not unique to the germ cells, but also occurred in the soma.
Larvae that were subjected to gon-1(RNAi), which compromises
germ-cell proliferation without affecting nongonadal cells (39),
retained significantly high levels of H3K4me3 despite a reduction
in the number of germ cells at this stage (Fig. 3C).
If the abnormal accumulation of H3K4me3 in the PGCs during
starvation is responsible for the fertility defects and reduced brood
size in the post-L1 diapause AMPK/aak-1/2 mutants, then reducing
their levels should suppress the reproductive defects seen in both
post-L1 diapause AMPK/aak-1/2 mutants and their descendants.
0
Fig. 3. H3K4me3 levels accumulate and persist over multiple generations in the primordial germ cells of post-L1 diapause AMPK mutants. (A) H3K4me3 is
increased in the PGCs of post-L1 diapause aak-1/2 mutants. Emergent wild-type and aak-1/2 L1 larvae were starved 3 d before fixation and immunostaining
with H3K4me3 (green) and the PGC-specific marker HTP-3 (red) antibodies. Dashed lines mark the PGCs; note that the PGCs undergo supernumerary divisions
in the aak-1/2 mutants, as previously described. (Scale bar, 5 μm.) (B) H3K4me3 is elevated through multiple generations in the progeny of post-L1 diapause
aak-1/2 L1 mutants. Quantification of H3K4me3 levels normalized to HTP-3 signal following immunostaining in the PGCs of nonstarved (−), 3 d starved (3d),
and in subsequent generations (F2, F4, and F6) of post-L1 diapause animals. n ≥ 8 different animals from which all PGCs were used for the quantification. *P <
0.05 (one-tailed t test). (C) The elevated H3K4me3 levels that arise in PGCs of starved aak-1/2 mutants occur in both the germ line and in the soma. The
modification persists into later stages of development and is dependent on the SET-2 histone methyltransferase. Wild-type, aak-1/2, set-2, and rbr-2 postL1 diapause larvae that spent 3 d in the diapause, or not (−), were recovered to replete plates and collected at the mid-L4 stage for immunoblot analysis using
an anti-H3K4me3 antibody. Forty percent more post-L1 diapause (*) aak-1/2; gon-1(RNAi) animals were required to match the levels of the α-tubulin loading
control. (D and E) Increased COMPASS complex activity contributes to sterility and brood-size defects in post-L1 diapause aak-1/2 mutants. Compromise of
COMPASS complex components by dsRNA soaking during the period of starvation improves fertility (D), and the frequency of animals that exhibit brood size
defects (E) in the F1 descendants of post-L1 diapause aak-1/2 mutants. Control animals (CTL) are aak-1/2 L1 larvae maintained 3 d in M9 buffer containing GFP
dsRNA. Error bars: 95% CI; *P < 0.05 Fisher’s exact test, (D) n ≥ 150, from three independent experiments. (E) For P0 analysis n ≥ 40, and for the F1 analysis n ≥
120 from three independent experiments. (F) PGCs of starved aak-1/2 mutants have abnormally high levels of SET-2. Immunostaining of SET-2 after 3 d of
starvation in wild-type and aak-1/2 mutant L1 larvae. Animals were counterstained with DAPI. Dotted white lines delineate the boundaries of the PGC nuclei.
(Scale bar, 5 μm.)
E2694 | www.pnas.org/cgi/doi/10.1073/pnas.1616171114
Demoinet et al.
generating phosphorylated motifs that are recognized and bound
by 14-3-3 proteins (40, 41). The 14-3-3 proteins are involved in
many diverse biological processes and affect their targets by multiple mechanisms. If AMPK-dependent phosphorylation of SET-2
or other components of the COMPASS complex results in recognition of the phosphotargets by 14-3-3 proteins to mediate its
effects downstream of nutrient/energy stress, then loss of the
C. elegans 14-3-3 proteins should recapitulate the phenotypes
typical of AMPK compromise in post-L1 diapause animals. In
C. elegans there are two predicted 14-3-3 proteins: ftt-2 and par-5,
but only par-5 is expressed in the germ line (42, 43). Because par-5
is an essential gene we performed RNA soaking experiments with
dsRNA corresponding to par-5 during the L1 diapause. PostL1 diapause wild-type larvae subjected to par-5(RNAi) exhibited
pronounced sterility compared with control animals, and this effect
could not be compounded by removing AMPK function, indicating
Demoinet et al.
PNAS PLUS
Misregulated Transcriptional Elongation Contributes to the Sterility
of Post-L1 Diapause AMPK Mutants. The early stages of animal
development are critical for reading and writing new epigenetic
80
NS
60
40
20
0
*
*
C
*
100
80
60
40
20
WT
aak-1/2
60
40
20
0
0
-
80
-
F1
F2
WT
Fig. 4. The 14-3-3 protein PAR-5 acts with AMPK, but in parallel to the
COMPASS complex, to protect germ-line integrity. (A) Wild-type and aak-1/2
mutant L1 larvae were hatched then maintained in M9 containing par-5
dsRNA for 3 d before recovery on food. Adult fertility was assessed in control
nonstarved and par-5(RNAi) animals; n = 100. Error bars: 95% CI; *P < 0.05
Fisher’s exact test; NS, nonsignificant. (B) The 14-3-3 compromise results in
transgenerational reproductive defects. Starved wild-type L1 larvae subjected to par-5(RNAi) soaking or not (−) for 3 d were recovered and allowed
to produce F1 and F2 progeny. The number of fertile adult animals in each
generation was evaluated and represented; n ≥ 250. Errors bars: 95% CI;
*P < 0.05 Fisher’s exact test. (C) set-2 and par-5 function in parallel pathways
to establish or maintain post-L1 diapause germ-line integrity. Genetic analysis was performed by soaking set-2(bn129) mutants in dsRNA corresponding
to par-5, as described above.
PNAS | Published online March 13, 2017 | E2695
DEVELOPMENTAL
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B100
*
100
Fertile adults (%)
A
Fertile adults (%)
(
par-5 Acts with AMPK to Ensure Germ-Line Integrity in a Manner That Is
Independent of set-2. AMPK commonly regulates its targets by
that the two gene products likely work in a linear genetic pathway
to affect adult fertility following starvation (Fig. 4A). In addition,
the fertility of both the F1 and F2 generation descendants that were
derived from post-L1 diapause wild-type animals subjected to par5(RNAi) were also affected (Fig. 4B). Therefore, the effect of the
single par-5(RNAi) treatment during the L1 diapause spanned at
least two generations. The similarity of the par-5(RNAi) phenotype
to that of the observed AMPK post-L1 diapause mutants, and the
observation that the compound mutations are not additive, is
consistent with a role for PAR-5 in mediating the ability of AMPK
to protect germ-line integrity during periods of starvation.
If AMPK triggers a PAR-5–mediated response to modulate
COMPASS activity through its regulation of SET-2 accumulation in the PGC nuclei, then the complete removal of set-2 from
par-5–compromised starved animals should suppress any inappropriate H3K4me3 caused by accumulation of SET-2, and ultimately improve the fertility of post-L1 diapause mutant animals.
This is, however, not what we observe in the set-2; par-5(RNAi)
animals, which exhibit a substantially greater frequency of sterility
than either single mutant alone following a 3-d period in the
L1 diapause (Fig. 4C). These data strongly suggest that par-5 and
set-2 do not function in a linear genetic pathway, but rather the
two gene products work independently of one another, whereby
both are dependent on AMPK function. Therefore, in response to
acute starvation AMPK regulates at least two different pathways
that ultimately affect germ-line integrity, and its absence during
the L1 diapause has reproductive consequences that can persist
through multiple generations. One branch appears to be mediated
by the 14-3-3 protein PAR-5, which likely targets effectors that
have yet to be elucidated, whereas a second branch impinges on
the chromatin-writing COMPASS complex.
We propose that during starvation AMPK blocks the catalysis
of H3K4me3 in the two PGCs through its ability to modify one or
more of the COMPASS components (SET-16, SET-2, ASH-2,
WDR-5.1). In post-L1 diapause AMPK mutant larvae, the consequent increase of H3K4me3 in the PGCs is associated with a
progressive transgenerational deterioration in reproductive fitness.
Fertile adults (%)
Consistent with a misregulation of the COMPASS complex in
the observed starvation-dependent sterility, soaking emergent
L1 larvae in buffer containing double-stranded RNA (dsRNA)
corresponding to any one of the COMPASS components set-2, set16, ash-2, or wdr-5.1 for a 3-d period during the L1 diapause was
sufficient to partially suppress both the sterility of the postL1 diapause AMPK/aak-1/2 mutant larvae and the reduced brood
size observed in the F1 generation. Conversely, the same treatment
did not affect the fertility or the brood size of wild-type animals
(Fig. 3 D and E and Fig. S3 E and F). Furthermore, the compromise of set-2 was sufficient to dramatically reduce the global
H3K4me3 levels in the post-L1 diapause AMPK/aak-1/2 mutant
larvae (Fig. 3C).
Curiously, most of the vulval defects of starved AMPK/aak-1/2
mutants were not suppressed when L1 larvae were treated with
dsRNA corresponding to set-2 or set-16, suggesting that other
H3K4me3-independent pathways may be disrupted in the postL1 diapause AMPK/aak-1/2 larvae (Table S1). Although the
suppression that we achieve by removing components of the
COMPASS complex is partial, this may reflect our inability to
remove all of the target protein through RNAi. On the other hand
we cannot rule out that other factors that may contribute to the
observed defect that are not downstream of inappropriate histone
methyltransferase activity.
AMPK has been demonstrated to drive adaptive metabolic
adjustment to energy stress by phosphorylating key cellular proteins, ultimately blocking anabolic pathways while simultaneously
enhancing pathways involved in energy production or conservation. Bioinformatic analysis revealed that all of the members of the
COMPASS-like complex components, except DPY-30, possess
amino acid signatures that match the optimal AMPK substrate
motif (Fig. S3G) (40). This finding represents a significant enrichment of these sites within this complex, suggesting that AMPK
may regulate the activity of the complex during environmental
challenges. Despite our efforts we could never acquire the quantity of PGCs to confirm this possibility biochemically. Moreover,
the transient nature of the phosphorylation makes it unlikely that
it would be present in the germ cells of later-stage post-L1
diapause larvae/adults.
To determine if AMPK might affect COMPASS function within
the starved PGCs by altering its abundance, we examined the
levels of SET-2 in the PGCs in starved L1 mutant larvae to assess
if its stability or its localization might be affected in an AMPKdependent manner. We noted that SET-2 levels were consistently
higher in the PGCs of starved L1 larvae that lack AMPK compared with starved wild-type PGCs, indicating that AMPK compromise may affect its accumulation in the PGC nuclei and
consequently increase the levels of H3K4me3 in the germ cells of
the starved AMPK/aak-1/2 mutants (Fig. 3F).
Discussion
By regulating its various protein targets, AMPK ensures that the
PGCs respond efficiently to starvation during the L1 diapause by
eliciting both cell cycle and developmental/reproductive quiescence. In its absence, the nutrient/energy contingency is relaxed,
allowing a critical chromatin writer, such as the COMPASS
complex, to aberrantly activate a postembryonic transcriptional
program in the PGCs during a period when all cells should remain quiescent (Fig. 5C).
It is unclear why the chromatin modifications that arise in the
post-L1 diapause AMPK mutants cannot be resolved in subsequent generations, but instead accumulate to progressively extinguish the germ line. The heritable effects of set-2 mutations on
lifespan are largely resolved in a limited number of generations
(53). In contrast, the transgenerational defects of post-L1 diapause AMPK mutant descendants often worsen and cannot be
E2696 | www.pnas.org/cgi/doi/10.1073/pnas.1616171114
B
100
80
*
60
100
Reduced broo
od size (%)
A
Fertile adu
ults (%)
information directed by maternal and/or paternal instruction (44,
45). How this information is transduced to affect gene expression
in response to a given stimulus is currently speculative at best.
During starvation in C. elegans L1 larvae, a specific geneexpression program is initiated (46) and during this period
RNA polymerase II is observed in different configurations: in the
“docked” state RNA polymerase II is on proximal promoters
waiting for cues to initiate, whereas in the “paused” configuration the complex remains in a postinitiated state, presumably on
hold until a physiological or developmental contingency is satisfied (47, 48).
This developmental pausing is not exceptional and in many
organisms RNA polymerase II is maintained in a postinitiated
state where subsequent elongation is regulated by factors that
associate with the phosphorylated carboxyl-terminal domain of the
RNA polymerase II large subunit (49). In the PGCs the role of
carboxyl-terminal domain phosphorylation appears to be less
critical than in the somatic cells, whereas orthologs of some of the
critical known elongation regulators are absent from the C. elegans
genome, suggesting that regulation of elongation is independent
of many of the mechanisms described in somatic cells (50). The
COMPASS complex may nevertheless contribute to this transcriptional switch in the PGCs in a manner that is independent of
the soma-specific controls and therefore must be neutralized in
the PGCs during starvation.
To determine if the defects observed in AMPK-compromised
starved animals are related to the link between increased
H3K4me3 and aberrant resumption of transcriptional elongation, we blocked any elongating complexes during the period
of starvation using 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole
(DRB), a potent, highly specific, elongation inhibitor (51, 52) and
quantified the effects on fertility in the treated animals and on the
brood-size defects in subsequent generations. Treatment of starved
AMPK/aak-1/2 mutant L1 stage larvae with DRB partially suppressed the sterility observed in post-L1 diapause AMPK/aak-1/2
mutant adult hermaphrodites by 49% (n = 100) (Fig. 5A), without
affecting the fertility of treated post-L1 diapause wild-type.
Moreover, consistent with AMPK and PTEN affecting two independent pathways that control PGC integrity, DRB treatment
had no effect on the sterility observed in post-L1 diapause
PTEN/daf-18 mutants (Fig. S1H).
The brood-size defects typical of the F1 generation of postL1 diapause AMPK mutants were also reduced, but the effect in
the F2 generation is also accompanied by a significant number of
affected F1 progeny animals that resolve their brood-size defects,
making it difficult to conclude whether the transgenerational defects are indeed dependent on aberrant elongation in the starved
PGCs of AMPK mutants (Fig. 5B). Nevertheless, by specifically
targeting the aberrant elongating complexes we were able to
correct many of the reproductive defects typical of the post-L1
diapause AMPK/aak-1/2 mutants.
40
20
30μM
-
P0
F1
F2
60
40
20
0
-
*
80
0
30μM DRB
-
aak-1/2
WT
30μM
DRB
aak-1/2
C
OpƟmal
condiƟons
AMPK
Gene expression
SET-16
H2
H2
H2
SET-2
ASH-2
H3K4me3
H3K4me3
COMPASS complex
acƟve
H3K4me3
PGCs
?
StarvaƟon
PAR-5
AMPK
?
P
SET-16
P
?
?
P
SET-2
H2
H2
H2
ASH-2
H3K4
COMPASS complex
inacƟve
H3K4
H3K4
PGCs
Fig. 5. Inappropriate transcriptional elongation during periods of starvation
contributes to the reproductive defects seen in AMPK mutants. (A) The elongation inhibitor DRB can partially suppress the sterility observed in postL1 diapause AMPK mutant hermaphrodites. Emergent wild-type and aak-1/2
larvae were maintained in M9 buffer containing 30 μM DRB or not (−) for 3 d
before recovery on replete plates. The proportion of fertile adult animals is
represented, n ≥ 150. Error bars: 95% CI; *P < 0.05 Fisher’s exact test.
(B) Transgenerational brood-size defects are resolved more efficiently in the
progeny of post-L1 diapause AMPK mutant larvae that were treated with DRB
during the period of starvation. The frequency of animals with reduced brood
size in aak-1/2 mutants and in the subsequent generation (F1 and F2) is represented, n ≥ 50. Errors bars: 95% CI; *P < 0.05 by Fisher’s exact test. (C) AMPK
affects quiescence and integrity of the PGCs during acute starvation. During
replete conditions, AMPK is inactive permitting the COMPASS complex to actively methylate H3K4, thereby enhancing transcriptional elongation. During
periods of acute starvation, AMPK becomes activated and targets components
of the COMPASS complex, effectively blocking the histone methyltransferase
and ultimately inhibiting transcriptional elongation, and hence germ-line gene
expression. In addition, AMPK functions with 14-3-3/PAR-5 to affect germ-line
integrity through a SET-2–independent pathway.
corrected by crossing with mutants that were never starved. In
addition, the sterility observed in wild-type animals that were
subjected to an 11-d L1 diapause is completely corrected in the
next generation. However, if AMPK is reduced in these animals as
they grow to adulthood, the fertility of the next generation of
animals is visibly affected, albeit not significantly (Fig. S2I). This
finding suggests that similar epigenetic modifications can occur
even in wild-type animals after severe starvation, and AMPK may
be important for the resolution of these modifications in the
subsequent generation. In the absence of AMPK function, these
chromatin modifications become more abundant and can be
transmitted to subsequent generations. The germ-line defects we
describe herein resemble those seen in spr-5 mutants that have
compromised demethylase function, where the germ line becomes
mortalized over several generations (28, 54). Understanding the
mechanism through which AMPK functions with PAR-5 to ensure
PGC integrity may provide some insight regarding the potential
targets that exacerbate the progressive germ-line extinction that
occurs in the descendants of post-L1 diapause AMPK mutants.
We have shown that during periods of acute starvation in the
L1 stage, the absence of AMPK results in the inappropriate
Demoinet et al.
were allowed to develop to the L4, after which they were singled to plates
and were allowed to reproduce and were transferred daily throughout their
entire reproductive period (3 d). The number of F1 offspring that were
present on each plate that reached the L4/young adult stage was thereafter
counted, allowing us to infer whether the parent had a normal or reduced
brood size. Fifty to 150 animals were analyzed per genotype per time point
from three independent experiments.
The viability of post-L1 diapause hermaphrodites was scored every 48 h
following the L4 stage after varying durations in the diapause (0, 1, 3, 11 d);
100–300 animals were evaluated from three independent experiments.
PNAS PLUS
writing of epigenetic marks that compromise germ-line integrity
because of this change in gene expression. The L1 diapause, and
potentially other diapause states, such as the dauer, may be critical
developmental buffer zones, allowing the animal to couple appropriate stasis of the chromatin landscape to environmental
conditions. Our data suggest that AMPK is a pivotal molecular
link to couple the chromatin environment and consequent adaptive changes in gene expression, with the metabolic status of the
animal to ensure proper regulation of gene expression during this
extended developmental hiatus. Recent data have demonstrated
that starvation can induce the expression of specific RNAs that
may provide some selective advantage in subsequent generations
(10). It will be particularly exciting to determine if the expression
of this suite of RNAs may be coupled to AMPK function to trigger
a heritable adaptive change in the germ line during periods of
severe energy stress.
Our data delineate how an AMPK-mediated adaptation during
acute starvation includes the buffering of epigenetic writers and is
critical to preserve germ-line integrity. This is presumably achieved
by adjusting energy resources accordingly in part by limiting enzyme activities that could otherwise compromise the fitness of the
animal. How general this AMPK function may be is currently unknown, nor can we speculate on the limits of its buffering capacity.
At present, there are no polymorphisms that have been characterized that affect AMPK function and that are associated with
the affected Hongerwinter victims or their children. However, our
findings suggest that failure at any level of this complex cascade
downstream of AMPK activation could have far-reaching implications. Variations in an AMPK-mediated response could potentially account for the frequency and spectrum of observed genetic
and epigenetic anomalies that manifest in these individuals and in
other survivors of famine.
Progressive Brood-Size Reduction in Individual Parental Lineages. Ten independent post-L1 diapause aak-1/2 mutant lineages were analyzed independently. For each lineage, the entire population of F1 progeny from postL1 diapause aak-1/2 fertile parents (P0) was singled onto fresh plates with
food. Brood-size defects were quantified from individual fertile parents by
counting progeny. Plates with animals that exhibited a reduced brood size
were kept to continue the analysis and 100–200 individuals per parental
lineage were singled every generation onto replete plates. The transgenerational progeny obtained from individual starting lineages were maintained
separately for the duration of the analysis. In the final generations of several
lines, the animals became very sick and the proportions of plates that possessed animals that showed a Him, Lon, Dpy, or Sma were assessed.
Transgenerational Rescue. To determine the role of sperm in the postL1 diapause reproductive defects 3-d post-L1 diapause aak-1/2 L4 hermaphrodites were crossed with healthy nonstarved aak-1/2 males, and the reciprocal cross was also performed. The resulting F1 brood size was evaluated
and the percentage of parents that exhibit a reduced brood size was calculated from plates with successful crosses. A minimum of 40 successful
crosses was analyzed. To determine the ability of wild-type gene copies to
rescue observed reproductive defects in our transgenerational rescue experiments, L4 hermaphrodites from late-generation (F7 or F8) postL1 diapause aak-1/2 mutants that exhibited severe brood-size defects were
crossed with healthy nonstarved wild-type or aak-1/2 males. Reproductive
defects were determined in the resulting cross progeny, as described above.
Materials and Methods
Postrecovery Defects Following the L1 Diapause. The L1 starvation assay was
adapted from previously described protocols (56, 57). Briefly, animals were
fed with OP50 for at least five generations before any analysis. Gravid
hermaphrodites were treated with alkaline hypochlorite and the resulting
eggs were hatched and cultured in sterile M9 medium in 15-mL tubes with
rotation at 20 °C for the duration of the diapause. After 24 h, the density of
newly hatched L1 larvae was adjusted to 6–10 L1 larvae per microliter. To
determine viability, 10-μL aliquots were distributed on 6-cm nematode
growth medium (NGM) plates seeded with OP50 every 48 h and the number
of L1 larvae was counted (initial). The following day, the number of moving
animals was recorded as viable, and the survival rate was calculated as viable
animals per initial number seeded. This experiment was repeated twice per
time point. A minimum of three independent experiments was performed
per analysis; the average survival at each time point was determined and
used to generate the survival curve where error bars indicate SD.
Analysis of Fertility, Brood Size, and Viability. Newly hatched larvae were
maintained in the absence of food and every 48 h groups of L1 larvae were
transferred to OP50-seeded NGM plates and maintained at 20 °C until the
L2 stage, when they were singled onto seeded NGM plates and allowed to
grow to adulthood. Larval growth and other postembryonic developmental
phenotypes (Bag, Burst, Evl, Muv) were monitored. Fertility and other phenotypic abnormalities (presence of dead embryos, males, long adults, premature death) were assessed up until 1-wk post-L4 stage. All experiments
were performed at least three times; 200–800 recovered animals were analyzed per genotype per time point were monitored.
Brood-size analysis was performed by counting progeny born from fertile
parents of varying genotype. Fertile post-L1 diapause AMPK mutant adults
were either parents that generated a reduced brood size (fewer than 100 F1
progeny), or those parents that produced a normal brood size (more than
100 F1 progeny). To quantify brood size, post-L1 diapause hermaphrodites
Demoinet et al.
RNAi Soaking During the L1 Diapause. dsRNAs that corresponded to set-2, set16, ash-2, wdr-5, par-5, and GFP (as a control), were prepared as described
previously (58). Wild-type and aak-1/2 L1 larvae were obtained by alkaline
hypochlorite treatment and were maintained in M9 buffer containing
dsRNA of the query gene (1–1.5 μg/μL) during the 3-d diapause. L1 larvae
were then transferred to seeded NGM plates to recover to the L4 stage.
Subsequently, 50 larvae were collected for each dsRNA tested, maintained at
20 °C for 3 or 4 d, after which their fertility and brood size were scored.
Adults were sterile or fertile, whereas fertile animals could either be “reduced brood size” (<100 F1), or “normal brood size” (>100 F1). For transgenerational analysis (from F1 to F3), 50 F1 L4-stage larvae obtained from
parents with reduced brood size were singled to seeded NGM plates and
their fertility and brood size were scored as above. Each experiment was
performed three independent times.
DRB Treatment. We determined our working concentration of 30 μM DRB
through a typical LE50 drug dosage. Wild-type and aak-1/2 L1 larvae
obtained by alkaline hypochlorite treatment, were maintained in M9 buffer
containing 30 μM DRB, or not, during the 3-d duration in the diapause.
Following the starvation, postdiapause L1 larvae were transferred to seeded
NGM plates and when they reached the L4 stage 50 larvae per condition
were isolated and incubated at 20 °C for 3 or 4 d to assess their fertility and
brood size. The reproductive defects fall into three different categories:
sterile, fertile with reduced brood size (≤100 F1), or fertile with normal
brood size (>100 F1). For transgenerational analysis (from F1 to F3), 50 F1
animals at the L4 stage, obtained from parents with reduced brood size,
were singled onto NGM plates seeded with OP50, and their fertility and
brood size were scored as described above. Each experiment was performed
minimally three independent times.
ACKNOWLEDGMENTS. We thank Florence Couteau, Monique Zetka, Brandon
Faubert, Rusty Jones, Bill Kelly, Francesca Palladino, Susan Strome, Vincent
Calcagno, and Anne Brunet for strains, reagents, and discussion; and Victor
Ambros, Monique Zetka, and Stephanie Crane-Weber for critically reading the
manuscript. Many of the strains used here were generated or provided by the
Gene Knockout Consortium or the Caenorhabditis Genetics Center, respectively. This work was supported by funding from the Canadian Institutes of
Health Research.
PNAS | Published online March 13, 2017 | E2697
DEVELOPMENTAL
BIOLOGY
C. elegans Strains and Culture. C. elegans were cultured at 20 °C on OP50, as
previously described (55) unless otherwise indicated. Strains used in this
study include: Bristol N2 wild type, aak-1(tm1944)I, aak-2(ok524)X, daf-18
(ok480)IV, raga-1(ok386)II, age-1(hx546)II, set-2(ok952)III, set-2(n4589)III, set2(bn129)III, rbr-2(tm1231)IV.
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