AMPK blocks starvation-inducible transgenerational defects in Caenorhabditis elegans

Abstract

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.

Keywords: AMPK; C. elegans; COMPASS; epigenetics; histone methyltransferase.

Fig. S1.

AMPK and PTEN act through independent pathways to control PGC cell cycle quiescence, germ-line integrity, and survival during the L1 diapause. (A) AMPK (aak-1/2) and PTEN/daf-18 are required to survive the stress of the L1 diapause. Animals of the indicated genotypes were hatched in the absence of food and viability was determined every 24 h following hatching. “Days” represent duration of starvation. Data represent the mean ± SD of three independent experiments. (B) AMPK (aak-1/2) and PTEN/daf-18 act independently to ensure adult reproductive fitness in post-L1 diapause animals. Animals were maintained in the L1 diapause for varying durations (Days) followed by recovery on replete plates (with OP50) until adulthood and fertility was scored in all adult animals. “Days” represent duration of starvation; “–” indicates animals that were not starved. Error bars: 95% CI. (C) Prolonged duration in the L1 diapause affects brood size of fertile adult animals. L1 animals were maintained in the L1 diapause for varying durations, recovered to replete plates, and their total F₁ progeny number were counted and the distribution shown. The average brood size for each group is depicted by the horizontal bar; the dotted line represents the minimum threshold brood size used to define reduced brood size. (D) Distribution of aak-1/2 and wild-type brood sizes following varying durations in the L1 diapause. The duration of the diapause correlates negatively with the brood size of the animals tested. After a 3-d period in the diapause, ∼80% of the recovered post-L1 diapause aak-1/2 mutants produce broods of reduced size (fewer than 100 F₁ progeny). For better visualization, density distribution is smoothed by grouping values in bin widths of 12 (0–11; 12–23; 24–35; …; 389–400). Ctl (–) indicates animals that were not starved; n > 40 per genotype per condition. (E) Prolonged duration in the L1 diapause results in premature adult lethality. L1 larvae of wild-type, aak-1/2, and daf-18 were maintained 3 d (*) or 11 d (**) without food in the L1 diapause, or were not starved (−), before being singled to replete plates and allowed to grow and develop. Post-L4 stage survival was scored every 2 d; n ≥100 animals per point. (F) Both AMPK (aak-1/2) and PTEN (daf-18) are required for the maintenance of PGC cell-cycle quiescence during the L1 diapause, but through independent pathways. Germ-cell numbers were scored by counting HTP-3–stained cells in starved wild-type or mutant animals after either 1 or 3 d without food in the L1 diapause; n ≥ 50 animals. (G) Differential suppression of sterility between AMPK and PTEN/daf-18 mutants. Loss of raga-1 or age-1 suppresses the sterility of post-L1 diapause daf-18 mutants, but not aak-1/2 mutants. Error bars: 95% CI; *P < 0.05 by Fisher’s exact test; NS, nonsignficant; n ≥ 50. (H) Synchronized daf-18 L1 larvae were maintained in M9 buffer without food for 3 d with or without 30 µM DRB. Animals were then transferred to regular OP50-seeded NGM plates and fertility was assessed after they reached adulthood. Nonstarved daf-18 larvae were used as a control. Assays were performed three times and the data represent the mean ± SD, where n = 50 for each condition.

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), post-L1 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 OP50-seeded 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) wild-type 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 post-L1 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 post-L1 diapause aak-1/2 mutant males (♂^dia), or hermaphrodites (⚥^dia) 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.

Fig. S2.

Prolonged duration in the L1 diapause results in somatic and germ-line defects in post-L1 diapause AMPK mutants. (A) DIC images of young adult hermaphrodites that include (a) a wild-type adult recovered after a 3-d period in the L1 diapause, (b–d) recovered aak-1/2 adults that were previously subjected to 3 d in the L1 diapause. Dashed lines demarcate the gonad boundary. (Scale bar, 50 μm.) (B) aak-1/2 post-L1 diapause adults display a range of germ-line defects, disruption of meiotic progression resulting in defects in gamete differentiation. Extruded gonads from adult post-L1 diapause animals that were previously subjected to 3 d without food in the L1 diapause: wild-type (a) or aak-1/2 (b–d) at L4 + 24 h were stained with DAPI. The severity of the defects in aak-1/2 gonads is associated with the observed reproductive defects gonad extruded from a post-L1 diapause adults that had a (b) normal brood size, (c) a reduced brood size, or (d) a sterile hermaphrodite. The solid line indicates the transition zone (when present) based on nuclear morphology assessed by DAPI staining, and arrows indicate inappropriate proximal germ-cell divisions. B and C are composite micrograph images that were stitched together using Adobe Photoshop (B) or Microsoft Image Composite Editor (C). (Scale bars, 50 μm.) (C) Post-L1 diapause aak-1/2 adults that were subjected to 3 d in the diapause possess gonads with reduced sperm numbers based on DAPI staining (L4 + 24 h). (Scale bars, 10 μm.) (D) AMPK is critical for the maintenance of germ-line integrity through a pathway that is independent of its effects on essential postembryonic developmental processes. Animals that demonstrated any visible developmental defect were discarded before our brood-size analysis (Table S1) and only adults that survive 4 d post-L4 stage were compared in our brood-size analysis (“excluding early deaths”). “3”: 3-d duration in the L1 diapause, or “–”: not starved. Error bars: 95% CI. (E) Loss of AMPK signaling results in a reduction in brood size that becomes progressively worse with each subsequent generation following the acute starvation. This is not observed in 11-d starved wild-type or 3-d–starved daf-18 animals. Three different methods of selection (methods #1, #2, #3) were used (Materials and Methods) and under no circumstances did we observe transgenerational defects in the subsequent generations of post-L1 diapause wild-type or PTEN/daf-18 animals. (F) aak-1/2 mutants exhibit a progressive reduction in brood size with each successive generation, similar to a Mrt phenotype. Real brood-size quantification over multiple generations from seven independent post-L1 diapause aak-1/2 mutant parents (P₀-I to P₀-VII) that initially exhibited a reduced F₁ brood size phenotype (method #1). Lineage P₀-VI and P₀-VII were totally extinguished by the F₃ generation. (G) Brood-size defects typical of fertile post-L1 diapause AMPK mutant adults cannot be restored by crossing F₄ or F₅ post-L1 diapause aak-1/2 mutant lineage (P₀-VIII) with nonstarved aak-1/2 males. A partial rescue is however observed in the progeny (*) of these F₁ hermaphrodites generated from this generation is allowed to self-fertilize and yield an F₂ generation. Error bars: 95% CI. (H) Prolonged durations without food in the L1 diapause did not confer a survival advantage for wild-type or aak-1/2 mutants when resubjected to starvation during the L1 stage of the subsequent F₁ generations. F₁ generation L1 larvae obtained from fertile post-L1 diapause adults that were subjected to 3 d in the diapause. F₁* represents starved L1 obtained from post-L1 diapause aak-1/2 mutant parents that showed a reduced brood size phenotype. Mean ± SD of three independent experiments. Data were obtained from ≥three independent experiments, and ≥80 animals were scored for each experiment. (I) Wild-type (N2) L1 larvae were starved in M9 buffer for 11 d after which they were transferred to plates seeded with OP50 or bacteria expressing aak-2 dsRNA. Adult aak-2 (RNAi) animals were then singled to regular NGM plates with OP50. The fertility of the treated and nontreated parents (P₀) and their F₁ generation were then assessed. The assays was performed twice. Data represent the mean ± SD where n = 50 for each condition.

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 (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 (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.

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 post-L1 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.)

Fig. S3.

Post L1-diapause AMPK mutant animals accumulate H3K4me3 that is dependent on SET-2 histone methyltransferase. (A) Post-L1 diapause aak-1/2 L1 larvae have abnormally high levels of H3K4me3 in their PGCs. Wild-type and aak-1/2 L1 larvae were maintained either 1 or 3 d without food in the L1 diapause then immunostained with antibodies that recognize H3K4me3 and counterstained with DAPI. H3K4me3 levels were quantified and normalized to DNA content (DAPI) in the PGCs; n ≥ 15 different animals. *P < 0.05 (one-tailed t test). (B) H2B phosphorylation is induced by starvation and is AMPK-dependent. Western blot performed using antibodies against the H2 phosphorylated S36 epitope (ECM Biosciences) using whole-animal extracts from animals subjected to 3 d in the L1 diapause (“S” for starved) or nonstarved L1 extracts (“H” for healthy) treated with CIP phosphatase (+), or not (−), to show specificity for the phosphorylated isoform. (C and D) A misregulation of histone methyltransferase results in brood-size defects and premature lethality in post-L1 diapause AMPK mutants. L1 larvae of the following genotypes: set-2 (n4589) (a strong allele) or set-2 (ok952) (a weak allele), or the demethylase rbr-2 (tm1941), were subjected to varying periods in the L1 diapause then recovered on plates with food and grown to reproductive maturity. (C) The F₁ brood size of resulting fertile adults was scored and represented in box plot format; n ≥ 40 per genotype per condition. (D) rbr-2 mutants phenocopy the premature adult death of post-L1 diapause aak-1/2 animals. Wild-type, aak-1/2, set-2, and rbr-2 L1 larvae were maintained in the L1 diapause for 3 d (*) or not starved (–), then recovered to replete plates and their post-L4 stage survival was scored every 2 d; n ≥100 animals per point. (E and F) Reducing the COMPASS activity by soaking animals in dsRNA that corresponds to each of the COMPASS components during the 3-d period in the diapause does not affect (E) the fertility of wild-type animals either in post-L1 diapause animals or, (F) the brood size of their subsequent F₁ generation. Error bars: 95% CI; n ≥ 100 per condition from three independent experiments. (G) Two histone methyltransferases (SET-2 and SET-16) and associated components of the COMPASS complex are enriched for consensus AMPK phosphorylation sites. Peptide sequences were compared with the described AMPK consensus site described by Gwinn et al. (40) (AMPK motif) and aligned for comparison. There is a significant enrichment (Fisher exact test, P = 2.8 × 10⁻⁷) in proteins with at least one AMPK consensus phosphorylation recognition motif within the COMPASS complex. Red, phosphoacceptor site (Ser/Thr); yellow, hydrophobic residues; blue, basic residues; green, acidic residues. To perform Fisher’s exact test: 11,353 proteins were identified that have a single AMPK consensus site among a total of 20,405 Caenorhabditis elegans proteins (excluding isoforms) according to Wormbase release WS246. This enrichment is still valid even when protein size is taken into consideration: 24 of 11,353 AMPK consensus sites are found in COMPASS complex components (2.1%). This is significantly different from the number expected according to random distribution. All of the COMPASS components together contain 11,130 aa for a total of 8,378,701 aa (1.3%); exact binomial test P = 0.00211.

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.

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 post-L1 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.