A histone H4 lysine 20 methyltransferase couples environmental cues to sensory neuron control of developmental plasticity

Animals change developmental fates in response to external cues. In the nematode Caenorhabditis elegans, unfavorable environmental conditions induce a state of diapause known as dauer by inhibiting the conserved DAF-2 insulin-like signaling (ILS) pathway through incompletely understood mechanisms. We have previously established a role for the C. elegans dosage compensation protein DPY-21 in the control of dauer arrest and DAF-2 ILS. Here, we show that the histone H4 lysine 20 methyltransferase SET-4, which also influences dosage compensation, promotes dauer arrest in part by repressing the X-linked ins-9 gene, which encodes a new agonist insulin-like peptide (ILP) expressed specifically in the paired ASI sensory neurons that are required for dauer bypass. ins-9 repression in dauer-constitutive mutants requires DPY-21, SET-4 and the FoxO transcription factor DAF-16, which is the main target of DAF-2 ILS. By contrast, autosomal genes encoding major agonist ILPs that promote reproductive development are not repressed by DPY-21, SET-4 or DAF-16/FoxO. Our results implicate SET-4 as a sensory rheostat that reinforces developmental fates in response to environmental cues by modulating autocrine and paracrine DAF-2 ILS. Summary: The C. elegans histone methyltransferase SET-4 acts to link environmental signals to diapause through regulation of the X-linked insulin-like peptide gene ins-9 in sensory neurons.


INTRODUCTION
To maintain evolutionary fitness, organisms must react appropriately to environmental cues. The free-living nematode Caenorhabditis elegans has evolved a developmental strategy to optimize survival in changing environments. Under replete conditions, larvae progress through four stages (L1-L4) to become reproductive adults. In adverse conditions such as overcrowding, heat or food scarcity, larvae arrest in an alternative stage known as dauer. Adapted for survival in harsh environments, dauers are morphologically, metabolically and behaviorally distinct from reproductive L3 larvae. Improvement of ambient conditions induces dauer exit and resumption of reproductive development (Fielenbach and Antebi, 2008;Riddle, 1988). The dominant environmental cue that influences dauer arrest is a constitutively elaborated pheromone that indicates population density (Butcher et al., 2008;Golden and Riddle, 1982).
C. elegans dauer arrest has served as a useful paradigm for understanding the molecular basis of developmental plasticity. Genetic analysis has defined four conserved signaling pathways that promote reproductive development in favorable environments. The DAF-11 transmembrane guanylyl cyclase acts in chemosensory neurons to regulate dauer arrest through the cyclic nucleotide-gated channel subunits TAX-2 and TAX-4. Downstream of DAF-11, DAF-2 insulin receptor (InsR)-and DAF-7 transforming growth factor-β (TGFβ)-like pathways act in parallel to promote reproductive development by inhibiting the activities of the FoxO transcription factor DAF-16 and the SMAD transcription factor DAF-3, respectively. Distal to DAF-16/FoxO and DAF-3/SMAD, bile-acid-like steroid hormones known as dafachronic acids (DAs) promote reproductive development by regulating the activity of the conserved nuclear receptor DAF-12 (Fielenbach and Antebi, 2008).
Although genetic analysis has identified how components of the DAF-11, DAF-2/InsR, DAF-7/TGFβ and DAF-12 pathways interact to promote reproductive development in favorable conditions (Gottlieb and Ruvkun, 1994;Riddle et al., 1981;Thomas et al., 1993;Vowels and Thomas, 1992), the molecular nature of the upstream events that couple external cues to the activities of these pathways remains poorly understood. Laser ablation experiments demonstrated that the amphid sensory neurons are required for induction of dauer arrest by pheromone (Schackwitz et al., 1996;Vowels and Thomas, 1994). Indeed, the dauerinhibitory ASI sensory neurons (Bargmann and Horvitz, 1991) are specific sites of expression of three insulin-like peptides (ILPs) that promote reproductive development through DAF-2/InsR (INS-4, INS-6 and DAF-28) (Chen and Baugh, 2014;Cornils et al., 2011;Hung et al., 2014;Li et al., 2003), as well as the DAF-7 TGFβ-like ligand that promotes reproductive development (Ren et al., 1996;Schackwitz et al., 1996). Furthermore, crude dauer pheromone reduces the expression of DAF-28 and DAF-7 in ASI Schackwitz et al., 1996), suggesting that pheromone induces dauer arrest at least in part by reducing the expression of agonist ligands in sensory neurons that regulate DAF-2/InsR and TGFβ-like signaling. How pheromone represses these ligands remains a mystery.
We have previously reported an unforeseen role for the C. elegans dosage compensation protein DPY-21 in promoting dauer arrest through inhibition of the DAF-2/InsR pathway (Dumas et al., 2013). DPY-21 is a component of the condensin-like dosage compensation complex (DCC) that equalizes X-linked gene expression between males and hermaphrodites by binding to both hermaphrodite X chromosomes during embryogenesis and repressing gene expression approximately twofold (Meyer, 2010;Yonker and Meyer, 2003). Here, we show that the conserved histone H4 lysine 20 (H4K20) methyltransferase SET-4, which also influences dosage compensation (Kramer et al., 2015;Vielle et al., 2012;Wells et al., 2012), promotes dauer arrest in a sex-specific manner by synergizing with DAF-16/FoxO to repress ins-9, an X-linked gene that encodes an ILP expressed specifically in ASI neurons (Chen and Baugh, 2014;Pierce et al., 2001). These findings reveal a sexually dimorphic role for regulators of histone H4K20 methylation in broadening the dynamic range of sensory responses to environmental cues that control developmental plasticity.
As previous reports link H4K20 methylation status to dosage compensation (Vielle et al., 2012;Wells et al., 2012) and DPY-21 promotes dauer arrest through dosage compensation (Dumas et al., 2013), we hypothesized that SET-4 may also regulate dauer arrest through dosage compensation. To test this, we determined the effect of set-4 mutation on the dauer-constitutive phenotype of eak-7;akt-1 double mutant hermaphrodites and males. If SET-4 promotes dauer arrest through the same mechanism as dosage compensation, then set-4 mutation should suppress dauer in hermaphrodites but not in males, as the DCC is inactive in males (Meyer, 2010). dpy-21 and set-4 mutations suppressed the dauerconstitutive phenotype of eak-7;akt-1 hermaphrodites but did not affect dauer arrest in males (Fig. 1F). Therefore, SET-4 may act through dosage compensation to control dauer arrest. We verified the role of SET-4 in dosage compensation by showing that set-4 mutation suppressed lethality in xol-1 sex-1 mutant males, which die due to inappropriate activation of dosage compensation (Dawes et al., 1999) (Fig. S1G).
In order to determine whether SET-4 plays a role in regulating dauer entry in wild-type animals in response to physiologic stimuli, we tested the ability of dauer pheromone to induce dauer arrest in wild-type and set-4 mutant animals. Mutation of either set-4 or dpy-21 decreased the sensitivity of wild-type animals to pheromone (Fig. 1G). Therefore, SET-4 and DPY-21 promote dauer arrest in wild-type animals in response to increases in population density.

SET-4 is a H4K20 methyltransferase
The mammalian SET-4 ortholog SUV420H2 is a H4K20 methyltransferase (Schotta et al., 2004), and C. elegans SET-4 promotes H4K20 trimethylation (Vielle et al., 2012;Webster et al., 2013;Wells et al., 2012). We confirmed the requirement of SET-4 for H4K20 di-and trimethylation in vivo ( Fig. 2A). Immunoblots showed no detectable SET-4 protein in set-4(n4600) and set-4(ok1481) backgrounds, consistent with these being strong loss-of-function alleles. SET-4 protein levels in set-4(dp268) are comparable with wild type ( Fig. 2A). H4K20me2 and H4K20me3 levels are undetectable in all three set-4 mutant backgrounds ( Fig. 2A), suggesting that the S182F substitution in the SET domain abrogates catalytic activity. To test this possibility directly, we purified recombinant wild-type and mutant GST-SET-4 fusion proteins and tested their ability to methylate modified H4 peptides (H4K20me0, H4K20me1 and H4K20me2) in vitro. Mass spectrometry analysis revealed that both wild-type GST-SET-4 and GST-SUV420H2 were capable of converting H4K20me1 to H4K20me2 (Fig. 2B,C). Consistent with in vitro experiments using human SET-4 orthologs SUV420H1 and SUV420H2 (Southall et al., 2014;Wu et al., 2013), methylation was not detected with unmethylated or dimethylated substrates, nor were trimethylated products detected in any assay ( Fig. 2B,C). It is possible that an enzyme distinct from SET-4 catalyzes H4K20 trimethylation in vivo. Alternatively, conversion of H4K20me2 to H4K20me3 by SET-4 in vivo may require a co-factor that is absent from our in vitro reactions.
GST-SET-4(S182F) did not methylate H4K20me1, indicating that the S182F mutation abolishes catalytic activity (Fig. S2). Given that set-4(dp268) suppresses dauer to a similar extent to the two deletion alleles (Fig. 1B), these data are consistent with SET-4 influencing dauer arrest through its conserved role in methylating H4K20. Percentage methylation of H4K20 peptide substrates by GST proteins fused to wild-type SET-4, mutant SET-4(S182F) or human SET-4 ortholog SUV420H2 is shown. Data represent mean values from three biological replicates. (C) MALDI spectra illustrating conversion of H4K20me1 to H4K20me2 by GST-SET-4. Monoisotopic masses ( protonated) of peptide substrates are indicated with arrowheads. Spectra are representative of three independent experiments.

SET-4 acts in neurons to promote dauer arrest
To determine where SET-4 is expressed, we generated strains expressing reporter genes under the control of set-4 regulatory elements. Because two independent C-terminal SET-4::GFP translational fusions failed to rescue dauer arrest in set-4 mutants, we generated strains expressing a set-4p::GFP promoter fusion to determine the spatiotemporal activity of the set-4 promoter. set-4p:: GFP transgenic animals expressed GFP broadly in embryos (Fig.  S3). Post-embryonically, we detected fluorescence predominantly in the head and tail regions of the animal, in a pattern consistent with neuronal expression. To confirm this, we established a transgenic strain that co-expressed set-4p::GFP and mCherry driven by the pan-neuronal rab-3 promoter (Nonet et al., 1997). At all developmental stages interrogated, we observed colocalization of green and red fluorescence (Fig. 3A), consistent with somatic set-4p::GFP expression being predominantly neuronal. We did not observe significant GFP expression in intestine, body wall muscle or hypodermis.
As the amphid sensory neurons play a crucial role in regulating dauer arrest (Bargmann and Horvitz, 1991;Schackwitz et al., 1996;Vowels and Thomas, 1994), we interrogated them for expression of set-4p::GFP. Amphid neurons possess ciliary dendrites that are in direct contact with the environment and can be labeled with the lipophilic dye DiI (Starich et al., 1995). The extent of colocalization of green and red fluorescence in set-4p::GFP transgenic animals exposed to DiI (Fig. 3B) reveals that the set-4 promoter is active in amphid sensory neurons as well as in other cells.
To test whether neuronal expression of set-4 is functionally important for dauer arrest, we generated tissue-specific set-4 transgenes and tested them for the ability to rescue dauer arrest in set-4 mutants. The neuronal rab-3p::set-4 transgene rescued dauer formation to a similar extent to a set-4 transgene driven by its native promoter (Fig. 3C). By contrast, intestine-, hypodermis-and muscle-specific set-4 transgenes did not rescue dauer arrest to a greater extent than a transgene expressing the set-4(dp268) mutant. Taken together, these data indicate that SET-4 functions in the nervous system to promote dauer arrest.

Transcriptome-wide influences of DPY-21 and SET-4 on dauer regulation
We previously showed that the DCC component DPY-21 promotes DAF-16/FoxO activity (Dumas et al., 2013). To gain broader insight into how DPY-21 and SET-4 control dauer arrest, we performed whole-transcriptome profiling to compare genome-wide regulatory influences (henceforth referred to as the 'regulome') of DPY-21 and SET-4 to those of the key transcription factors controlling dauer arrest in eak-7;akt-1 animals, DAF-16/FoxO and the nuclear receptor DAF-12 (Alam et al., 2010). We identified genes differentially expressed between wild-type and eak-7;akt-1 double mutant animals [fold change ≥1.5 and false discovery rate (FDR) <0.05]. We then compared the transcriptomes of eak-7;akt-1 double mutants with those of eak-7;akt-1 animals harboring mutations in dpy-21, set-4, daf-16/FoxO or daf-12, and identified genes that are differentially expressed in the opposite direction as in wild-type relative to eak-7;akt-1 (Table S1). Regulomes were validated by comparison with published data where possible (see below).
To validate the DPY-21 regulome, we found significant overlap between the set of 700 X-linked genes differentially expressed in eak-7;akt-1 dpy-21 versus eak-7;akt-1 animals with the 374 Xlinked genes subject to dosage compensation in embryos (Jans et al., 2009) (119 genes; Fig. S4A and Table S2; P=2.1e −26 ). Three hundred and eight of the 333 genes that make up the SET-4 dauer regulome (92.5%) are also part of the DPY-21 dauer regulome ( Fig. 4A and Table S1), suggesting that a functional relationship between DPY-21 and SET-4 may exist in post-embryonic dauer regulation.
The X-linked ins-9 gene is repressed by DPY-21, SET-4 and DAF-16/FoxO Based on genetic epistasis experiments (Dumas et al., 2013) (Fig. 1), we hypothesized that DPY-21 and SET-4 influence DAF-16/FoxO activity through repression of X-linked genes. Moreover, in light of the neuronal site of action of SET-4 (Fig. 3C) and its expression in amphid sensory neurons (Fig. 3B), we speculated that key dauer regulatory genes subject to dosage compensation might function in a signaling capacity in the nervous system, upstream of DAF-12. Therefore, we examined the set of X-linked genes common to DPY-21 and DAF-16/FoxO regulomes that were not regulated by DAF-12, which acts downstream in the dauer regulatory cascade (Fielenbach and Antebi, 2008;Schaedel et al., 2012). This filtering strategy defined a set of 47 X-linked genes coordinately regulated by DPY-21 and DAF-16/FoxO but not influenced by daf-12 mutation ( Fig. 4B; Table S5).

ins-9 is expressed specifically in a single pair of amphid neurons
Previous studies using reporters driven by the ins-9 promoter suggested that ins-9 is expressed in the ASI and ASJ amphid neurons, as well as in additional tissues (Chen and Baugh, 2014;Pierce et al., 2001;Ritter et al., 2013). By contrast, in transgenic L2 larvae expressing ins-9::SL2::mNG, we consistently observed green fluorescence solely in one pair of sensory neurons. In animals in  Table S5. Data represent the aggregate of five biological replicate samples, each from thousands of progeny with no fewer than 200 animals per sample.
which neuronal morphology was discernable, we identified the fluorescent cells as the ASI amphid neurons (Fig. 5C). We did not observe fluorescence in more than one pair of amphid neurons in any animal, nor did we detect fluorescence in other neurons or tissues. As ins-9::SL2::mNG contains genomic elements from the ins-9 locus that are missing from other reporters in the literature (Chen and Baugh, 2014;Pierce et al., 2001;Ritter et al., 2013), these observed patterns of expression are likely to be physiologically relevant.
ins-9 and akt-2 are required for suppression of dauer arrest by set-4 mutation To determine the extent to which ins-9 derepression contributes to dauer suppression in set-4 mutants, we tested the ability of set-4 to suppress the dauer-constitutive phenotype of daf-2/InsR mutants in wild-type and ins-9 loss-of-function backgrounds. We generated strong loss-of-function ins-9 alleles using CRISPR/Cas9 genome editing (Paix et al., 2015). Two probable null alleles, dp675 and dp677, have nonsense mutations in the F-peptide region of ins-9 that lies N-terminal to the functional B and A peptides (Pierce et al., 2001) (Fig. S5). Although neither allele induced dauer arrest in a wild-type background, both ins-9(dp675) and ins-9(dp677) partially rescued dauer arrest in set-4;daf-2 double mutants (Fig. 5D), indicating that dauer suppression caused by set-4 mutation is due in part to de-repression of ins-9.
We previously showed that the X-linked gene akt-2 is required for dauer suppression caused by dpy-21 mutation (Dumas et al., 2013).
The autosomal ins-7 gene contributes to suppression of dauer arrest by set-4 mutation As the only X-linked ins gene, ins-9 is the sole ins gene subject to direct regulation by dosage compensation or other X-chromosomespecific mechanisms of gene regulation. However, it is conceivable that other ins genes could contribute to dauer regulation through indirect effects on their expression. Genes encoding three agonist ILPs, INS-4, INS-6 and DAF-28, are expressed in the ASI sensory neurons and have established roles in inhibiting dauer arrest and promoting reproductive development (Cornils et al., 2011;Hung et al., 2014;Li et al., 2003). To determine whether regulation of ins-4, ins-6 and/or daf-28 contributes to dauer suppression in this context, we measured ins-4, ins-6 and daf-28 transcript levels in wild-type, eak-7;akt-1 double mutant and eak-7;akt-1 triple mutants with reduced DPY-21 or SET-4 activity. None of these genes was repressed in eak-7;akt-1 double mutants compared with wild-type animals, nor did loss of set-4 or dpy-21 cause significant increases in their expression (Fig. S6B-D). Therefore, neither DPY-21 nor SET-4 influences dauer arrest through regulation of ins-4, ins-6 and daf-28 expression.

DISCUSSION
Although much is known about the conserved signaling pathways that control C. elegans dauer arrest, how these pathways are regulated by upstream inputs is poorly understood. In the present study, we have established a framework for understanding how DPY-21 and SET-4 promote dauer arrest in the context of reduced DAF-2 ILS. Specifically, we have discovered that the conserved H4K20 methyltransferase SET-4 acts in the nervous system to promote dauer arrest. It does so, in part, by synergizing with DAF-16/FoxO to repress the X-linked insulin-like peptide gene ins-9. We hypothesize that SET-4 and DPY-21 act similarly to repress ins-9 and akt-2 directly, thus attenuating DAF-2 ILS and promoting dauer arrest through activation of DAF-16/FoxO (Fig. 6).
Although  has been predicted to function as an agonist ILP based both on structural models that indicate similarity to the agonist ILPs INS-4, INS-6 and DAF-28 (Pierce et al., 2001) and on expression changes upon starvation and feeding of larvae (Chen and Baugh, 2014), analysis of existing ins-9 mutants has not revealed phenotypes consistent with this (Fernandes de Abreu et al., 2014). This may be due to ins-9(tm3618) not being a strong loss-offunction allele. By contrast, our analysis of transgenic animals overexpressing ins-9 (Fig. 5B) and mutant animals harboring nonsense ins-9 alleles (Fig. 5D-F) provides the first experimental evidence demonstrating that INS-9 is an agonist ILP.
Several ILPs have been implicated in dauer regulation (Cornils et al., 2011;Fernandes de Abreu et al., 2014;Hung et al., 2014;Li et al., 2003;Murphy et al., 2003;Pierce et al., 2001). However, the mechanistic basis for how environmental cues regulate ILPs remains obscure. The initial events that control dauer arrest through DAF-2 ILS likely take place in the amphid sensory neurons, which are required for dauer formation in response to pheromone (Schackwitz et al., 1996;Vowels and Thomas, 1994). Indeed, genes encoding INS-4, INS-6 and DAF-28, which collectively play a major role in promoting reproductive development through DAF-2 ILS (Cornils et al., 2011;Hung et al., 2014;Li et al., 2003), are expressed in ASI (Chen and Baugh, 2014;Cornils et al., 2011;Hung et al., 2014;Li et al., 2003), and the transcription of ins-6 and daf-28 is inhibited by dauer pheromone through unknown mechanisms (Cornils et al., 2011;Li et al., 2003). Our finding that DPY-21 and SET-4 influence dauer arrest in part by repressing the X-linked ins-9 gene provides a potential mechanistic link between a dauer-regulatory environmental cue and an ILP expressed in sensory neurons that control the dauer decision.
An intriguing but incompletely understood aspect of dauer morphogenesis is the mechanistic basis for the commitment of larvae to either the reproductive or dauer developmental fate. Assays in which larvae are shifted between favorable and unfavorable conditions at different times after hatching indicate the existence of commitment points beyond which animals develop reproductively or arrest as dauers regardless of ambient conditions (Golden and Riddle, 1984;Schaedel et al., 2012). Commitment to reproductive development correlates temporally with the activation of a feedforward loop amplifying organismal DA biosynthesis through DAdependent induction of DAF-9 expression in the hypodermis (Schaedel et al., 2012). However, the XXX cells (Ohkura et al., 2003), which are thought to be the sole source of DA biosynthesis prior to the commitment point (Schaedel et al., 2012), are not in direct contact with the environment. Therefore, they must receive upstream inputs from sensory neurons that convey information about ambient conditions.
Our finding that DPY-21 and SET-4 synergize with DAF-16/ FoxO to repress ins-9 is consistent with a hypothetical model in which INS-9 may function as a key node in an autocrine feedforward loop in the ASI sensory neurons that reinforces levels of its own expression in response to changing environments, upstream of DA biosynthesis in the XXX cells and hypodermis. In replete conditions, ins-9 expression in ASI is expected to lead to activation of DAF-2 ILS and inhibition of DAF-16/FoxO. As DAF-16/FoxO inhibits ins-9 expression, decreased DAF-16/FoxO activity would lead to increased ins-9 expression, which would presumably lead to further activation of DAF-2 ILS and inhibition of DAF-16/FoxO, both in an autocrine fashion in ASI as well as in other cells that express DAF-2/InsR. In the context of increased population density, pheromone would promote ins-9 repression through DPY-21 and SET-4, and reduce autocrine and paracrine engagement of DAF-2/ InsR, resulting in DAF-16/FoxO activation, further repression of ins-9 and dauer arrest (Fig. 6). The effect of DPY-21 and SET-4 would not be limited to sensory neurons, as they would also act in other cells responding to INS-9 to control their sensitivity to ILPs by repressing akt-2 (Dumas et al., 2013) (Fig. 6 and Fig. S6A). In addition, ins-9 regulation may also be amplified through other ILPs such as INS-7, which functions in a feed-forward loop in adults to coordinate DAF-16/FoxO activity throughout the animal (Murphy et al., 2007).

C. elegans strains and maintenance
Mutant alleles are listed in the supplementary Materials and Methods. Compound mutants were generated using standard protocols. All animals were maintained on nematode growth media (NGM) plates seeded with E. coli OP50 using standard techniques. Strains are available upon request.

Dauer arrest assays
Dauer arrest assays were performed as previously described (Hu et al., 2006). daf-9(dh6) mutant animals were propagated on NGM plates supplemented with 10 nM Δ 7 -DA, then transferred to NGM plates for egglays as previously described (Dumas et al., 2013). For male dauer assays, males were crossed to isogenic L4 hermaphrodites, and the gender of dauer progeny was determined after dauer exit.

Dauer pheromone assays
Dauer pheromone was prepared as previously described (Golden and Riddle, 1982;Schroeder and Flatt, 2014). Details are provided in the supplementary Materials and Methods.

Generation of transgenic strains
Details pertaining to the generation of reporter constructs and transgenic strains are provided in the supplementary Materials and Methods.

RNA isolation
Greater than 200 gravid hermaphrodites were allowed to lay eggs for 6 h at 20°C and then removed. Eggs were transferred to 25°C for 24 h. Larvae were harvested, washed once in M9 buffer and once in water, and resuspended in TRIzol (Invitrogen). After five sequential freeze-thaws, RNA was extracted using chloroform. Extracted RNA was purified using a Direct-zol RNA Miniprep Kit (Zymo Research).
qPCR cDNA was synthesized with oligo-dT priming using the SuperScript III First Strand Synthesis Kit (Invitrogen). The equivalent of 10 ng of starting RNA was used as template in a 15 μl reaction using the Quantitect SYBRgreen qPCR Kit (Qiagen). Reactions were performed in a RotorGene 6000 (Corbett Research) and results analyzed using RotorGene 6000 Software (version 1.7). Samples were normalized to pmp-3 expression prior to comparison between groups (Hoogewijs et al., 2008). See Table S8 for primer sequences. Relative expression was calculated as described (Nolan et al., 2006).

Confocal microscopy
Animals were mounted on slides layered with a thin 3% agarose pad containing 25 mM sodium azide. Images were captured on a Leica Inverted SP5X Confocal Microscope (Leica) using LAS AF software.

RNA-seq analysis
Whole-transcriptome profiling was performed by the University of Michigan DNA Sequencing Core as previously described (Chen et al., 2015) using 100 ng input RNA per sample. Samples were barcoded and multiplexed, and 100-nucleotide paired-end sequencing was performed using an Illumina HiSeq 2000 sequencer and Version 4 reagents. Five experimental replicates were analyzed. Correlation coefficients between replicates and genotypes are shown in Table S9.
The significance of overlap with dosage-compensated X-linked genes (Jans et al., 2009), strongly regulated dauer genes (Liu et al., 2004) and DAF-16 targets in the daf-2(e1370) background (Chen et al., 2015) was calculated using a hypergeometric distribution, assuming 5863 X-linked transcripts and 46233 genome-wide transcripts in C. elegans detected in our RNA-seq analysis (by either the UCSCce10 reference transcriptome or de novo transcript assembly). If necessary, common WormBase Gene identifiers were downloaded from WormBase version WS250 (intermine. wormbase.org).

Immunoblotting and antibodies
To generate protein lysates, animals were washed in M9, then in sterile water. Pelleted animals were resuspended in equal volumes of worm lysis buffer (Webster et al., 2013), incubated at 85°C for 5 min, then sonicated on ice for two cycles of 30 s each at 70% power using a Sonic Dismembrator Model 100 (Fisher Scientific). Homogenates were quantified using a DC Protein Quantification Kit (BioRad). Protein (50 μg per lane) was loaded using Criterion systems (BioRad) and transferred to Immobilon Psq (Millipore). Details pertaining to antibodies are provided in the supplementary Materials and Methods. Membranes were blocked with 5% milk in TBS+0.5% Tween 20. Antibodies were diluted in Western Blocking Solution (Sigma) prior to incubation with membranes. Blots were washed with TBS+0.5% Tween 20. Signal was detected by ECL (Pierce).
Histone methyltransferase assay set-4 cDNAs were amplified from RNA isolated from wild-type or set-4(dp268) mutant animals. Human Suv420H2 cDNA was obtained from Origene. Clones were ligated into pGEX4T1 vector (GE Healthcare). Protein expression was induced overnight at 16°C with 0.1 mM IPTG in BL21-CodonPlus(DE3)-RIPL cells (Agilent Technologies) grown in Terrific Broth (Invitrogen)+4% glycerol (Sigma). Cells were disrupted using a Sonic Dismembrator Model 100 sonicator (Fisher Scientific), with four cycles of ten 1 s on/off pulses of 10-30% intensity. Lysates were cleared by centrifugation at 20,000 g for 5 min and incubated with Glutathione-Sepharose beads (GE Healthcare) rotating overnight at 4°C. Expression of recombinant protein was confirmed by Coomassie staining and anti-GST immunoblot. Beads bound to recombinant protein were incubated in 10 mM Tris ( pH 8.0), 2 μM β-mercaptoethanol, and 7 mM S-adenosylmethionine (Sigma) in the presence of 2 mM substrate peptide corresponding to amino acids 8-30 of C. elegans histone H4 (AnaSpec) rotating for 4 h at 30°C. Reactions were analyzed using a Waters MicroMass MALDI-TOF mass spectrometer and analyzed with MassLynx software. Ratios of peak heights corresponding to reactant and product peptide were calculated to define percent conversion.

Male rescue assay
Mated gravid hermaphrodites were placed on NGM plates to lay eggs for 24 h at 20°C. Egglayers were removed, and the numbers of hatchlings and eggs were counted. After 72 h, the animals were moved to 4°C to slow their movement. Male rescue was calculated as the ratio of live males to total eggs laid. Each experiment was performed in triplicate.

Statistics
Two-tailed Student's t-test was used to measure significance in experiments unless otherwise noted. Data are presented as the average±standard error of the mean (s.e.m.) of at least three biological replicates, each replicate performed in triplicate. n values for dauer assays are listed from left to right.