Multicopy transgene arrays remain widely used in C. elegans research. It is usually assumed that they behave neutrally, not impacting the phenotype under investigation. Here, we reveal that a previously reported heterochronic extra-molt phenotype associated with myrf-1(mg412) depends on the presence of the integrated molting reporter mgIs49 . Nanopore long-read sequencing shows that mgIs49 is a massive 8.8-Mb insertion - around 50% of the size of its host chromosome - which disrupts the prmt-9 gene. Both mgIs49 and another array, maIs105 , cause dysregulation of the transcriptome and accumulation of reads mapping to the promoter sequences used as components of the array. We identify additional arrays exceeding 4 Mb and show that variable molting defects occur across different transgenic lines when combined with myrf-1(mg412) , implicating array size or composition in the synthetic phenotype. Our results underscore the necessity of replacing multicopy reporters in developmental studies with single-copy insertions or endogenous tagging whenever possible.

The RNA-binding protein (RBP) LIN28 is a key regulator of temporal cell fates, promoting stem cell identity and suppressing differentiation. It regulates the processing of the let-7 miRNA but additionally binds thousands of mRNAs, suggesting a model of coordinated regulation across many targets. Yet, direct evidence for such a network function is scarce for both LIN28 and other broadly binding RBPs. Here, we show that in Caenorhabditis elegans larvae, loss of LIN28 leaves the levels of most LIN28-bound transcripts unchanged. The only exceptions are the lin-46 mRNA and the let-7 miRNA. Comprehensive genetic analyses identify these two as the functionally essential LIN28 targets: lin-28; lin-46; let-7 triple mutant animals exhibit complete suppression of the lin-28(0) precocious heterochronic phenotypes and recapitulate the retarded lin-46; let-7 double mutant phenotypes. Conversely, simultaneous overexpression of lin-46 and let-7 mimics LIN28 loss. Unexpectedly, LIN28 controls not only cell identity but also the tempo of development, using the same two targets. These findings establish a close functional link between the heterochronic pathway and the clock regulating developmental tempo and demonstrate that an RBP can achieve complex biological outcomes through a small set of targets.

Proteases are enzymes that catalyze the hydrolysis of peptide bonds in proteins for their functional modification or degradation. Members of the Dipeptidyl Peptidase IV (DPPIV) family are exopeptidases that cleave dipeptides off the N-termini of their substrate peptides, typically after proline or alanine. Recently, we showed that human DPP4 and Caenorhabditis elegans DPF-3 have a larger target repertoire in vitro, permitting cleavage after additional amino acids. Here, we use terminal amine isotopic labeling of substrates (TAILS) to identify DPF-3 targets in vivo and observe cleavage of MEP-1 after threonine, confirming a broader substrate specificity of DPF-3 also in vivo. Demonstrating physiological relevance, we show that rendering MEP-1 resistant to cleavage disrupts its stability, leading to developmental abnormalities such as defective gonadal migration and reproductive issues. Collectively, our findings highlight a previously unappreciated complexity in the substrate specificity of DPPIV family proteases and suggest that their physiological roles may extend beyond what is currently known.

Genetic oscillators drive precisely timed gene expression, crucial for development and physiology. Using the C. elegans molting clock as a model, we investigate how oscillators can schedule the orderly expression of thousands of genes. Single-cell RNA sequencing reveals a broad peak phase dispersion in individual tissues, mirrored by rhythmic changes in chromatin accessibility at thousands of regulatory elements identified by time-resolved ATAC-seq. We develop a linear model to predict chromatin dynamics based on the binding of >200 transcription factors. This identifies nine key regulators acting additively to determine the peak phase and amplitude of each regulatory element. Strikingly, these factors can also generate constitutive, non-rhythmic activity through destructive interference. Validating its power, the model accurately predicts the impact of GRH-1/Grainyhead perturbation on both chromatin and transcript dynamics. This work provides a conceptual framework for understanding how combinatorial, non-cooperative transcription factor binding schedules complex gene expression patterns in development and other dynamic biological processes.

MicroRNAs (miRNAs) are essential regulators involved in multiple biological processes. To achieve their gene repression function, they are loaded in miRNA-specific Argonautes to form the miRNA-induced silencing complex (miRISC). Mammals and C. elegans possess more than one paralog of miRNA-specific Argonautes, but the dynamic between them remains unclear. Here, we report the conserved dipeptidyl peptidase DPF-3 as an interactor of the miRNA-specific Argonaute ALG-1 in C. elegans. Knockout of dpf-3 increases ALG-2 levels and miRISC formation in alg-1 loss-of-function animals, thereby compensating for ALG-1 loss and rescuing miRNA-related defects observed. DPF-3 can cleave an ALG-2 N-terminal peptide in vitro but does not appear to rely on this catalytic activity to regulate ALG-2 in vivo. This study uncovers the importance of DPF-3 in the miRNA pathway and provides insights into how multiple miRNA Argonautes contribute to achieving proper miRNA-mediated gene regulation in animals.

Abstract The mammalian protein PERIOD (PER) and its C. elegans orthologue LIN-42 have been proposed to constitute an evolutionary link between two distinct, circadian and developmental, timing systems. While the function of PER in animal circadian rhythms is well understood molecularly and mechanistically, this is not true for LIN-42’s function in timing rhythmic development, reflected in C. elegans molting cycles. We observed arrhythmic molts upon combined deletion of a region comprising two distinct sequence elements previously termed SYQ and LT. This region functions as a casein kinase I (CK1)-binding domain (CK1BD) mediating stable binding to KIN-20, the C. elegans CK1δ/ε orthologue. CK1 phosphorylates LIN-42, and the CK1BD sub-domains SYQ/CKBD-A and LT/CKBD-B play distinct roles in controlling CK1-binding and kinase activity in vitro. KIN-20 and the LIN-42 CK1BD are required for proper molt timing in vivo, and loss of LIN-42 binding or of the phosphorylated LIN-42 tail impairs nuclear accumulation of KIN-20. These findings indicate that LIN-42/PER and KIN-20/CK1 form a functionally conserved signaling module of two distinct chronobiological systems.

Substrate specificity determines protease functions in physiology and in clinical and biotechnological applications, yet quantitative cleavage information is often unavailable, biased, or limited to a small number of events. Here, we develop qPISA (quantitative Protease specificity Inference from Substrate Analysis) to study Dipeptidyl Peptidase Four (DPP4), a key regulator of blood glucose levels. We use mass spectrometry to quantify >40,000 peptides from a complex, commercially available peptide mixture. By analyzing changes in substrate levels quantitatively instead of focusing on qualitative product identification through a binary classifier, we can reveal cooperative interactions within DPP4's active pocket and derive a sequence motif that predicts activity quantitatively. qPISA distinguishes DPP4 from the related C. elegans DPF-3 (a DPP8/9-orthologue), and we relate the differences to the structural features of the two enzymes. We demonstrate that qPISA can direct protein engineering efforts like the stabilization of GLP-1, a key DPP4 substrate used in the treatment of diabetes and obesity. Thus, qPISA offers a versatile approach for profiling protease and especially exopeptidase specificity, facilitating insight into enzyme mechanisms and biotechnological and clinical applications.

Temporally and spatially controlled accumulation underlies the functions of microRNAs (miRNAs) in various developmental processes. In Caenorhabditis elegans, this is exemplified by the temporal patterning miRNAs lin-4 and let-7, but for most miRNAs, developmental expression patterns remain poorly resolved. Indeed, experimentally observed long half-lives may constrain possible dynamics. Here, we profile miRNA expression throughout C. elegans postembryonic development at high temporal resolution, which identifies dynamically expressed miRNAs. We use mathematical models to explore the underlying mechanisms. For let-7, we can explain, and experimentally confirm, a striking stepwise accumulation pattern through a combination of rhythmic transcription and stage-specific regulation of precursor processing by the RNA-binding protein LIN-28. By contrast, the dynamics of several other miRNAs cannot be explained by regulation of production rates alone. Specifically, we show that a combination of oscillatory transcription and rhythmic decay drive rhythmic accumulation of miR-235, orthologous to miR-92 in other animals. We demonstrate that decay of miR-235 and additional miRNAs depends on EBAX-1, previously implicated in target-directed miRNA degradation (TDMD). Taken together, our results provide insight into dynamic miRNA decay and establish a resource to studying both the developmental functions of, and the regulatory mechanisms acting on, miRNAs.

C. elegans develops through four larval stages that are rhythmically terminated by molts, that is, the synthesis and shedding of a cuticular exoskeleton. Each larval cycle involves rhythmic accumulation of thousands of transcripts, which we show here relies on rhythmic transcription. To uncover the responsible gene regulatory networks (GRNs), we screened for transcription factors that promote progression through the larval stages and identified GRH-1, BLMP-1, NHR-23, NHR-25, MYRF-1, and BED-3. We further characterize GRH-1, a Grainyhead/LSF transcription factor, whose orthologues in other animals are key epithelial cell-fate regulators. We find that GRH-1 depletion extends molt durations, impairs cuticle integrity and shedding, and causes larval death. GRH-1 is required for, and accumulates prior to, each molt, and preferentially binds to the promoters of genes expressed during this time window. Binding to the promoters of additional genes identified in our screen furthermore suggests that we have identified components of a core molting-clock GRN. Since the mammalian orthologues of GRH-1, BLMP-1 and NHR-23, have been implicated in rhythmic homeostatic skin regeneration in mouse, the mechanisms underlying rhythmic C. elegans molting may apply beyond nematodes.

Substrate specificity determines protease functions in physiology and in clinical and biotechnological application. However, affordable and unbiased assays for large-scale quantification of substrate cleavage have been lacking. Here, we develop hiMAPS (high-throughput mapping of protease cleavage sites), a cheap, mass spectrometry-based assay, to derive cleavage motifs for human Dipeptidyl Peptidase Four (DPP4), a key regulator of blood glucose levels. We recapitulate the known benefit of proline in the penultimate (P1) position from the substrate N-terminus, extend it to additional residues, and identify and quantify combinatorial interactions of P1 with its neighbors. These findings reveal extensive cooperativity among the enzyme’s active site subsites and allow us to derive a sequence motif that predicts substrate turnover. We show that this information provides new opportunities to engineer stabilized versions of a key substrate of DPP4, the small peptide hormone GLP-1, whose derivatives are used for clinical treatment of type 2 diabetes and obesity. Finally, structural and biochemical characterization of a DPP4 homologue, C. elegans DPF-3, reveal specific subsite differences and a distinct cleavage motif, providing insight into the mechanistic basis of the observed specificity. Collectively, our findings present a broadly applicable framework to high-throughput mapping of protease cleavage sites, extend our understanding of protease specificity, and provide a chemical space for substrate engineering of a clinically relevant family of exopeptidases.### Competing Interest StatementThe authors have declared no competing interest.

The RNA-binding protein TRIM71/LIN-41 is a phylogenetically conserved developmental regulator that functions in mammalian stem cell reprogramming, brain development, and cancer. TRIM71 recognizes target mRNAs through hairpin motifs and silences them through molecular mechanisms that await identification. Here, we uncover that TRIM71 represses its targets through RNA-supported interaction with TNRC6/ GW182, a core component of the miRNA-induced silencing complex (miRISC). We demonstrate that AGO2, TRIM71, and UPF1 each recruit TNRC6 to specific sets of transcripts to silence them. As cellular TNRC6 levels are limiting, competition occurs among the silencing pathways, such that the loss of AGO proteins or of AGO binding to TNRC6 enhances the activities of the other pathways. We conclude that a miRNA-like silencing activity is shared among different mRNA silencing pathways and that the use of TNRC6 as a central hub provides a means to integrate their activities.

Argonaute proteins are at the core of the microRNA-mediated gene silencing pathway essential for animals. In C. elegans, the microRNA-specific Argonautes ALG-1 and ALG-2 regulate multiple processes required for proper animal developmental timing and viability. Here we identified a phosphorylation site on ALG-1 that modulates microRNA association. Mutating ALG-1 serine 642 into a phospho-mimicking residue impairs microRNA binding and causes embryonic lethality and post-embryonic phenotypes that are consistent with alteration of microRNA functions. Monitoring microRNA levels in alg-1 phosphorylation mutant animals shows that microRNA passenger strands increase in abundance but are not preferentially loaded into ALG-1, indicating that the miRNA binding defects could lead to microRNA duplex accumulation. Our genetic and biochemical experiments support protein kinase A (PKA) KIN-1 as the putative kinase that phosphorylates ALG-1 serine 642. Our data indicate that PKA triggers ALG-1 phosphorylation to regulate its microRNA association during C. elegans development.

Molting, that is, the synthesis and shedding of a cuticular exoskeleton, is a defining characteristic of ecdysozoa. In nematodes such as C. elegans , molts rhythmically terminate each of four larval stages. The molting cycle is tightly coupled to the rhythmic accumulation of thousands of transcripts. Here, using chromatin immunoprecipitation coupled to sequencing (ChIP-seq) and quantitative reporter assays, we show that these dynamic gene expression patterns rely on rhythmic transcription. To gain insight into the relevant gene regulatory networks (GRNs), we performed an RNAi-based screen for transcription factors required for molting to identify potential components of a molting clock. We find that depletion of GRH-1, BLMP-1, NHR-23, NHR-25, MYRF-1 or BED-3 impairs progression through the molting cycle. We characterize GRH-1, a Grainyhead/LSF transcription factor whose orthologues in other animals are key epithelial cell fate regulators. We show that GRH-1 depletion causes a dose-dependent extension of molt duration, defects in cuticle formation and shedding, and larval death. Coincident with its rhythmic accumulation, GRH-1 is required repetitively for each molt, during specific time windows preceding lethargus. These findings are consistent with a function of GRH-1 in a molting cycle GRN. As its mammalian orthologues, as well as those of BLMP-1 and NHR-23, have been implicated in rhythmic homeostatic skin regeneration in mouse, the mechanisms underlying rhythmic C. elegans molting may apply beyond nematodes.

Animals increase by orders of magnitude in volume during development. Therefore, small variations in growth rates among individuals could amplify to a large heterogeneity in size. By live imaging of C. elegans, we show that amplification of size heterogeneity is prevented by an inverse coupling of the volume growth rate to the duration of larval stages and does not involve strict size thresholds for larval moulting. We perturb this coupling by changing the developmental tempo through manipulation of a transcriptional oscillator that controls the duration of larval development. As predicted by a mathematical model, this perturbation alters the body volume. Model analysis shows that an inverse relation between the period length and the growth rate is an intrinsic property of genetic oscillators and can occur independently of additional complex regulation. This property of genetic oscillators suggests a parsimonious mechanism that counteracts the amplification of size differences among individuals during development.

During C. elegans larval development, thousands of genes, accounting for >20% of the transcriptome, exhibit oscillatory expression with large amplitudes. The time of peaking varies for different genes, but expression generally peaks once per larval stage, with both the oscillation period and larval stage duration varying in concert with temperature. This and other evidence support the existence of a gene expression oscillator that functions as a developmental clock. In this article, we review what is known about the biology, architecture and possible mechanisms of this clock. We compare it to other oscillators, and highlight tools and approaches suited to its study. Finally, we point out implications of these wide-spread and dynamic changes of gene expression on any type of gene expression profiling experiment in C. elegans larvae and how such experiments need to be controlled.

Small RNA pathways defend the germlines of animals against selfish genetic elements, yet pathway activities need to be contained to prevent silencing of self genes. Here, we reveal a proteolytic mechanism that controls endogenous small interfering (22G) RNA activity in the Caenorhabditis elegans germline to protect genome integrity and maintain fertility. We find that DPF-3, a P-granule-localized N-terminal dipeptidase orthologous to mammalian dipeptidyl peptidase (DPP) 8/9, processes the unusually proline-rich N termini of WAGO-1 and WAGO-3 Argonaute (Ago) proteins. Without DPF-3 activity, these WAGO proteins lose their proper complement of 22G RNAs. Desilencing of repeat-containing and transposon-derived transcripts, DNA damage, and acute sterility ensue. These phenotypes are recapitulated when WAGO-1 and WAGO-3 are rendered resistant to DPF-3-mediated processing, identifying them as critical substrates of DPF-3. We conclude that N-terminal processing of Ago proteins regulates their activity and promotes silencing of selfish genetic elements by ensuring Ago association with appropriate small RNAs.

Gene expression oscillators can coordinate developmental events in space and time. In C. elegans , a gene expression oscillator directs rhythmic accumulation of ∼25% of the transcriptome, and thus thousands of transcripts, presumably to control molting, a process of rhythmic skin regeneration. Recently, a reverse genetic screen identified several transcription factors important for molting. Here, we characterize one of these, BLMP-1, orthologous to the mammalian transcription repressor PRDM1. We find it to be important for timely molting, and oscillatory gene expression. We propose a dual function for BLMP-1 in shaping oscillatory gene expression and coupling it to a set of direct targets, which ensures cuticular integrity. With mammalian PRDM1/BLIMP1 promoting regular cycles of postnatal hair follicle regeneration, our findings point to the possible existence of a fundamentally conserved clock mechanism in control of rhythmic skin regeneration.

The developmental role of histone H3K9 methylation (H3K9me), which typifies heterochromatin, remains unclear. In Caenorhabditis elegans , loss of H3K9me leads to a highly divergent upregulation of genes with tissue and developmental-stage specificity. During development H3K9me is lost from differentiated cell type-specific genes and gained at genes expressed in earlier developmental stages or other tissues. The continuous deposition of H3K9me2 by the SETDB1 homolog MET-2 after terminal differentiation is necessary to maintain repression. In differentiated tissues, H3K9me ensures silencing by restricting the activity of a defined set of transcription factors at promoters and enhancers. Increased chromatin accessibility following the loss of H3K9me is neither sufficient nor necessary to drive transcription. Increased ATAC-seq signal and gene expression correlate at a subset of loci positioned away from the nuclear envelope, while derepressed genes at the nuclear periphery remain poorly accessible despite being transcribed. In conclusion, H3K9me deposition can confer tissue-specific gene expression and maintain the integrity of terminally differentiated muscle by restricting transcription factor activity.

Animals increase by orders of magnitude in their volume during development. Hence, even small differences in the growth rates between individuals could generate large differences in their adult body size. Yet, such volume divergence among individuals is usually not observed in nature. We combined theory and experiment to understand the mechanisms of body size uniformity. Using live imaging, we measured the volume growth of hundreds of individuals of C. elegans over the entire span of their postembryonic development. We find that C. elegans grows exponentially in volume with a coefficient of variation of the growth rate of ∼7%, but that individuals diverge much less in volume than expected from this heterogeneity. The mechanism counteracting size divergence does not involve size thresholds for developmental milestones. Instead, an inverse coupling of the growth rate and the duration of development produces a constant volume fold change per larval stage. The duration of larval stages of C. elegans is determined by the period of a developmental oscillator. Using mathematical modelling, we show that an anti-correlation between the growth rate and the oscillatory period emerges as an intrinsic property of a genetic oscillator. We propose that the robustness of body volume fold change is a hard-wired characteristic of the oscillatory circuit and does not require elaborate mechanisms of size control by cellular signalling. Indeed, the coupling of growth and development was unaltered by mutation of canonical pathways of growth control. This novel concept of size homeostasis may broadly apply to other multicellular systems controlled by genetic oscillators.