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In this study, we describe the engineering of sub-100 nm nanomicelles (DTX-PC NMs) derived from phosphocholine derivative of docetaxel (DTX)-conjugated lithocholic acid (DTX-PC) and poly(ethylene glycol)-tethered lithocholic acid. Administration of DTX-PC NMs decelerate tumor progression and increase the mice survivability compared to Taxotere (DTX-TS), the FDA-approved formulation of DTX. Unlike DTX-TS, DTX-PC NMs do not cause any systemic toxicity and slow the decay rate of plasma DTX concentration in rodents and non-rodent species including non-human primates. We further demonstrate that DTX-PC NMs target demethylation of CpG islands of Sparcl1 (a tumor suppressor gene) by suppressing DNA methyltransferase activity, and increase the expression of Sparcl1 that leads to tumor regression. Therefore, this unique system has the potential to improve the quality of life in cancer patients, and can be translated as a next-generation chemotherapeutic.

Diet regulates complex life‐history traits such as longevity. For optimal lifespan, organisms employ intricate adaptive mechanisms whose molecular underpinnings are less known. We show that Caenorhabditis elegans FLR‐4 kinase prevents lifespan differentials on the bacterial diet having higher Vitamin B12 levels. The flr‐4 mutants are more responsive to the higher B12 levels of Escherichia coli HT115 diet, and consequently, have enhanced flux through the one‐carbon cycle. Mechanistically, a higher level of B12 transcriptionally downregulates the phosphoethanolamine methyltransferase pmt‐2 gene, which modulates phosphatidylcholine (PC) levels. Pmt‐2 downregulation activates cytoprotective gene expression through the p38‐MAPK pathway, leading to increased lifespan only in the mutant. Evidently, preventing bacterial B12 uptake or inhibiting one‐carbon metabolism reverses all the above phenotypes. Conversely, supplementation of B12 to E. coli OP50 or genetically reducing PC levels in the OP50‐fed mutant extends lifespan. Together, we reveal how worms maintain adaptive capacity to diets having varying micronutrient content to ensure a normal lifespan., Multiple genes interact to maintain physiological homeostasis when Caenorhabditis elegans feeds on the varied diet it encounters in its ecological niche. Worms harboring a kinase‐dead version of the FLR‐4 protein are more responsive to the higher dietary Vitamin B12 present in Escherichia coli HT115. This boosts the flux through the one‐carbon metabolism, leading to reduction in phosphatidylcholine levels. Since the mutant FLR‐4 fails to prevent p38‐MAPK activation, these animals have better health and life span.

Significance The endoplasmic reticulum (ER) deteriorates with age and fails to mount an effective stress response against misfolded proteins (UPRER), leading to protein folding disorders. However, preconditioning the ER using a mild ER stress (ER hormesis) can protect against future insults. We show that dietary restriction, an intervention that protects against protein misfolding disorders and increases life span across species, uses ER hormesis as a mechanism of action. Simply mimicking the ER hormesis in Caenorhabditis elegans by transient treatment with pharmacological reagents leads to delayed age-onset failure of UPRER, better capacity to process misfolded proteins, and increased life span. We also show that this process may be conserved in a mammalian cellular model of neurodegenerative disease., Unfolded protein response (UPR) of the endoplasmic reticulum (UPRER) helps maintain proteostasis in the cell. The ability to mount an effective UPRER to external stress (iUPRER) decreases with age and is linked to the pathophysiology of multiple age-related disorders. Here, we show that a transient pharmacological ER stress, imposed early in development on Caenorhabditis elegans, enhances proteostasis, prevents iUPRER decline with age, and increases adult life span. Importantly, dietary restriction (DR), that has a conserved positive effect on life span, employs this mechanism of ER hormesis for longevity assurance. We found that only the IRE-1–XBP-1 branch of UPRER is required for the longevity effects, resulting in increased ER-associated degradation (ERAD) gene expression and degradation of ER resident proteins during DR. Further, both ER hormesis and DR protect against polyglutamine aggregation in an IRE-1–dependent manner. We show that the DR-specific FOXA transcription factor PHA-4 transcriptionally regulates the genes required for ER homeostasis and is required for ER preconditioning-induced life span extension. Finally, we show that ER hormesis improves proteostasis and viability in a mammalian cellular model of neurodegenerative disease. Together, our study identifies a mechanism by which DR offers its benefits and opens the possibility of using ER-targeted pharmacological interventions to mimic the prolongevity effects of DR.

Dietary restriction (DR) increases life span and improves health in most model systems tested, including non-human primates. In C. elegans, as in other models, DR leads to reprogramming of metabolism, improvements in mitochondrial health, large changes in expression of cytoprotective genes and better proteostasis. Understandably, multiple global transcriptional regulators like transcription factors FOXO/DAF-16, FOXA/PHA-4, HSF1/HSF-1 and NRF2/SKN-1 are important for DR longevity. Considering the wide-ranging effects of p53 on organismal biology, we asked whether the C. elegans ortholog, CEP-1 is required for DR-mediated longevity assurance. We employed the widely-used TJ1 strain of cep-1(gk138). We show that cep-1(gk138) suppresses the life span extension of two genetic paradigms of DR, but two non-genetic modes of DR remain unaffected in this strain. We find that two aspects of DR, increased autophagy and up-regulation of the expression of cytoprotective xenobiotic detoxification program (cXDP) genes, are dampened in cep-1(gk138). Importantly, we find that background mutation(s) in the strain may be the actual cause for the phenotypic differences that we observed and cep-1 may not be directly involved in genetic DR-mediated longevity assurance in worms. Identifying these mutation(s) may reveal a novel regulator of longevity required specifically by genetic modes of DR.

The metabolic state of an organism instructs gene expression modalities, leading to changes in complex life history traits, such as longevity. Dietary restriction (DR), which positively affects health and life span across species, leads to metabolic reprogramming that enhances utilisation of fatty acids for energy generation. One direct consequence of this metabolic shift is the upregulation of cytoprotective (CyTP) genes categorized in the Gene Ontology (GO) term of “Xenobiotic Detoxification Program” (XDP). How an organism senses metabolic changes during nutritional stress to alter gene expression programs is less known. Here, using a genetic model of DR, we show that the levels of polyunsaturated fatty acids (PUFAs), especially linoleic acid (LA) and eicosapentaenoic acid (EPA), are increased following DR and these PUFAs are able to activate the CyTP genes. This activation of CyTP genes is mediated by the conserved p38 mitogen-activated protein kinase (p38-MAPK) pathway. Consequently, genes of the PUFA biosynthesis and p38-MAPK pathway are required for multiple paradigms of DR-mediated longevity, suggesting conservation of mechanism. Thus, our study shows that PUFAs and p38-MAPK pathway function downstream of DR to help communicate the metabolic state of an organism to regulate expression of CyTP genes, ensuring extended life span., Metabolic reprogramming during Dietary Restriction (DR) activates cytoprotective gene expression. Here the authors show that PUFAs generated during DR signal via the p38-MAPK pathway to enhance cytoprotective gene expression, contributing to increased longevity.

SummaryReductive stress leads to the loss of disulfide bond formation and induces the unfolded protein response of the endoplasmic reticulum (UPRER), necessary to regain proteostasis in the compartment. Here we show that peroxide accumulation during reductive stress attenuates UPRER amplitude by altering translation without any discernible effect on transcription. Through a comprehensive genetic screen in Saccharomyces cerevisiae, we identify modulators of reductive stress-induced UPRER and demonstrate that oxidative quality control (OQC) genes modulate this cellular response in the presence of chronic but not acute reductive stress. Using a combination of microarray and relative quantitative proteomics, we uncover a non-canonical translation attenuation mechanism that acts in a bipartite manner to selectively downregulate highly expressed proteins, decoupling the cell’s transcriptional and translational response during reductive ER stress. Finally, we demonstrate that PERK, a canonical translation attenuator in higher eukaryotes, helps in bypassing a ROS-dependent, non-canonical mode of translation attenuation.

Summary Insulin/IGF-1-like signalling (IIS) and dietary restriction (DR) are the two major modulatory pathways controlling longevity across species. Here, we show that both pathways license a common chromatin modifier, ZFP-1/AF10. The downstream transcription factors of the IIS and select DR pathways, DAF-16/FOXO or PHA-4/FOXA, respectively, both transcriptionally regulate the expression of zfp-1. ZFP-1, in turn, negatively regulates the expression of DAF-16/FOXO and PHA-4/FOXA target genes, apparently forming feed-forward loops that control the amplitude as well as the duration of gene expression. We show that ZFP-1 mediates this regulation by negatively influencing the recruitment of DAF-16/FOXO and PHA-4/FOXA to their target promoters. Consequently, zfp-1 is required for the enhanced longevity observed during DR and on knockdown of IIS. Our data reveal how two distinct sensor pathways control an overlapping set of genes, using different downstream transcription factors, integrating potentially diverse and temporally distinct nutritional situations.

Chromatin immunoprecipitation (ChIP) coupled to quantitative real-time PCR (ChIP-qPCR) or Next-Generation Sequencing (ChIP-seq) enables us to study the dynamics of chromatin recruitment of transcription factors (TFs). The popular model system Caenorhabditis elegans has provided us with fundamental understanding of the role of Insulin/IGF-1-like signaling (IIS) in metabolism and aging. The FOXO TF DAF-16 is the major output of the pathway that regulates most of the phenotypes associated with the IIS pathway. Here, we describe a ChIP protocol to study FOXO recruitment dynamics in whole C. elegans extracts. We discuss detailed practical procedures, including optimization, growth, harvesting, formaldehyde fixation, sonication of worms, TF immunoprecipitation for further downstream processing using qPCR as well as NGS for the analysis of FOXO-bound DNA.

// Neeraj Kumar 1,2 , Vaibhav Jain 1,* , Anupama Singh 1,* , Urmila Jagtap 1 , Sonia Verma 1 and Arnab Mukhopadhyay 1 1 Molecular Aging Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India 2 Current address: Centre for Human Genetics and Molecular Medicine, School of Health Sciences , Central University of Punjab, Bathinda, India * These authors have contributed equally to this work Correspondence to: Neeraj Kumar, email: // Arnab Mukhopadhyay, email: // Keywords : DAF-16, FOXO, ChIP-seq, C. elegans, transcription, Gerotarget Received : August 31, 2015 Accepted : October 20, 2015 Published : November 02, 2015 Abstract Lowering insulin-IGF-1-like signalling (IIS) activates FOXO transcription factors (TF) to extend life span across species. To study the dynamics of FOXO chromatin occupancy under this condition in C. elegans , we report the first recruitment profile of endogenous DAF-16 and show that the response is conserved. DAF-16 predominantly acts as a transcriptional activator and binding within the 0.5 kb promoter-proximal region results in maximum induction of downstream targets that code for proteins involved in detoxification and longevity. Interestingly, genes that are activated under low IIS already have higher DAF-16 recruited to their promoters in WT. DAF-16 binds to variants of the FOXO consensus sequence in the promoter proximal regions of genes that are exclusively targeted during low IIS. We also define a set of ‘core’ direct targets, after comparing multiple studies, which tend to co-express and contribute robustly towards IIS-associated phenotypes. Additionally, we show that nuclear hormone receptor DAF-12 as well as zinc-finger TF EOR-1 may bind DNA in close proximity to DAF-16 and distinct TF classes that are direct targets of DAF-16 may be instrumental in regulating its indirect targets. Together, our study provides fundamental insights into the transcriptional biology of FOXO/DAF-16 and gene regulation downstream of the IIS pathway.

Advanced glycation end products (AGEs) are formed when glucose reacts nonenzymatically with proteins; these modifications are implicated in aging and pathogenesis of many age-related diseases including type II diabetes, atherosclerosis, and neurodegenerative disorders. Thus, pharmaceutical interventions that can reduce AGEs may delay age-onset diseases and extend lifespan. Using LC-MS(E), we show that rifampicin (RIF) reduces glycation of important cellular proteins in vivo and consequently increases lifespan in Caenorhabditis elegans by up to 60%. RIF analog rifamycin SV (RSV) possesses similar properties, while rifaximin (RMN) lacks antiglycation activity and therefore fails to affect lifespan positively. The efficacy of RIF and RSV as potent antiglycating agents may be attributed to the presence of a p-dihydroxyl moiety that can potentially undergo spontaneous oxidation to yield highly reactive p-quinone structures, a feature absent in RMN. We also show that supplementing rifampicin late in adulthood is sufficient to increase lifespan. For its effect on longevity, rifampicin requires DAF-18 (nematode PTEN) as well as JNK-1 and activates DAF-16, the FOXO homolog. Interestingly, the drug treatment modulates transcription of a different subset of DAF-16 target genes, those not controlled by the conserved Insulin-IGF-1-like signaling pathway. RIF failed to increase the lifespan of daf-16 null mutant despite reducing glycation, showing thereby that DAF-16 may not directly affect AGE formation. Together, our data suggest that the dual ability to reduce glycation in vivo and activate prolongevity processes through DAF-16 makes RIF and RSV effective lifespan-extending interventions.

Diet profoundly affects metabolism and incidences of age-related diseases. Animals adapt their physiology to different food-types, modulating complex life-history traits like aging. The molecular mechanisms linking adaptive capacity to diet with aging are less known. We identify FLR-4 kinase as a novel modulator of aging in C. elegans, depending on bacterial diet. FLR-4 functions to prevent differential activation of the p38MAPK pathway in response to diverse food-types, thereby maintaining normal life span. In a kinase-dead flr-4 mutant, E. coli HT115 (K12 strain), but not the standard diet OP50 (B strain), is able to activate p38MAPK, elevate expression of cytoprotective genes through the nuclear hormone receptor NHR-8 and enhance life span. Interestingly, flr-4 and dietary restriction utilize similar pathways for longevity assurance, suggesting cross-talks between cellular modules that respond to diet quality and quantity. Together, our study discovers a new C. elegans gene-diet pair that controls the plasticity of aging., Author summary For animals living in the wild, being able to utilize a wide range of diet is evolutionarily advantageous as they can survive even when their optimal diet is depleted. Since diet is known to influence the rate of aging, animals seem to have evolved intricate mechanisms to maintain homeostasis and normal life span, but the molecular mechanisms are less understood. Using a small nematode, C. elegans as a model, we show that the adaptive capacity to different diet is maintained by a kinase gene. When this gene is mutated, worms start living longer on one strain of bacterial diet but not on the other. We identify the molecular cascade required for this food-type-dependent longevity. We show that this cascade of events significantly overlaps with the pathway that determine food quantity-dependent life span enhancement. Our study thus elucidates a part of the molecular monitoring system that regulates longevity dependent on the available quality and quantity of diet.

Alternative splicing (AS) coupled to nonsense-mediated decay (AS-NMD) is a conserved mechanism for post-transcriptional gene regulation. Here we show that, during dietary restriction (DR), AS is enhanced in Caenorhabditis elegans and mice. A splicing mediator hrpu-1 regulates a significant part of these AS events in C. elegans; knocking it down suppresses DR-mediated longevity. Concurrently, due to increased AS, NMD pathway genes are upregulated and knocking down UPF1 homologue smg-2 suppresses DR lifespan. Knockdown of NMD during DR significantly increases the inclusion of PTC-containing introns and the lengths of the 3′UTRs. Finally, we demonstrate that PHA-4/FOXA transcriptionally regulates the AS-NMD genes. Our study suggests that DR uses AS to amplify the proteome, supporting physiological remodelling required for enhanced longevity. This increases the dependence on NMD, but also helps fine-tune the expression of metabolic and splicing mediators. AS-NMD may thus provide an energetically favourable level of dynamic gene expression control during dietary restriction., Alternative splicing coupled to nonsense-mediated decay (AS-NMD) is a conserved mechanism for post-transcriptional gene regulation. Here, the authors provide evidence that AS-NMD is enhanced during dietary restriction (DR) and is required for DR-mediated longevity assurance in C. elegans.

Dietary restriction (DR) increases life span and delays the onset of age-related diseases across species. However, the molecular mechanisms have remained relatively unexplored in terms of gene regulation. In C. elegans, a popular model for aging studies, the FOXA transcription factor PHA-4 is a robust genetic regulator of DR, although little is known about how it regulates gene expression. We profiled the transcriptome and miRNAome of an eat-2 mutant, a genetic surrogate of DR, by Next Generation sequencing and find that most of the miRNAs are upregulated in the young-adult worms, none significantly downregulated. Interestingly, PHA-4 can potentially regulate the expression of most of these miRNA genes. Remarkably, many of the PHA-4-regulated genes that are induced during DR are also targets of the PHA-4-upregulated miRNAs, forming a large feed-forward gene regulatory network. The genes targeted by the feed-forward loops (FFLs) are enriched for functions related to ubiquitin-mediated decay, lysosomal autophagy, cellular signalling, protein folding etc., processes that play critical roles in DR and longevity. Together our data provides a framework for understanding the complex and unique regulatory network employed during DR, suggesting that PHA-4 employs such FFLs to fine-tune gene expression and instil robustness in the system during energy crisis.

Although dietary restriction (DR) is known to extend lifespan across species, from yeast to mammals, the signalling events downstream of food/nutrient perception are not well understood. In Caenorhabditis elegans, DR is typically attained either by using the eat-2 mutants that have reduced pharyngeal pumping leading to lower food intake or by feeding diluted bacterial food to the worms. In this study, we show that knocking down a mammalian MEKK3-like kinase gene, mekk-3 in C. elegans, initiates a process similar to DR without compromising food intake. This DR-like state results in upregulation of beta-oxidation genes through the nuclear hormone receptor NHR-49, a HNF-4 homolog, resulting in depletion of stored fat. This metabolic shift leads to low levels of reactive oxygen species (ROS), potent oxidizing agents that damage macromolecules. Increased beta-oxidation, in turn, induces the phase I and II xenobiotic detoxification genes, through PHA-4/FOXA, NHR-8 and aryl hydrocarbon receptor AHR-1, possibly to purge lipophilic endotoxins generated during fatty acid catabolism. The coupling of a metabolic shift with endotoxin detoxification results in extreme longevity following mekk-3 knock-down. Thus, MEKK-3 may function as an important nutrient sensor and signalling component within the organism that controls metabolism. Knocking down mekk-3 may signal an imminent nutrient crisis that results in initiation of a DR-like state, even when food is plentiful.

Signal transduction pathways are tightly regulated by phosphorylation-dephosphorylation cycles and yet the mammalian genome contains far more genes that encode for protein kinases than protein phosphatases. Therefore, to target specific substrates, many phosphatases associate with distinct regulatory subunits and thereby modulate multiple cellular processes. One such example is the C. elegans PP2A regulatory subunit PPTR-1 that negatively regulates the insulin/insulin-like growth factor signaling pathway to modulate longevity, dauer diapause, fat metabolism and stress resistance. PPTR-1, as well as its mammalian homolog B56beta, specifically target the PP2A enzyme to AKT and mediate the dephosphorylation of this important kinase at a conserved threonine residue. In C. elegans, the major consequence of this modulation is activation of the FOXO transcription factor homolog DAF-16, which in turn regulates transcription of its many target genes involved in longevity and stress resistance. Understanding the function of B56 subunits may have important consequences in diseases such as Type 2 diabetes and cancer where the balance of Akt phosphorylation is deregulated.

In Caenorhabditis elegans, the insulin/IGF-1 signaling pathway controls many biological processes such as life span, fat storage, dauer diapause, reproduction and stress response . This pathway is comprised of many genes including the insulin/IGF-1 receptor (DAF-2) that signals through a conserved PI 3-kinase/AKT pathway and ultimately down-regulates DAF-16, a forkhead transcription factor (FOXO). DAF-16 also receives input from several other pathways that regulate life span such as the germline and the JNK pathway [Hsin, H., Kenyon, C., 1999. Signals from the reproductive system regulate the lifespan of C. elegans. Nature 399, 362-366; Oh, S.W., Mukhopadhyay, A., Svrzikapa, N., Jiang, F., Davis, R.J., Tissenbaum, H.A., 2005. JNK regulates lifespan in Caenorhabditis elegans by modulating nuclear translocation of forkhead transcription factor/DAF-16. Proc. Natl. Acad. Sci. USA 102, 4494-4499]. Therefore, DAF-16 integrates signals from multiple pathways and regulates its downstream target genes to control diverse processes. Here, we discuss the signals to and from DAF-16, with a focus on life span regulation.

Gene families expand by gene duplication, and resulting paralogs diverge through mutation. Functional diversification can include neofunctionalization as well as subfunctionalization of ancestral functions. In addition, redundancy in which multiple genes fulfill overlapping functions is often maintained. Here, we use the family of 40 Caenorhabditis elegans insulins to gain insight into the balance between specificity and redundancy. The insulin/insulin-like growth factor (IIS) pathway comprises a single receptor, DAF-2. To date, no single insulin-like peptide recapitulates all DAF-2-associated phenotypes, likely due to redundancy between insulin-like genes. To provide a first-level annotation of potential patterns of redundancy, we comprehensively delineate the spatiotemporal and conditional expression of all 40 insulins in living animals. We observe extensive dynamics in expression that can explain the lack of simple patterns of pairwise redundancy. We propose a model in which gene families evolve to attain differential alliances in different tissues and in response to a range of environmental stresses.

One of the current challenges of neurodegenerative disease research is to determine whether signaling pathways that are essential to cellular homeostasis might contribute to neuronal survival and modulate the pathogenic process in human disease. InCaenorhabditis elegans,sir-2.1/SIRT1 overexpression protects neurons from the early phases of expanded polyglutamine (polyQ) toxicity, and this protection requires the longevity-promoting factordaf-16/FOXO. Here, we show that this neuroprotective effect also requires the DAF-16/FOXO partnerbar-1/β-catenin and putative DAF-16-regulated geneucp-4, the sole mitochondrial uncoupling protein (UCP) in nematodes. These results fit with a previously proposed mechanism in which the β-catenin FOXO and SIRT1 proteins may together regulate gene expression and cell survival. Knockdown of β-catenin enhanced the vulnerability to cell death of mutant-huntingtin striatal cells derived from the HdhQ111 knock-in mice. In addition, this effect was compensated by SIRT1 overexpression and accompanied by the modulation of neuronal UCP expression levels, further highlighting a cross-talk between β-catenin and SIRT1 in the modulation of mutant polyQ cytoxicity. Taken together, these results suggest that integration of β-catenin, sirtuin and FOXO signaling protects from the early phases of mutant huntingtin toxicity.

The tubby loci provide a unique opportunity to study adult-onset obesity. Mutation in either mammalian tubby or its homologue in Caenorhabditis elegans, tub-1, results in increased fat storage. Previously, we have shown that TUB-1 interacts with a new Rab GTPase-activating protein, RBG-3, for the regulation of fat storage. To understand further the molecular mechanism of TUB-1, we identified the Rab GTPase downstream of RBG-3. We found that RBG-3 preferentially stimulates the intrinsic GTPase activity of RAB-7 in both human and C. elegans. Importantly, either mutation or RNA interference knockdown in rab-7 reduces stored fat in wild type and tub-1 mutants. In addition, the small GTPase rab-5 and genes that regulate Rab membrane localization and nucleotide recycling are required for the regulation of fat storage, thereby defining a role for endocytic recycling in this process. We propose that TUB-1 controls receptor or sensory molecule degradation in neurons by regulating a RAB-7-mediated endocytic pathway.

What is the relationship between reproduction and longevity? Evolutionary biology suggests that reproduction exacts a cost in somatic maintenance, a cost that reduces longevity. The frequent occurrence of this tradeoff between life span and fecundity, both due to experimental manipulations as well as natural variation, suggest that the mechanism might be conserved during evolution. Until recently, little was known about the mechanistic details of how reproduction might regulate life span. Here we discuss recent advances in our understanding of the regulation of life span by reproductive signaling, focusing on studies using Caenorhabditis elegans.

Transcription regulatory networks consist of physical and functional interactions between transcription factors (TFs) and their target genes. The systematic mapping of TF-target gene interactions has been pioneered in unicellular systems, using "TF-centered" methods (e.g., chromatin immunoprecipitation). However, metazoan systems are less amenable to such methods. Here, we used "gene-centered" high-throughput yeast one-hybrid (Y1H) assays to identify 283 interactions between 72 C. elegans digestive tract gene promoters and 117 proteins. The resulting protein-DNA interaction (PDI) network is highly connected and enriched for TFs that are expressed in the digestive tract. We provide functional annotations for approximately 10% of all worm TFs, many of which were previously uncharacterized, and find ten novel putative TFs, illustrating the power of a gene-centered approach. We provide additional in vivo evidence for multiple PDIs and illustrate how the PDI network provides insights into metazoan differential gene expression at a systems level.

DAF-16, a forkhead transcription factor, is a key regulator of longevity, metabolism and dauer diapause in Caenorhabditis elegans. The precise mechanism by which DAF-16 regulates multiple functions, however, is poorly understood. Here, we used chromatin immunoprecipitation (ChIP) to identify direct targets of DAF-16. We cloned 103 target sequences containing consensus DAF-16 binding sites and selected 33 targets for further analysis. Expression of most of these genes is regulated in a DAF-16–dependent manner, and inactivation of more than half of these genes significantly altered DAF-16–dependent functions, including life span, fat storage and dauer formation. Our results show that the ChIP-based cloning strategy leads to greater enrichment for DAF-16 target genes than previous screening strategies. We also demonstrate that DAF-16 is recruited to multiple promoters to coordinate regulation of its downstream targets. The large number of target genes discovered provides insight into how DAF-16 controls diverse biological functions.

DAF-16/forkhead transcription factor, the downstream target of the insulin-like signaling in Caenorhabditis elegans , is indispensable for both lifespan regulation and stress resistance. Here, we demonstrate that c-Jun N-terminal kinase (JNK) is a positive regulator of DAF-16 in both processes. Our genetic analysis suggests that the JNK pathway acts in parallel with the insulin-like signaling pathway to regulate lifespan and both pathways converge onto DAF-16. We also show that JNK-1 directly interacts with and phosphorylates DAF-16. Moreover, in response to heat stress, JNK-1 promotes the translocation of DAF-16 into the nucleus. Our findings define an interaction between two well conserved proteins, JNK-1 and DAF-16, and provide a mechanism by which JNK regulates longevity and stress resistance.

SummaryIn C. elegans, similar to in mammals, mutations in the tubby homolog, tub-1, promote increased fat deposition. Here, we show that mutation in tub-1 also leads to life span extension dependent on daf-16/FOXO. Interestingly, function of tub-1 in fat storage is independent of daf-16. A yeast two-hybrid screen identified a novel TUB-1 interaction partner (RBG-3); a RabGTPase-activating protein. Both TUB-1 and RBG-3 localize to overlapping neurons. Importantly, RNAi of rbg-3 decreases fat deposition in tub-1 mutants but does not affect life span. We demonstrate that TUB-1 is expressed in ciliated neurons and undergoes both dendritic and ciliary transport. Additionally, tub-1 mutants are chemotaxis defective. Thus, tub-1 may regulate fat storage either by modulating transport, sensing, or responding to signals in ciliated neurons. Taken together, we define a role for tub-1 in regulation of life span and show that tub-1 regulates life span and fat storage by two independent mechanisms.

The C. elegans insulin/IGF-1 signaling (IIS) cascade plays a central role in the regulation of lifespan, dauer diapause, metabolism and stress response. The major regulatory control of IIS is through phosphorylation of its components by serine/threonine-specific protein kinases. In a RNAi screen for serine/threonine protein phosphatases that counter-balance the effect of the kinases in the IIS pathway, we identified pptr-1, a B56 regulatory subunit of the PP2A holoenzyme. Modulation of pptr-1 affects phenotypes associated with the IIS pathway including lifespan, dauer, stress resistance and fat storage. We show that PPTR-1 functions by regulating worm AKT-1 phosphorylation at Thr 350. With striking conservation, mammalian B56β regulates Akt phosphorylation at Thr 308 in 3T3-L1 adipocytes. In C. elegans, this modulation ultimately leads to changes in subcellular localization and transcriptional activity of the forkhead transcription factor DAF-16. This study reveals a conserved role for the B56 regulatory subunit in modulating insulin signaling through AKT dephosphorylation and thereby has widespread implications in cancer and diabetes research.