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1.  Juvenile Hormone-Receptor Complex Acts on Mcm4 and Mcm7 to Promote Polyploidy and Vitellogenesis in the Migratory Locust 
PLoS Genetics  2014;10(10):e1004702.
Juvenile hormone (JH), a sesquiterpenoid produced by the corpora allata, coordinates insect growth, metamorphosis, and reproduction. While JH action for the repression of larval metamorphosis has been well studied, the molecular basis of JH in promoting adult reproduction has not been fully elucidated. Methoprene-tolerant (Met), the JH receptor, has been recently shown to mediate JH action during metamorphosis as well as in vitellogenesis, but again, the precise mechanism underlying the latter has been lacking. We have now demonstrated using Met RNAi to phenocopy a JH-deprived condition in migratory locusts, that JH stimulates DNA replication and increases ploidy in preparation for vitellogenesis. Mcm4 and Mcm7, two genes in the DNA replication pathway were expressed in the presence of JH and Met. Depletion of Mcm4 or Mcm7 inhibited de novo DNA synthesis and polyploidization, and resulted in the substantial reduction of vitellogenin mRNA levels as well as severely impaired oocyte maturation and ovarian growth. By using luciferase reporter and electrophoretic mobility shift assays, we have shown that Met directly regulates the transcription of Mcm4 and Mcm7 by binding to upstream consensus sequences with E-box or E-box-like motifs. Our work suggests that the JH-receptor complex acts on Mcm4 and Mcm7 to regulate DNA replication and polyploidy for vitellogenesis and oocyte maturation.
Author Summary
Vitellogenesis, a hormonally-regulated process for the synthesis of yolk proteins by the fat body or liver and their sequestration in developing oocytes, takes place in all oviparous animals except mammals. Polyploidy has also been implicated in hormone-regulated development and reproduction. Although juvenile hormone (JH) is known to regulate polyploidy in insect models and also plays a pivotal role in stimulating insect vitellogenesis, the molecular mechanisms remain poorly understood. In the migratory locust, Locusta migratoria, vitellogenesis is dependent on JH, and JH stimulates DNA replication and increases ploidy in the fat body. Here, we show that a JH-receptor complex comprised of Methoprene-tolerant (Met) and a steroid receptor co-activator activates the transcription of two mini-chromosome maintenance (Mcm) genes, Mcm4 and Mcm7. Knockdown of Mcm4 or Mcm7 via RNAi can phenocopy JH-deprivation and Met-depletion, resulting in reduced ploidy, blocked vitellogenin (Vg) expression, as well as arrested oocyte maturation and ovarian growth. This study provides evidence that JH acts through its receptor on the Mcm machinery to replicate the genome of fat body cells in preparation for the massive synthesis of Vg and possibly other proteins required for oocyte maturation and egg production.
PMCID: PMC4207617  PMID: 25340846
2.  Genome-Wide Analysis of Germline Signaling Genes Regulating Longevity and Innate Immunity in the Nematode Pristionchus pacificus 
PLoS Pathogens  2012;8(8):e1002864.
Removal of the reproductive system of many animals including fish, flies, nematodes, mice and humans can increase lifespan through mechanisms largely unknown. The abrogation of the germline in Caenorhabditis elegans increases longevity by 60% due to a signal emitted from the somatic gonad. Apart from increased longevity, germline-less C. elegans is also resistant to other environmental stressors such as feeding on bacterial pathogens. However, the evolutionary conservation of this pathogen resistance, its genetic basis and an understanding of genes involved in producing this extraordinary survival phenotype are currently unknown. To study these evolutionary aspects we used the necromenic nematode Pristionchus pacificus, which is a genetic model system used in comparison to C. elegans. By ablation of germline precursor cells and subsequent feeding on the pathogen Serratia marcescens we discovered that P. pacificus shows remarkable resistance to bacterial pathogens and that this response is evolutionarily conserved across the Genus Pristionchus. To gain a mechanistic understanding of the increased resistance to bacterial pathogens and longevity in germline-ablated P. pacificus we used whole genome microarrays to profile the transcriptional response comparing germline ablated versus un-ablated animals when fed S. marcescens. We show that lipid metabolism, maintenance of the proteasome, insulin signaling and nuclear pore complexes are essential for germline deficient phenotypes with more than 3,300 genes being differentially expressed. In contrast, gene expression of germline-less P. pacificus on E. coli (longevity) and S. marcescens (immunity) is very similar with only 244 genes differentially expressed indicating that longevity is due to abundant gene expression also involved in immunity. By testing existing mutants of Ppa-DAF-16/FOXO and the nuclear hormone receptor Ppa-DAF-12 we show a conserved function of both genes in resistance to bacterial pathogens and longevity. This is the first study to show that the influence of the reproductive system on extending lifespan and innate immunity is conserved in evolution.
Author Summary
Removal of the germline in the nematode Caenorhabditis elegans can increase lifespan and resistance to bacterial pathogens. Currently there is no information on what genes are regulated to produce this resistance phenotype in other nematodes and whether they are the same as genes involved in lifespan regulation. We used the necromenic nematode, Pristionchus pacificus, a species that diverged from C. elegans 250–400 MYA, ablated its germline and found increased resistance to the pathogens Serratia marcescens and Xenorhabdus nematophila. In a novel manner we performed cell ablation of the germline, exposure to bacterial pathogens and used whole genome microarrays of the same animals to find that this resistance is due to expression of genes involved in insulin signaling, nuclear pore complexes, ribosomal translation and lipid production. Furthermore, we see little difference between germline ablated lifespan and immunity leading us to believe that living longer is due to an abundance of genes also being involved with immunity. We could also show that, similar to C. elegans, the transcription factor DAF-16/FOXO and nuclear hormone receptor DAF-12, are integral for this response. Our study is the first to understand how the reproductive system regulates both lifespan and innate immunity transcriptionally and offers insights into the signaling cascades involved with resisting pathogen attack.
PMCID: PMC3415453  PMID: 22912581
3.  The Somatic Reproductive Tissues of C. elegans Promote Longevity through Steroid Hormone Signaling 
PLoS Biology  2010;8(8):e1000468.
Removal of the germ cells of C. elegans extends lifespan in part because signals from the somatic reproductive tissues activate the nuclear hormone receptor DAF-12.
In Caenorhabditis elegans and Drosophila melanogaster, removing the germline precursor cells increases lifespan. In worms, and possibly also in flies, this lifespan extension requires the presence of somatic reproductive tissues. How the somatic gonad signals other tissues to increase lifespan is not known. The lifespan increase triggered by loss of the germ cells is known to require sterol hormone signaling, as reducing the activity of the nuclear hormone receptor DAF-12, or genes required for synthesis of the DAF-12 ligand dafachronic acid, prevents germline loss from extending lifespan. In addition to sterol signaling, the FOXO transcription factor DAF-16 is required to extend lifespan in animals that lack germ cells. DAF-12/NHR is known to assist with the nuclear accumulation of DAF-16/FOXO in these animals, yet we find that loss of DAF-12/NHR has little or no effect on the expression of at least some DAF-16/FOXO target genes. In this study, we show that the DAF-12-sterol signaling pathway has a second function to activate a distinct set of genes and extend lifespan in response to the somatic reproductive tissues. When germline-deficient animals lacking somatic reproductive tissues are given dafachronic acid, their expression of DAF-12/NHR-dependent target genes is restored and their lifespan is increased. Together, our findings indicate that in C. elegans lacking germ cells, the somatic reproductive tissues promote longevity via steroid hormone signaling to DAF-12.
Author Summary
Reproductive tissues are known to generate important intercellular signals. For example, in mammals, the reproductive tissues produce steroid hormones such as estrogen and testosterone that have profound effects on development and physiology. Studies of the nematode C. elegans and other organisms have shown that the reproductive system can also affect the rate at which an animal ages. Removal of C. elegans' germ cells extends lifespan but this effect is not simply due to sterility, as removal of both the somatic reproductive tissues and the germ cells does not extend lifespan. Instead, loss of the germ cells extends lifespan by activating a pathway that requires input from the somatic gonad. In this study, we demonstrate that the somatic reproductive tissues promote longevity by controlling the activity of a steroid signaling pathway that regulates the DAF-12 nuclear hormone receptor.
PMCID: PMC2930862  PMID: 20824162
4.  A Transcription Elongation Factor That Links Signals from the Reproductive System to Lifespan Extension in Caenorhabditis elegans 
PLoS Genetics  2009;5(9):e1000639.
In Caenorhabditis elegans and Drosophila melanogaster, the aging of the soma is influenced by the germline. When germline-stem cells are removed, aging slows and lifespan is increased. The mechanism by which somatic tissues respond to loss of the germline is not well-understood. Surprisingly, we have found that a predicted transcription elongation factor, TCER-1, plays a key role in this process. TCER-1 is required for loss of the germ cells to increase C. elegans' lifespan, and it acts as a regulatory switch in the pathway. When the germ cells are removed, the levels of TCER-1 rise in somatic tissues. This increase is sufficient to trigger key downstream events, as overexpression of tcer-1 extends the lifespan of normal animals that have an intact reproductive system. Our findings suggest that TCER-1 extends lifespan by promoting the expression of a set of genes regulated by the conserved, life-extending transcription factor DAF-16/FOXO. Interestingly, TCER-1 is not required for DAF-16/FOXO to extend lifespan in animals with reduced insulin/IGF-1 signaling. Thus, TCER-1 specifically links the activity of a broadly deployed transcription factor, DAF-16/FOXO, to longevity signals from reproductive tissues.
Author Summary
The reproductive status and longevity of animals are strongly interlinked. Increasing age influences the reproductive capacities of most animals. However, little is known about how reproductive status might affect lifespan. Experiments in worms and flies have shown that removing cells that give rise to gametes, the “germ cells”, makes them live longer. We know very little about the genes and molecules that are involved in this process. In this study, we have identified a gene called tcer-1 that promotes the longevity of the roundworm Caenorhabditis elegans when its germ cells are removed. The gene tcer-1 codes for a protein, TCER-1, that is predicted to function as a “transcription elongation factor” (it allows the completion of RNA synthesis during the process of gene expression). Our experiments imply that when the germ cells of worms are removed, TCER-1 collaborates with a transcription factor called DAF-16/FOXO to express genes that contribute to increased longevity. DAF-16/FOXO can extend lifespan in response to other physiological cues besides loss of germ cells. However, TCER-1 specifically helps this widely used longevity protein to respond to signals that reflect the reproductive status. Counterparts of DAF-16/FOXO are known to control aging in other organisms, including humans, so the identification of TCER-1 may lead to a better understanding of the relationship between reproduction and aging in other species, too.
PMCID: PMC2729384  PMID: 19749979
5.  Revealing genes associated with vitellogenesis in the liver of the zebrafish (Danio rerio) by transcriptome profiling 
BMC Genomics  2009;10:141.
In oviparous vertebrates, including fish, vitellogenesis consists of highly regulated pathways involving 17β-estradiol (E2). Previous studies focused on a relatively small number of hepatic expressed genes during vitellogenesis. This study aims to identify hepatic genes involved in vitellogenesis and regulated by E2, by using zebrafish microarray gene expression profiling, and to provide information on functional distinctive genes expressed in the liver of a vitellogenic female, using zebrafish as a model fish.
Genes associated with vitellogenesis were revealed by the following paired t-tests (SAM) comparisons: a) two-month old vitellogenic (Vit2) females were compared with non-vitellogenic (NV) females, showing 825 differentially expressed transcripts during early stages of vitellogenesis, b) four-month old vitellogenic (Vit4) females were compared with NV females, showing 1,046 differentially expressed transcripts during vitellogenesis and c) E2-treated males were compared with control males, showing 1,828 differentially expressed transcripts regulated by E2. A Venn diagram revealed 822 common transcripts in the three groups, indicating that these transcripts were involved in vitellogenesis and putatively regulated by E2. In addition, 431 transcripts were differentially expressed in Vit2 and Vit4 females but not in E2-treated males, indicating that they were putatively not up-regulated by E2. Correspondence analysis showed high similarity in expression profiles of Vit2 with Vit4 and of NV females with control males. The E2-treated males differed from the other groups. The repertoire of genes putatively regulated by E2 in vitellogenic females included genes associated with protein synthesis and reproduction. Genes associated with the immune system processes and biological adhesion, were among the genes that were putatively not regulated by E2. E2-treated males expressed a large array of transcripts that were not associated with vitellogenesis.
The study revealed several genes that were not reported before as being regulated by E2. Also, the hepatic expression of several genes was reported here for the first time.
Gene expression profiling of liver samples revealed 1,046 differentially expressed transcripts during vitellogenesis of which at least ~64% were regulated by E2. The results raise the question on the regulation pattern and temporal pleiotropic expression of hepatic genes in vitellogenic females.
PMCID: PMC2678157  PMID: 19335895
6.  Fatty Acid Desaturation Links Germ Cell Loss to Longevity Through NHR-80/HNF4 in C. elegans 
PLoS Biology  2011;9(3):e1000599.
Lifespan extension induced by germline ablation in C. elegans is regulated by the nuclear hormone receptor NHR-80 in a process that requires the production of oleic acid by activation of the lipid desaturase FAT-6/SCD1.
Preventing germline stem cell proliferation extends lifespan in nematodes and flies. So far, studies on germline-longevity signaling have focused on daf-16/FOXO and daf-12/VDR. Here, we report on NHR-80/HNF4, a nuclear receptor that specifically mediates longevity induced by depletion of the germ line through a mechanism that implicates fatty acid monodesaturation.
Methods and Findings
nhr-80/HNF4 is induced in animals lacking a germ line and is specifically required for their extended longevity. Overexpressing nhr-80/HNF4 increases the lifespan of germline-less animals. This lifespan extension can occur in the absence of daf-16/FOXO but requires the presence of the nuclear receptor DAF-12/VDR. We show that the fatty acid desaturase, FAT-6/SCD1, is a key target of NHR-80/HNF4 and promotes germline-longevity by desaturating stearic acid to oleic acid (OA). We find that NHR-80/HNF4 and OA must work in concert to promote longevity.
Taken together, our data indicate that the NHR-80 pathway participates in the mechanism of longevity extension through depletion of the germ line. We identify fat-6 and OA as essential downstream elements although other targets must also be present. Thus, NHR-80 links fatty acid desaturation to lifespan extension through germline ablation in a daf-16/FOXO independent manner.
Author Summary
Reproduction and aging are two processes that seem to be closely intertwined. Experiments in Caenorhabditis elegans and Drosophila have shown that depletion of the germ line increases lifespan and that this process depends on insulin and lipophilic-hormone signaling. Recently, it was demonstrated that when germline stem cells (GSCs) cease to proliferate, fat metabolism is altered and this affects longevity. In this study, we have identified a nuclear hormone receptor, NHR-80, that mediates longevity through depletion of the germ line by promoting fatty acid desaturation. The nhr-80 gene is up-regulated at the mRNA and protein levels in germline-less animals, leading to the transcription of the gene, fat-6, and the production of oleic acid (OA). Our experiments also show that the NHR-80/FAT-6/OA pathway does not require the presence of DAF-16 but instead, depends fully on the presence of DAF-12, a steroid receptor that affects lifespan. We provide evidence that other NHR-80 targets must be present concomitantly. Our results reinforce the notion that fat metabolism is profoundly altered in response to GSC proliferation, and the data contribute to a better understanding of the molecular relationship between reproduction, fat metabolism, and aging.
PMCID: PMC3057950  PMID: 21423649
7.  daf-31 Encodes the Catalytic Subunit of N Alpha-Acetyltransferase that Regulates Caenorhabditis elegans Development, Metabolism and Adult Lifespan 
PLoS Genetics  2014;10(10):e1004699.
The Caenorhabditis elegans dauer larva is a facultative state of diapause. Mutations affecting dauer signal transduction and morphogenesis have been reported. Of these, most that result in constitutive formation of dauer larvae are temperature-sensitive (ts). The daf-31 mutant was isolated in genetic screens looking for novel and underrepresented classes of mutants that form dauer and dauer-like larvae non-conditionally. Dauer-like larvae are arrested in development and have some, but not all, of the normal dauer characteristics. We show here that daf-31 mutants form dauer-like larvae under starvation conditions but are sensitive to SDS treatment. Moreover, metabolism is shifted to fat accumulation in daf-31 mutants. We cloned the daf-31 gene and it encodes an ortholog of the arrest-defective-1 protein (ARD1) that is the catalytic subunit of the major N alpha-acetyltransferase (NatA). A daf-31 promoter::GFP reporter gene indicates daf-31 is expressed in multiple tissues including neurons, pharynx, intestine and hypodermal cells. Interestingly, overexpression of daf-31 enhances the longevity phenotype of daf-2 mutants, which is dependent on the forkhead transcription factor (FOXO) DAF-16. We demonstrate that overexpression of daf-31 stimulates the transcriptional activity of DAF-16 without influencing its subcellular localization. These data reveal an essential role of NatA in controlling C. elegans life history and also a novel interaction between ARD1 and FOXO transcription factors, which may contribute to understanding the function of ARD1 in mammals.
Author Summary
The development of a living organism is influenced by the environmental conditions such as nutrient availability. Under starvation conditions, the C. elegans larvae will enter a special developmental stage called dauer larva. An insulin-like signaling pathway controls dauer formation as well as adult lifespan by inhibiting the activity of FOXO transcription factor DAF-16 that regulates expression of stress-resistant genes. Here we isolate a new gene called daf-31; this gene encodes a protein that regulates C. elegans larval development, metabolism and adult lifespan. This protein has been found in other species to be part of an enzyme that functions to modify other proteins. We show that overexpression of our newly discovered protein stimulates the transcriptional activity of DAF-16. Interestingly, abnormal regulation of human proteins similar to DAF-31 results in tumor formation. It is known that human FOXO proteins prevent tumorigenesis. Therefore, it is possible that abnormal DAF-31 activity may lead to tumor growth by reducing DAF-16 activity. Thus, the present study may not only contribute to understanding the role of a universal enzyme in controlling development, metabolism and lifespan in other organisms besides worms but may also shed light on the mechanisms of tumorigenesis in humans.
PMCID: PMC4199510  PMID: 25330189
8.  Insulin/IGF-1-mediated longevity is marked by reduced protein metabolism 
Quantitative proteomics, lifespan analysis, and biochemical assays were utilized to show that Insulin/IGF-1-mediated longevity in C. elegans is strongly associated with a daf-16 dependent global reduction in protein metabolism.
A daf-16 dependent global reduction in protein translation is observed in daf-2 long-lived mutant.The reduction in active translation is independent of germline activityA role for protein metabolism is identified in the Insulin/IGF-1-mediated extension of life.
Mutations in the daf-2 gene of the conserved Insulin/Insulin-like Growth Factor (IGF-1) pathway double the lifespan of the nematode Caenorhabditis elegans. This phenotype is completely suppressed by deletion of Forkhead transcription factor daf-16. To uncover regulatory mechanisms coordinating this extension of life, we employed a quantitative proteomics strategy with daf-2 mutants in comparison with N2 and daf-16; daf-2 double mutants. This revealed a remarkable longevity-specific decrease in proteins involved in mRNA processing and transport, the translational machinery, and protein metabolism. Correspondingly, the daf-2 mutants display lower amounts of mRNA and 20S proteasome activity, despite maintaining total protein levels equal to that observed in wild types. Polyribosome profiling in the daf-2 and daf-16;daf-2 double mutants confirmed a daf-16-dependent reduction in overall translation, a phenotype reminiscent of Dietary Restriction-mediated longevity, which was independent of germline activity. RNA interference (RNAi)-mediated knockdown of proteins identified by our approach resulted in modified C. elegans lifespan confirming the importance of these processes in Insulin/IGF-1-mediated longevity. Together, the results demonstrate a role for the metabolism of proteins in the Insulin/IGF-1-mediated extension of life.
PMCID: PMC3734508  PMID: 23820781
ageing; high-throughput analysis; metabolism; protein metabolism; translation
9.  CAMKII and Calcineurin regulate the lifespan of Caenorhabditis elegans through the FOXO transcription factor DAF-16 
eLife  2013;2:e00518.
The insulin-like signaling pathway maintains a relatively short wild-type lifespan in Caenorhabditis elegans by phosphorylating and inactivating DAF-16, the ortholog of the FOXO transcription factors of mammalian cells. DAF-16 is phosphorylated by the AKT kinases, preventing its nuclear translocation. Calcineurin (PP2B phosphatase) also limits the lifespan of C. elegans, but the mechanism through which it does so is unknown. Herein, we show that TAX-6•CNB-1 and UNC-43, the C. elegans Calcineurin and Ca2+/calmodulin-dependent kinase type II (CAMKII) orthologs, respectively, also regulate lifespan through DAF-16. Moreover, UNC-43 regulates DAF-16 in response to various stress conditions, including starvation, heat or oxidative stress, and cooperatively contributes to lifespan regulation by insulin signaling. However, unlike insulin signaling, UNC-43 phosphorylates and activates DAF-16, thus promoting its nuclear localization. The phosphorylation of DAF-16 at S286 by UNC-43 is removed by TAX-6•CNB-1, leading to DAF-16 inactivation. Mammalian FOXO3 is also regulated by CAMKIIA and Calcineurin.
eLife digest
Although aging might seem to be a passive process—resulting simply from wear and tear over a lifetime—it can actually be accelerated or slowed down by genetic mutations. This phenomenon has been most thoroughly studied in the nematode worm, Caenorhabditis elegans. Normally, this worm lives for just two or three weeks, but genetic mutations that reduce the activity of certain enzymes in a series of biochemical reactions known as the insulin/IGF-1 signalling pathway can extend its lifespan by up to a factor of ten, and similar effects have been seen in flies and mice. Lifespans can also be increased by blocking other signalling pathways or restricting the intake of calories.
This increase in lifespan associated with the insulin/IGF-1 signalling pathway is known to involve a protein called DAF-16 and two kinases called AKT-1 and AKT-2. Under normal conditions the AKT kinases add several phosphate groups to the DAF-16, which prevents it from travelling to the nucleus of the cell. However, when genetic techniques are used to block the insulin/IGF-1 signalling pathway, the AKT kinases are unable to add the phosphate groups; this leaves the DAF-16 free to enter the nucleus, where it activates a network of genes that promotes longevity.
In addition to kinases, the insulin/IGF-1 signalling pathway also involves enzymes called phosphatases that remove the phosphate groups from other proteins. In particular, a phosphatase called calcineurin is known to be involved in the regulation of lifespan, but the details of this process are not fully understood.
Now, Tao et al. have carried out a series of genetic and biochemical experiments to determine how phosphatases exert their influence on aging. The results show that calcineurin targets DAF-16, the same protein that is targeted by the AKT kinases. Moreover, another kinase also targets DAF-16 when the worm is exposed to heat, starvation or some other form of stress: this kinase, which is not involved in the insulin/IGF-1 signalling pathway, is called CAMKII.
Tao et al. show that these kinases act on DAF-16 in different ways: CAMKII activates it by adding the phosphate group at a specific site known as S286, whereas the AKT kinases deactivate DAF-16 because they add phosphate groups at different sites, thereby preventing it from entering the nucleus. Calcineurin neutralizes the effect of CAMKII by removing the phosphate group at S286 to deactivate the DAF-16.
In addition to shedding new light on the regulation of lifespan in C. elegans, the new results could improve our understanding of aging in humans, and also the development of diabetes and other age-related diseases, because the equivalent molecules in mammalian cells are regulated in similar ways.
PMCID: PMC3691573  PMID: 23805378
aging; lifespan; FOXO; CAMKII; calcineurin; DAF-16; C. elegans
10.  Cell-Nonautonomous Signaling of FOXO/DAF-16 to the Stem Cells of Caenorhabditis elegans 
PLoS Genetics  2012;8(8):e1002836.
In Caenorhabditis elegans (C. elegans), the promotion of longevity by the transcription factor DAF-16 requires reduced insulin/IGF receptor (IIR) signaling or the ablation of the germline, although the reason for the negative impact of germ cells is unknown. FOXO/DAF-16 activity inhibits germline proliferation in both daf-2 mutants and gld-1 tumors. In contrast to its function as a germline tumor suppressor, we now provide evidence that somatic DAF-16 in the presence of IIR signaling can also result in tumorigenic activity, which counteracts robust lifespan extension. In contrast to the cell-autonomous IIR signaling, which is required for larval germline proliferation, activation of DAF-16 in the hypodermis results in hyperplasia of the germline and disruption of the surrounding basement membrane. SHC-1 adaptor protein and AKT-1 kinase antagonize, whereas AKT-2 and SGK-1 kinases promote, this cell-nonautonomous DAF-16 function. Our data suggest that a functional balance of DAF-16 activities in different tissues determines longevity and reveals a novel, cell-nonautonomous role of FOXO/DAF-16 to affect stem cells.
Author Summary
Previous studies have shown that DAF–16/FOXO transcription factor promotes longevity and stress resistance and inhibits tumor progression in the absence of insulin signaling. Here we show that active DAF-16 in the epidermis can shorten lifespan by promoting a tumorous germline phenotype. In contrast to the known inhibitory effect of insulin signaling upon DAF-16, an active insulin and PI3K signaling are required for DAF-16–mediated signaling to the germline. In addition, AKT-1– and SHC-1–mediated JNK signaling antagonize AKT-2 and SGK-1 to affect the reproductive system. This is to our knowledge the first report about a detrimental effect of DAF-16 on lifespan. Furthermore it emphasizes that DAF-16 activity is highly dependent on the cellular context and communication between different tissues.
PMCID: PMC3420913  PMID: 22916022
11.  DAF-2/Insulin-Like Signaling in C. elegans Modifies Effects of Dietary Restriction and Nutrient Stress on Aging, Stress and Growth 
PLoS ONE  2007;2(11):e1240.
Dietary restriction (DR) and reduced insulin/IGF-I-like signaling (IIS) are two regimens that promote longevity in a variety of organisms. Genetic analysis in C. elegans nematodes has shown that DR and IIS couple to distinct cellular signaling pathways. However, it is not known whether these pathways ultimately converge on overlapping or distinct targets to extend lifespan.
Principal Findings
We investigated this question by examining additional effects of DR in wildtype animals and in daf-2 mutants with either moderate or severe IIS deficits. Surprisingly, DR and IIS had opposing effects on these physiological processes. First, DR induced a stress-related change in intestinal vesicle trafficking, termed the FIRE response, which was suppressed in daf-2 mutants. Second, DR did not strongly affect expression of a daf-2- and stress-responsive transcriptional reporter. Finally, DR-related growth impairment was suppressed in daf-2 mutants.
These findings reveal that an important biological function of DAF-2/IIS is to enhance growth and survival under nutrient-limited conditions. However, we also discovered that levels of DAF-2 pathway activity modified the effects of DR on longevity. Thus, while DR and IIS clearly affect lifespan through independent targets, there may also be some prolongevity targets that are convergently regulated by these pathways.
PMCID: PMC2080776  PMID: 18043747
12.  Positive Feedback between Transcriptional and Kinase Suppression in Nematodes with Extraordinary Longevity and Stress Resistance 
PLoS Genetics  2009;5(4):e1000452.
Insulin/IGF-1 signaling (IIS) regulates development and metabolism, and modulates aging, of Caenorhabditis elegans. In nematodes, as in mammals, IIS is understood to operate through a kinase-phosphorylation cascade that inactivates the DAF-16/FOXO transcription factor. Situated at the center of this pathway, phosphatidylinositol 3-kinase (PI3K) phosphorylates PIP2 to form PIP3, a phospholipid required for membrane tethering and activation of many signaling molecules. Nonsense mutants of age-1, the nematode gene encoding the class-I catalytic subunit of PI3K, produce only a truncated protein lacking the kinase domain, and yet confer 10-fold greater longevity on second-generation (F2) homozygotes, and comparable gains in stress resistance. Their F1 parents, like weaker age-1 mutants, are far less robust—implying that maternally contributed trace amounts of PI3K activity or of PIP3 block the extreme age-1 phenotypes. We find that F2-mutant adults have <10% of wild-type kinase activity in vitro and <60% of normal phosphoprotein levels in vivo. Inactivation of PI3K not only disrupts PIP3-dependent kinase signaling, but surprisingly also attenuates transcripts of numerous IIS components, even upstream of PI3K, and those of signaling molecules that cross-talk with IIS. The age-1(mg44) nonsense mutation results, in F2 adults, in changes to kinase profiles and to expression levels of multiple transcripts that distinguish this mutant from F1 age-1 homozygotes, a weaker age-1 mutant, or wild-type adults. Most but not all of those changes are reversed by a second mutation to daf-16, implicating both DAF-16/ FOXO–dependent and –independent mechanisms. RNAi, silencing genes that are downregulated in long-lived worms, improves oxidative-stress resistance of wild-type adults. It is therefore plausible that attenuation of those genes in age-1(mg44)-F2 adults contributes to their exceptional survival. IIS in nematodes (and presumably in other species) thus involves transcriptional as well as kinase regulation in a positive-feedback circuit, favoring either survival or reproduction. Hyperlongevity of strong age-1(mg44) mutants may result from their inability to reset this molecular switch to the reproductive mode.
Author Summary
Insulin/IGF-1 signaling (IIS) impacts development, metabolism, and longevity in Caenorhabditis elegans. It has been viewed as a cascade of kinase reactions, chiefly phosphorylation of other kinases, leading to inactivation of the DAF-16/FOXO transcription factor. PI3K, a phosphatidylinositol kinase at the center of this pathway, converts PIP2 to PIP3, instrumental to kinase docking and activation. Here we show that PI3K deficiency elicits transcriptional inhibition of many kinases, including those of IIS itself. This creates a positive-feedback loop, wherein DAF-16/FOXO silences expression of the very kinases that would have inactivated it. In the resulting “flip-flop” genetic switch, either kinase signaling or transcriptional silencing may predominate. We discovered the transcriptional arm of this switch in infertile age-1(mg44) mutants, defective for PI3K activity. The absence of PIP3 and PIP3-dependent kinase activity gives free rein to gene silencing by DAF-16/FOXO. This two-tiered response could scarcely have evolved for the benefit of a sterile mutant; some components presumably serve regulatory functions in normal animals, reinforcing a switch responsive to environmental and internal signals. In age-1(mg44) mutants, complete inactivation of PI3K “fuses” the switch, locking worms into longevity mode. With signaling profoundly silenced, they cannot resume reproduction, but instead acquire a remarkable capacity for individual survival.
PMCID: PMC2661368  PMID: 19360094
13.  DAF-16/FoxO Directly Regulates an Atypical AMP-Activated Protein Kinase Gamma Isoform to Mediate the Effects of Insulin/IGF-1 Signaling on Aging in Caenorhabditis elegans 
PLoS Genetics  2014;10(2):e1004109.
The DAF-16/FoxO transcription factor controls growth, metabolism and aging in Caenorhabditis elegans. The large number of genes that it regulates has been an obstacle to understanding its function. However, recent analysis of transcript and chromatin profiling implies that DAF-16 regulates relatively few genes directly, and that many of these encode other regulatory proteins. We have investigated the regulation by DAF-16 of genes encoding the AMP-activated protein kinase (AMPK), which has α, β and γ subunits. C. elegans has 5 genes encoding putative AMP-binding regulatory γ subunits, aakg-1-5. aakg-4 and aakg-5 are closely related, atypical isoforms, with orthologs throughout the Chromadorea class of nematodes. We report that ∼75% of total γ subunit mRNA encodes these 2 divergent isoforms, which lack consensus AMP-binding residues, suggesting AMP-independent kinase activity. DAF-16 directly activates expression of aakg-4, reduction of which suppresses longevity in daf-2 insulin/IGF-1 receptor mutants. This implies that an increase in the activity of AMPK containing the AAKG-4 γ subunit caused by direct activation by DAF-16 slows aging in daf-2 mutants. Knock down of aakg-4 expression caused a transient decrease in activation of expression in multiple DAF-16 target genes. This, taken together with previous evidence that AMPK promotes DAF-16 activity, implies the action of these two metabolic regulators in a positive feedback loop that accelerates the induction of DAF-16 target gene expression. The AMPK β subunit, aakb-1, also proved to be up-regulated by DAF-16, but had no effect on lifespan. These findings reveal key features of the architecture of the gene-regulatory network centered on DAF-16, and raise the possibility that activation of AMP-independent AMPK in nutritionally replete daf-2 mutant adults slows aging in C. elegans. Evidence of activation of AMPK subunits in mammals suggests that such FoxO-AMPK interactions may be evolutionarily conserved.
Author Summary
Aging is an important problem for human health and is regulated by complex gene regulatory networks. In a simple nematode worm (Caenorhabditis elegans) mutation of the insulin/IGF-1 receptor daf-2 dramatically extends lifespan. This is due to the increased activity of DAF-16, a FoxO transcription factor, leading to altered expression of genes, many encoding other regulatory proteins. We have focused on one such protein, AMP-activated protein kinase (AMPK), that is important for regulating cellular homeostasis under conditions of low energy availability (e.g. starvation). We find that DAF-16 binds to the promoter of aakg-4 (a gene encoding an atypical γ subunit of AMPK) and increases its expression. Inhibition of aakg-4 leads to down-regulation of multiple DAF-16 target genes and shortens the life of daf-2 mutants. Taken together with a previous report showing that AMPK activates DAF-16, this suggests that AAKG-4 and DAF-16 are involved in a positive feedback loop which accelerates effects of DAF-16 on gene expression, and could contribute to longevity. This study defines a new part of the complex gene regulatory network in which DAF-16 acts to control aging. FoxO-AMPK interactions are present in higher animals, where they could potentially also influence aging.
PMCID: PMC3916255  PMID: 24516399
14.  Long-term starvation and ageing induce AGE-1/PI 3-kinase-dependent translocation of DAF-16/FOXO to the cytoplasm 
BMC Biology  2006;4:1.
The provision of stress resistance diverts resources from development and reproduction and must therefore be tightly regulated. In Caenorhabditis elegans, the switch to increased stress resistance to promote survival through periods of starvation is regulated by the DAF-16/FOXO transcription factor. Reduction-of-function mutations in AGE-1, the C. elegans Class IA phosphoinositide 3-kinase (PI3K), increase lifespan and stress resistance in a daf-16 dependent manner. Class IA PI3Ks downregulate FOXOs by inducing their translocation to the cytoplasm. However, the circumstances under which AGE-1 is normally activated are unclear. To address this question we used C. elegans first stage larvae (L1s), which when starved enter a developmentally-arrested diapause stage until food is encountered.
We find that in L1s both starvation and daf-16 are necessary to confer resistance to oxidative stress in the form of hydrogen peroxide. Accordingly, DAF-16 is localised to cell nuclei after short-term starvation. However, after long-term starvation, DAF-16 unexpectedly translocates to the cytoplasm. This translocation requires functional age-1. H2O2 treatment can replicate the translocation and induce generation of the AGE-1 product PIP3. Because feeding reduces to zero in ageing adult C. elegans, these animals may also undergo long-term starvation. Consistent with our observation in L1s, DAF-16 also translocates to the cytoplasm in old adult worms in an age-1-dependent manner.
DAF-16 is activated in the starved L1 diapause. The translocation of DAF-16 to the cytoplasm after long-term starvation may be a feedback mechanism that prevents excessive expenditure on stress resistance. H2O2 is a candidate second messenger in this feedback mechanism. The lack of this response in age-1(hx546) mutants suggests a novel mechanism by which this mutation increases longevity.
PMCID: PMC1403811  PMID: 16457721
15.  p38 MAPK Regulates Expression of Immune Response Genes and Contributes to Longevity in C. elegans 
PLoS Genetics  2006;2(11):e183.
The PMK-1 p38 mitogen-activated protein kinase pathway and the DAF-2–DAF-16 insulin signaling pathway control Caenorhabditis elegans intestinal innate immunity. pmk-1 loss-of-function mutants have enhanced sensitivity to pathogens, while daf-2 loss-of-function mutants have enhanced resistance to pathogens that requires upregulation of the DAF-16 transcription factor. We used genetic analysis to show that the pathogen resistance of daf-2 mutants also requires PMK-1. However, genome-wide microarray analysis indicated that there was essentially no overlap between genes positively regulated by PMK-1 and DAF-16, suggesting that they form parallel pathways to promote immunity. We found that PMK-1 controls expression of candidate secreted antimicrobials, including C-type lectins, ShK toxins, and CUB-like genes. Microarray analysis demonstrated that 25% of PMK-1 positively regulated genes are induced by Pseudomonas aeruginosa infection. Using quantitative PCR, we showed that PMK-1 regulates both basal and infection-induced expression of pathogen response genes, while DAF-16 does not. Finally, we used genetic analysis to show that PMK-1 contributes to the enhanced longevity of daf-2 mutants. We propose that the PMK-1 pathway is a specific, indispensable immunity pathway that mediates expression of secreted immune response genes, while the DAF-2–DAF-16 pathway appears to regulate immunity as part of a more general stress response. The contribution of the PMK-1 pathway to the enhanced lifespan of daf-2 mutants suggests that innate immunity is an important determinant of longevity.
The innate immune system provides the first line of defense against pathogen infection and relies upon pathways conserved across mammals, insects, and nematodes. Here, the authors have analyzed the transcriptional response of the nematode Caenorhabditis elegans to infection by the human pathogen Pseudomonas aeruginosa. They investigated this transcriptional response in the context of two conserved pathways involved in pathogen defense: the PMK-1 p38 mitogen-activated protein kinase (p38 MAPK) pathway and the DAF-2–DAF-16 insulin-signaling pathway. Specifically, the authors found that the p38 MAPK pathway plays a critical role in the infection-induced expression of secreted immune response genes. These genes include C-type lectins, lysozymes, and antimicrobial peptides that fight off infection in many species. In contrast, they found that the DAF-16 pathway is not required for immune response gene expression and may regulate immunity as part of a general stress response that functions in parallel to p38 MAPK. In addition, the authors observed that p38 MAPK contributes to the enhanced longevity of daf-2 mutants, implicating p38 MAPK signaling in the regulation of longevity, possibly through its role in immunity.
PMCID: PMC1635533  PMID: 17096597
16.  Genes That Act Downstream of Sensory Neurons to Influence Longevity, Dauer Formation, and Pathogen Responses in Caenorhabditis elegans 
PLoS Genetics  2012;8(12):e1003133.
The sensory systems of multicellular organisms are designed to provide information about the environment and thus elicit appropriate changes in physiology and behavior. In the nematode Caenorhabditis elegans, sensory neurons affect the decision to arrest during development in a diapause state, the dauer larva, and modulate the lifespan of the animals in adulthood. However, the mechanisms underlying these effects are incompletely understood. Using whole-genome microarray analysis, we identified transcripts whose levels are altered by mutations in the intraflagellar transport protein daf-10, which result in impaired development and function of many sensory neurons in C. elegans. In agreement with existing genetic data, the expression of genes regulated by the transcription factor DAF-16/FOXO was affected by daf-10 mutations. In addition, we found altered expression of transcriptional targets of the DAF-12/nuclear hormone receptor in the daf-10 mutants and showed that this pathway influences specifically the dauer formation phenotype of these animals. Unexpectedly, pathogen-responsive genes were repressed in daf-10 mutant animals, and these sensory mutants exhibited altered susceptibility to and behavioral avoidance of bacterial pathogens. Moreover, we found that a solute transporter gene mct-1/2, which was induced by daf-10 mutations, was necessary and sufficient for longevity. Thus, sensory input seems to influence an extensive transcriptional network that modulates basic biological processes in C. elegans. This situation is reminiscent of the complex regulation of physiology by the mammalian hypothalamus, which also receives innervations from sensory systems, most notably the visual and olfactory systems.
Author Summary
The senses provide animals with information about their environment, which affects not only their behavior but also their internal state and physiological outputs. How this information is processed is still unclear. In this study, we used mutant C. elegans roundworms that had defective sensory neurons to investigate how changes in sensation alter the expression of genes and regulate physiology, specifically the worms' choice to hibernate during growth and their longevity as fully-grown adults. We showed that defects in sensory neurons change the pattern of gene expression and regulate these outputs through known hormonal pathways, including insulin/IGF-1 and steroid pathways. We also identified a new regulator of longevity, MCT-1, that is predicted to transport small metabolites and hormones in the body. Unexpectedly, we found that sensory impairment altered yet another physiological output, the response to infectious agents. It prevented the worms from avoiding infectious bacteria and reduced the expression of potentially protective factors, but also increased the worms' resistance to infection, suggesting a complex network of responses to environmental stimuli. Understanding how sensory information is relayed in this relatively simple organism may inform our understanding of sensory processing in higher organisms like mammals.
PMCID: PMC3527274  PMID: 23284299
17.  Insulin/IGF-1 and Hypoxia Signaling Act in Concert to Regulate Iron Homeostasis in Caenorhabditis elegans 
PLoS Genetics  2012;8(3):e1002498.
Iron plays an essential role in many biological processes, but also catalyzes the formation of reactive oxygen species (ROS), which can cause molecular damage. Iron homeostasis is therefore a critical determinant of fitness. In Caenorhabditis elegans, insulin/IGF-1 signaling (IIS) promotes growth and reproduction but limits stress resistance and lifespan through inactivation of the DAF-16/FoxO transcription factor (TF). We report that long-lived daf-2 insulin/IGF-1 receptor mutants show a daf-16–dependent increase in expression of ftn-1, which encodes the iron storage protein H-ferritin. To better understand the regulation of iron homeostasis, we performed a TF–limited genetic screen for factors influencing ftn-1 gene expression. The screen identified the heat-shock TF hsf-1, the MAD bHLH TF mdl-1, and the putative histone acetyl transferase ada-2 as activators of ftn-1 expression. It also revealed that the HIFα homolog hif-1 and its binding partner aha-1 (HIFβ) are potent repressors of ftn-1 expression. ftn-1 expression is induced by exposure to iron, and we found that hif-1 was required for this induction. In addition, we found that the prolyl hydroxylase EGL-9, which represses HIF-1 via the von Hippel-Lindau tumor suppressor VHL-1, can also act antagonistically to VHL-1 in regulating ftn-1. This suggests a novel mechanism for HIF target gene regulation by these evolutionarily conserved and clinically important hydroxylases. Our findings imply that the IIS and HIF pathways act together to regulate iron homeostasis in C. elegans. We suggest that IIS/DAF-16 regulation of ftn-1 modulates a trade-off between growth and stress resistance, as elevated iron availability supports growth but also increases ROS production.
Author Summary
Iron plays a role in many biological processes, including energy generation and DNA replication. But to maintain health, levels of cellular iron must be just right: too much or too little iron can cause illnesses, such as anemia and hemochromatosis, respectively. Animals therefore carefully control their iron levels by regulating of iron uptake, transport, and storage within protein capsules called ferritins. But how do they coordinate this? Using the model organism C. elegans, we have discovered a network of genes and pathways that control iron homeostasis. We find that ferritin is regulated by insulin/IGF-1 signaling, which also controls growth and resistance to oxidative stress in response to harsh environmental conditions. Ferritin is also regulated by the hypoxia signaling pathway, which responds to oxygen and iron levels as well as to metabolic cues. We find that the hypoxia pathway acts as an iron sensor, a role it may also play in humans. Our work defines a network of signaling pathways that can adjust iron availability in response to a range of environmental cues. Understanding this network in C. elegans can help us to understand the causes of iron dyshomeostasis in humans, which can profoundly affect health.
PMCID: PMC3291539  PMID: 22396654
18.  C. elegans SIRT6/7 Homolog SIR-2.4 Promotes DAF-16 Relocalization and Function during Stress 
PLoS Genetics  2012;8(9):e1002948.
FoxO transcription factors and sirtuin family deacetylases regulate diverse biological processes, including stress responses and longevity. Here we show that the Caenorhabditis elegans sirtuin SIR-2.4—homolog of mammalian SIRT6 and SIRT7 proteins—promotes DAF-16–dependent transcription and stress-induced DAF-16 nuclear localization. SIR-2.4 is required for resistance to multiple stressors: heat shock, oxidative insult, and proteotoxicity. By contrast, SIR-2.4 is largely dispensable for DAF-16 nuclear localization and function in response to reduced insulin/IGF-1-like signaling. Although acetylation is known to regulate localization and activity of mammalian FoxO proteins, this modification has not been previously described on DAF-16. We find that DAF-16 is hyperacetylated in sir-2.4 mutants. Conversely, DAF-16 is acetylated by the acetyltransferase CBP-1, and DAF-16 is hypoacetylated and constitutively nuclear in response to cbp-1 inhibition. Surprisingly, a SIR-2.4 catalytic mutant efficiently rescues the DAF-16 localization defect in sir-2.4 null animals. Acetylation of DAF-16 by CBP-1 in vitro is inhibited by either wild-type or mutant SIR-2.4, suggesting that SIR-2.4 regulates DAF-16 acetylation indirectly, by preventing CBP-1-mediated acetylation under stress conditions. Taken together, our results identify SIR-2.4 as a critical regulator of DAF-16 specifically in the context of stress responses. Furthermore, they reveal a novel role for acetylation, modulated by the antagonistic activities of CBP-1 and SIR-2.4, in modulating DAF-16 localization and function.
Author Summary
Sensing and responding appropriately to environmental insults is a challenge facing all organisms. In the roundworm C. elegans, the FoxO protein DAF-16 moves to the nucleus in response to stress, where it regulates gene expression and plays a key role in ensuring organismal survival. In this manuscript, we characterize SIR-2.4 as a novel factor that promotes DAF-16 function during stress. SIR-2.4 is a member of a family of proteins called sirtuins, some of which promote increased lifespan in model organisms. Worms lacking SIR-2.4 show impaired DAF-16 nuclear recruitment, DAF-16–dependent gene expression, and survival in response to a variety of stressors. SIR-2.4 regulates DAF-16 by indirectly affecting levels of a modification called acetylation on DAF-16. Overall, our work has revealed SIR-2.4 to be a key new factor in stress resistance and DAF-16 regulation in C. elegans. Future studies will address whether mammalian SIR-2.4 homologs SIRT6 and SIRT7 act similarly towards mammalian FoxO proteins.
PMCID: PMC3441721  PMID: 23028355
19.  Germline Signals Deploy NHR-49 to Modulate Fatty-Acid β-Oxidation and Desaturation in Somatic Tissues of C. elegans 
PLoS Genetics  2014;10(12):e1004829.
In C. elegans, removal of the germline extends lifespan significantly. We demonstrate that the nuclear hormone receptor, NHR-49, enables the response to this physiological change by increasing the expression of genes involved in mitochondrial β-oxidation and fatty-acid desaturation. The coordinated augmentation of these processes is critical for germline-less animals to maintain their lipid stores and to sustain de novo fat synthesis during adulthood. Following germline ablation, NHR-49 is up-regulated in somatic cells by the conserved longevity determinants DAF-16/FOXO and TCER-1/TCERG1. Accordingly, NHR-49 overexpression in fertile animals extends their lifespan modestly. In fertile adults, nhr-49 expression is DAF-16/FOXO and TCER-1/TCERG1 independent although its depletion causes age-related lipid abnormalities. Our data provide molecular insights into how reproductive stimuli are integrated into global metabolic changes to alter the lifespan of the animal. They suggest that NHR-49 may facilitate the adaptation to loss of reproductive potential through synchronized enhancement of fatty-acid oxidation and desaturation, thus breaking down some fats ordained for reproduction and orchestrating a lipid profile conducive for somatic maintenance and longevity.
Author Summary
Much is known about how increasing age impairs fertility but we know little about how reproduction influences rate of aging in animals. Studies in model organisms such as worms and flies have begun to shed light on this relationship. In worms, removing germ cells that give rise to sperm and oocytes extends lifespan, increases endurance and elevates fat. Fat metabolism and hormonal signals play major roles in this lifespan augmentation but the genetic mechanisms involved are poorly understood. We show that a gene, nhr-49, enhances worm lifespan following germ-cell removal. NHR-49 is increased in animals that lack germ cells by conserved longevity proteins, DAF-16 and TCER-1. NHR-49, in turn, increases levels of genes that help burn fat and convert saturated fats into unsaturated forms. Through synchronized enhancement of these processes, NHR-49 helps eliminate excess fat delegated for reproduction and converts lipids into forms that favor a long life. NHR-49 impacts these processes during aging in normal animals too, but using different regulatory mechanisms. Our data helps understand how normal lipid metabolic processes can be harnessed to adapt to physiological fluctuations brought on by changes in the reproductive status of animals.
PMCID: PMC4256272  PMID: 25474470
20.  DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO 
Insulin/IGF-1 signaling controls metabolism, stress resistance and aging in Caenorhabditis elegans by regulating the activity of the DAF-16/FoxO transcription factor (TF). However, the function of DAF-16 and the topology of the transcriptional network that it crowns remain unclear. Using chromatin profiling by DNA adenine methyltransferase identification (DamID), we identified 907 genes that are bound by DAF-16. These were enriched for genes showing DAF-16-dependent upregulation in long-lived daf-2 insulin/IGF-1 receptor mutants (P=1.4e−11). Cross-referencing DAF-16 targets with these upregulated genes (daf-2 versus daf-16; daf-2) identified 65 genes that were DAF-16 regulatory targets. These 65 were enriched for signaling genes, including known determinants of longevity, but not for genes specifying somatic maintenance functions (e.g. detoxification, repair). This suggests that DAF-16 acts within a relatively small transcriptional subnetwork activating (but not suppressing) other regulators of stress resistance and aging, rather than directly regulating terminal effectors of longevity. For most genes bound by DAF-16∷DAM, transcriptional regulation by DAF-16 was not detected, perhaps reflecting transcriptionally non-functional TF ‘parking sites'. This study demonstrates the efficacy of DamID for chromatin profiling in C. elegans.
PMCID: PMC2950082  PMID: 20706209
aging; C. elegans; DAF-16/FoxO; DamID chromatin profiling; transcriptional networks
21.  The DAF-16 FOXO Transcription Factor Regulates natc-1 to Modulate Stress Resistance in Caenorhabditis elegans, Linking Insulin/IGF-1 Signaling to Protein N-Terminal Acetylation 
PLoS Genetics  2014;10(10):e1004703.
The insulin/IGF-1 signaling pathway plays a critical role in stress resistance and longevity, but the mechanisms are not fully characterized. To identify genes that mediate stress resistance, we screened for C. elegans mutants that can tolerate high levels of dietary zinc. We identified natc-1, which encodes an evolutionarily conserved subunit of the N-terminal acetyltransferase C (NAT) complex. N-terminal acetylation is a widespread modification of eukaryotic proteins; however, relatively little is known about the biological functions of NATs. We demonstrated that loss-of-function mutations in natc-1 cause resistance to a broad-spectrum of physiologic stressors, including multiple metals, heat, and oxidation. The C. elegans FOXO transcription factor DAF-16 is a critical target of the insulin/IGF-1 signaling pathway that mediates stress resistance, and DAF-16 is predicted to directly bind the natc-1 promoter. To characterize the regulation of natc-1 by DAF-16 and the function of natc-1 in insulin/IGF-1 signaling, we analyzed molecular and genetic interactions with key components of the insulin/IGF-1 pathway. natc-1 mRNA levels were repressed by DAF-16 activity, indicating natc-1 is a physiological target of DAF-16. Genetic studies suggested that natc-1 functions downstream of daf-16 to mediate stress resistance and dauer formation. Based on these findings, we hypothesize that natc-1 is directly regulated by the DAF-16 transcription factor, and natc-1 is a physiologically significant effector of the insulin/IGF-1 signaling pathway that mediates stress resistance and dauer formation. These studies identify a novel biological function for natc-1 as a modulator of stress resistance and dauer formation and define a functionally significant downstream effector of the insulin/IGF-1 signaling pathway. Protein N-terminal acetylation mediated by the NatC complex may play an evolutionarily conserved role in regulating stress resistance.
Author Summary
What are the mechanisms used by animals to cope with stressful environments that inflict damage or restrict essential processes such as growth, development, and reproduction? One strategy is changes in physiology that increase stress resistance, and an extreme version of this strategy is diapause, an alternative developmental state that is enduring and stress resistant. In the nematode C. elegans, stress tolerance and entry into a diapause state called dauer larvae are mediated by the conserved insulin/IGF-1 pathway. Specifically, the FOXO transcription factor DAF-16 promotes stress tolerance and dauer larvae development. However, the targets of DAF-16 that mediate these processes remain largely elusive. Using an unbiased forward genetic screen to discover new mediators of stress tolerance, we identified natc-1, a novel target of DAF-16 and the insulin/IGF-1 pathway. natc-1 encodes a conserved subunit of the N-terminal acetyltransferase C (NAT) complex. The NatC complex modifies target proteins by acetylating the N-terminus. We demonstrated that natc-1 mediates diapause entry and stress tolerance. Furthermore, we elucidated regulation of NatC by demonstrating that natc-1 is a direct transcriptional target that is repressed by DAF-16. These findings may be relevant to other animals because both the insulin/IGF-1 signaling pathway and the NAT system are conserved during evolution.
PMCID: PMC4199503  PMID: 25330323
22.  Roles of the Developmental Regulator unc-62/Homothorax in Limiting Longevity in Caenorhabditis elegans 
PLoS Genetics  2013;9(2):e1003325.
The normal aging process is associated with stereotyped changes in gene expression, but the regulators responsible for these age-dependent changes are poorly understood. Using a novel genomics approach, we identified HOX co-factor unc-62 (Homothorax) as a developmental regulator that binds proximal to age-regulated genes and modulates lifespan. Although unc-62 is expressed in diverse tissues, its functions in the intestine play a particularly important role in modulating lifespan, as intestine-specific knockdown of unc-62 by RNAi increases lifespan. An alternatively-spliced, tissue-specific isoform of unc-62 is expressed exclusively in the intestine and declines with age. Through analysis of the downstream consequences of unc-62 knockdown, we identify multiple effects linked to aging. First, unc-62 RNAi decreases the expression of yolk proteins (vitellogenins) that aggregate in the body cavity in old age. Second, unc-62 RNAi results in a broad increase in expression of intestinal genes that typically decrease expression with age, suggesting that unc-62 activity balances intestinal resource allocation between yolk protein expression and fertility on the one hand and somatic functions on the other. Finally, in old age, the intestine shows increased expression of several aberrant genes; these UNC-62 targets are expressed predominantly in neuronal cells in developing animals, but surprisingly show increased expression in the intestine of old animals. Intestinal expression of some of these genes during aging is detrimental for longevity; notably, increased expression of insulin ins-7 limits lifespan by repressing activity of insulin pathway response factor DAF-16/FOXO in aged animals. These results illustrate how unc-62 regulation of intestinal gene expression is responsible for limiting lifespan during the normal aging process.
Author Summary
The normal aging process in Caenorhabditis elegans involves coordinated changes in expression of about a thousand genes. In order to find upstream regulators responsible for these aging genes, we used a genomics approach to screen for transcription factors that bind to them. We focused on one such regulator, unc-62/Homothorax, that encodes a co-factor for a Hox transcription factor. Although essential for development, expression of unc-62 in adults limits lifespan. We find that unc-62 is a transcriptional activator of yolk protein genes, which are synthesized in the intestine and encode essential nutrients for progeny but accumulate to toxic levels with age. Additionally, analysis of unc-62 knockdown indicates that when vitellogenin transcription is decreased, transcription of intestinal genes with somatic functions increases. Thus, activation of yolk protein gene expression by unc-62 is both a burden on maternal health and a necessary resource for embryonic growth. Surprisingly, we also found that the intestine of old worms has novel expression of non-intestinal proteins that are toxic when expressed in the intestine and are activated by unc-62 in old age. Our work on transcriptional regulation of intrinsic gene expression differences during normal aging has revealed insights into the mechanisms that limit lifespan.
PMCID: PMC3585033  PMID: 23468654
23.  TATN-1 Mutations Reveal a Novel Role for Tyrosine as a Metabolic Signal That Influences Developmental Decisions and Longevity in Caenorhabditis elegans 
PLoS Genetics  2013;9(12):e1004020.
Recent work has identified changes in the metabolism of the aromatic amino acid tyrosine as a risk factor for diabetes and a contributor to the development of liver cancer. While these findings could suggest a role for tyrosine as a direct regulator of the behavior of cells and tissues, evidence for this model is currently lacking. Through the use of RNAi and genetic mutants, we identify tatn-1, which is the worm ortholog of tyrosine aminotransferase and catalyzes the first step of the conserved tyrosine degradation pathway, as a novel regulator of the dauer decision and modulator of the daf-2 insulin/IGF-1-like (IGFR) signaling pathway in Caenorhabditis elegans. Mutations affecting tatn-1 elevate tyrosine levels in the animal, and enhance the effects of mutations in genes that lie within the daf-2/insulin signaling pathway or are otherwise upstream of daf-16/FOXO on both dauer formation and worm longevity. These effects are mediated by elevated tyrosine levels as supplemental dietary tyrosine mimics the phenotypes produced by a tatn-1 mutation, and the effects still occur when the enzymes needed to convert tyrosine into catecholamine neurotransmitters are missing. The effects on dauer formation and lifespan require the aak-2/AMPK gene, and tatn-1 mutations increase phospho-AAK-2 levels. In contrast, the daf-16/FOXO transcription factor is only partially required for the effects on dauer formation and not required for increased longevity. We also find that the controlled metabolism of tyrosine by tatn-1 may function normally in dauer formation because the expression of the TATN-1 protein is regulated both by daf-2/IGFR signaling and also by the same dietary and environmental cues which influence dauer formation. Our findings point to a novel role for tyrosine as a developmental regulator and modulator of longevity, and support a model where elevated tyrosine levels play a causal role in the development of diabetes and cancer in people.
Author Summary
In people, elevated blood levels of the amino acid tyrosine are seen in obese individuals, and these elevations represent a novel risk factor for the development of diabetes. The enzyme tyrosine aminotransferase, which removes tyrosine from the body, has also been identified as a tumor suppressor gene, and this enzyme normally acts to prevent the development of liver cancer. In our work, we identify tyrosine aminotransferase as a regulator of larval development and adult longevity in the non-parasitic worm Caenorhabditis elegans. Worms with mutations impairing tyrosine aminotransferase activity show elevated levels of tyrosine, are prone to arresting development in a larval stage called a dauer, and show increased longevity. Part of the effect of tyrosine aminotransferase is due to inhibitory effects on an insulin-like signaling pathway in the worms. Our work suggests that levels of the amino acid tyrosine are sensed and can lead to changes in cell signaling. These results may provide insights into how tyrosine could be involved in obesity, diabetes, and cancer in people.
PMCID: PMC3868569  PMID: 24385923
24.  Unexpected Role for Dosage Compensation in the Control of Dauer Arrest, Insulin-Like Signaling, and FoxO Transcription Factor Activity in Caenorhabditis elegans 
Genetics  2013;194(3):619-629.
During embryogenesis, an essential process known as dosage compensation is initiated to equalize gene expression from sex chromosomes. Although much is known about how dosage compensation is established, the consequences of modulating the stability of dosage compensation postembryonically are not known. Here we define a role for the Caenorhabditis elegans dosage compensation complex (DCC) in the regulation of DAF-2 insulin-like signaling. In a screen for dauer regulatory genes that control the activity of the FoxO transcription factor DAF-16, we isolated three mutant alleles of dpy-21, which encodes a conserved DCC component. Knockdown of multiple DCC components in hermaphrodite and male animals indicates that the dauer suppression phenotype of dpy-21 mutants is due to a defect in dosage compensation per se. In dpy-21 mutants, expression of several X-linked genes that promote dauer bypass is elevated, including four genes encoding components of the DAF-2 insulin-like pathway that antagonize DAF-16/FoxO activity. Accordingly, dpy-21 mutation reduced the expression of DAF-16/FoxO target genes by promoting the exclusion of DAF-16/FoxO from nuclei. Thus, dosage compensation enhances dauer arrest by repressing X-linked genes that promote reproductive development through the inhibition of DAF-16/FoxO nuclear translocation. This work is the first to establish a specific postembryonic function for dosage compensation in any organism. The influence of dosage compensation on dauer arrest, a larval developmental fate governed by the integration of multiple environmental inputs and signaling outputs, suggests that the dosage compensation machinery may respond to external cues by modulating signaling pathways through chromosome-wide regulation of gene expression.
PMCID: PMC3697968  PMID: 23733789
Caenorhabditis elegans; dosage compensation; dauer; insulin signaling; DAF-16/FoxO
25.  Pseudomonas aeruginosa Suppresses Host Immunity by Activating the DAF-2 Insulin-Like Signaling Pathway in Caenorhabditis elegans 
PLoS Pathogens  2008;4(10):e1000175.
Some pathogens have evolved mechanisms to overcome host immune defenses by inhibiting host defense signaling pathways and suppressing the expression of host defense effectors. We present evidence that Pseudomonas aeruginosa is able to suppress the expression of a subset of immune defense genes in the animal host Caenorhabditis elegans by activating the DAF-2/DAF-16 insulin-like signaling pathway. The DAF-2/DAF-16 pathway is important for the regulation of many aspects of organismal physiology, including metabolism, stress response, longevity, and immune function. We show that intestinal expression of DAF-16 is required for resistance to P. aeruginosa and that the suppression of immune defense genes is dependent on the insulin-like receptor DAF-2 and the FOXO transcription factor DAF-16. By visualizing the subcellular localization of DAF-16::GFP fusion protein in live animals during infection, we show that P. aeruginosa–mediated downregulation of a subset of immune genes is associated with the ability to translocate DAF-16 from the nuclei of intestinal cells. Suppression of DAF-16 is mediated by an insulin-like peptide, INS-7, which functions upstream of DAF-2. Both the inhibition of DAF-16 and downregulation of DAF-16–regulated genes, such as thn-2, lys-7, and spp-1, require the P. aeruginosa two-component response regulator GacA and the quorum-sensing regulators LasR and RhlR and are not observed during infection with Salmonella typhimurium or Enterococcus faecalis. Our results reveal a new mechanism by which P. aeruginosa suppresses host immune defense.
Author Summary
Bacterial pathogens have evolved mechanisms to overcome the immune defenses that animals and plants deploy against them. In some cases, this involves directly interfering with the proper functioning of the immune system. Because pathogens that employ these strategies are often the most deadly and difficult to treat, it is important to understand how they are able to suppress the immune system in the context of the whole organism. In this paper, we show that Pseudomonas aeruginosa, a bacterial pathogen that is a major contributor to hospital-borne infections such as pneumonia, suppresses an immune defense pathway during infection of the simple animal host Caenorhabditis elegans. Using genetic modifications of both the pathogen and host, we identify components of the signaling pathways required to suppress host immune defenses. We find that P. aeruginosa employs the cell-to-cell communication system known as quorum sensing, which coordinates the expression of virulence factors to suppress host immune defense. In the host, an evolutionarily conserved insulin-like signaling pathway is affected by P. aeruginosa, resulting in the suppression of genes that are required for defense against infection in the intestinal epithelial cells. These findings suggest the possibility that P. aeruginosa may exploit similar mechanisms when causing infections of human epithelium, such as the epithelial lining of the lungs.
PMCID: PMC2568960  PMID: 18927620

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