Innate immune defense relies on post-translational modifications (PTMs) to protect against viral infections. SUMOylation plays complex roles in viral replication and antiviral defenses in mammals and has been implicated in age-associated diseases. Whether PTMs and SUMOylation contribute to age-induced immunosenescence is unknown. We find that antiviral defense in Caenorhabditis elegans is regulated through SUMOylation of DRH-1, ortholog of the cytosolic pattern recognition receptor RIG-I. The SUMO isopeptidase ULP-4 is essential for deSUMOylation of DRH-1 and activation of the intracellular pathogen response (IPR) after exposure to Orsay virus (OV). ULP-4 stabilizes DRH-1, which translocates to the mitochondria to activate the IPR. Loss of drh-1 or ulp-4 compromises antiviral defense; mutant animals fail to clear OV and develop intestinal pathogenesis. During aging, ulp-4 expression decreases, which promotes DRH-1 proteosomal degradation and IPR loss. Mutating the DRH-1 SUMOylated lysines partially rescued the age-associated lost inducibility of the IPR. Our work establishes that aging results in dysregulated SUMOylation and loss of DRH-1, which compromises antiviral defense and creates a physiological shift to favor chronic pathological infection in older animals.

Innate host defense mechanisms require posttranslational modifications (PTM) to protect against viral infection. Age-associated immunosenescence results in increased pathogenesis and mortality in the elderly, but the contribution of altered PTM regulation to immunosenescence is unknown. SUMOylation is a rapid and reversible post-translational modification that has been implicated in age-associated disease and plays conflicting roles in viral replication and antiviral defenses in mammals. We have discovered in Caenorhabditis elegans that induction of antiviral defense is regulated through SUMOylation of DRH-1, the ortholog of the DEAD/H-box helicase and cytosolic pattern recognition receptor RIG-I, and that this regulation breaks down during aging. We find the SUMO isopeptidase ULP-4 is essential for deSUMOylation of DRH-1 and activation of the intracellular pathogen response (IPR) after exposure to Orsay virus (OV), a natural enteric C. elegans pathogen. ULP-4 promotes stabilization of DRH-1, which translocates to the mitochondria to activate the IPR in young animals exposed to virus. Loss of either drh-1 or ulp-4 compromises antiviral defense resulting in a failure to clear the virus and signs of intestinal pathogenesis. During aging, expression of ulp-4 decreases, which results in increased proteosomal degradation of DRH-1 and loss of the IPR. Mutating the DRH-1 SUMOylated lysines resulted in the constitutive activation of the IPR in young animals and partially rescued the age-associated lost inducibility of the IPR. Our work establishes that aging results in dysregulated SUMOylation and loss of DRH-1, which compromises antiviral defense and creates a physiological shift to favor chronic pathological infection in older animals.

Dietary restriction (DR), the process of decreasing overall food consumption over an extended period of time, has been shown to increase longevity across evolutionarily diverse species and delay the onset of age-associated diseases in humans. In Caenorhabditis elegans, the Myc-family transcription factors (TFs) MXL-2 (Mlx) and MML-1 (MondoA/ChREBP), which function as obligate heterodimers, and PHA-4 (orthologous to FOXA) are both necessary for the full physiological benefits of DR. However, the adaptive transcriptional response to DR and the role of MML-1::MXL-2 and PHA-4 remains elusive. We identified the transcriptional signature of C. elegans DR, using the eat-2 genetic model, and demonstrate broad changes in metabolic gene expression in eat-2 DR animals, which requires both mxl-2 and pha-4. While the requirement for these factors in DR gene expression overlaps, we found many of the DR genes exhibit an opposing change in relative gene expression in eat-2;mxl-2 animals compared to wild-type, which was not observed in eat-2 animals with pha-4 loss. Surprisingly, we discovered more than 2000 genes synthetically dysregulated in eat-2;mxl-2, out of which the promoters of down-regulated genes were substantially enriched for PQM-1 and ELT-1/3 GATA TF binding motifs. We further show functional deficiencies of the mxl-2 loss in DR outside of lifespan, as eat-2;mxl-2 animals exhibit substantially smaller brood sizes and lay a proportion of dead eggs, indicating that MML-1::MXL-2 has a role in maintaining the balance between resource allocation to the soma and to reproduction under conditions of chronic food scarcity. While eat-2 animals do not show a significantly different metabolic rate compared to wild-type, we also find that loss of mxl-2 in DR does not affect the rate of oxygen consumption in young animals. The gene expression signature of eat-2 mutant animals is consistent with optimization of energy utilization and resource allocation, rather than induction of canonical gene expression changes associated with acute metabolic stress, such as induction of autophagy after TORC1 inhibition. Consistently, eat-2 animals are not substantially resistant to stress, providing further support to the idea that chronic DR may benefit healthspan and lifespan through efficient use of limited resources rather than broad upregulation of stress responses, and also indicates that MML-1::MXL-2 and PHA-4 may have distinct roles in promotion of benefits in response to different pro-longevity stimuli.

Aging and the age-associated decline of the proteome is determined in part through neuronal control of evolutionarily conserved transcriptional effectors, which safeguard homeostasis under fluctuating metabolic and stress conditions by regulating an expansive proteostatic network. We have discovered the Caenorhabditis elegans h omeodomain-interacting p rotein k inase (HPK-1) acts as a key transcriptional effector to preserve neuronal integrity, function, and proteostasis during aging. Loss of hpk-1 results in drastic dysregulation in expression of neuronal genes, including genes associated with neuronal aging. During normal aging hpk-1 expression increases throughout the nervous system more broadly than any other kinase. Within the aging nervous system, hpk-1 induction overlaps with key longevity transcription factors, which suggests hpk-1 expression mitigates natural age-associated physiological decline. Consistently, pan-neuronal overexpression of hpk-1 extends longevity, preserves proteostasis both within and outside of the nervous system, and improves stress resistance. Neuronal HPK-1 improves proteostasis through kinase activity. HPK-1 functions cell non-autonomously within serotonergic and GABAergic neurons to improve proteostasis in distal tissues by specifically regulating distinct components of the proteostatic network. Increased serotonergic HPK-1 enhances the heat shock response and survival to acute stress. In contrast, GABAergic HPK-1 induces basal autophagy and extends longevity, which requires mxl-2 (MLX), hlh-30 (TFEB), and daf-16 (FOXO). Our work establishes hpk-1 as a key neuronal transcriptional regulator critical for preservation of neuronal function during aging. Further, these data provide novel insight as to how the nervous system partitions acute and chronic adaptive response pathways to delay aging by maintaining organismal homeostasis.

The nervous system systemically coordinates proteostasis to delay organismal aging. However, the neuronal regulatory mechanisms that coordinate cellular anti-aging programs across tissue and cell-types are relatively unknown. In this work, we identify the h omeodomain-interacting p rotein ki nase (HPK-1), a transcriptional cofactor, as a novel neuronal component of the proteostatic network: its overexpression produces a paracrine signal to hyper-induce molecular chaperones and a neuroendocrine signal to induce autophagy in peripheral tissues. Neuronal HPK-1 signaling improves proteostasis in distal tissues through neurotransmitters. These pro-longevity modalities are independently regulated within serotonergic and GABAergic neurons, respectively, through distinct adaptive responses, either of which improve proteostasis in a cell non-autonomous manner. Serotonergic HPK-1 activity amplifies the heat shock response and protects the proteome from acute stress, without altering longevity. Conversely, increased GABAergic HPK-1 activity is sufficient to induce autophagy and extend longevity, without altering acute stress survival. Consistently, GABAergic neurons, but not serotonin, is essential for the cell non-autonomous induction of autophagy by neuronal HPK-1. These findings provide novel insight into how the nervous system partitions and coordinates unique adaptive response pathways to delay organismal aging, and reveals a key role for neuronal HPK-1 in regulating the proteostatic network throughout an intact metazoan animal. Significance Statement Aging and the age-associated decline of the proteome is determined in part through neuronal control of evolutionarily conserved transcriptional effectors, which safeguard homeostasis under fluctuating metabolic and stress conditions by regulating an expansive proteostatic network in peripheral tissues. How neuronal signaling mechanisms are primed, relayed through an organism, and specific responses are initiated in receiving cell types remain poorly understood. We have discovered that the Caenorhabditis elegans homeodomain-interacting protein kinase (HPK-1) is a novel transcriptional effector that functions within two distinct neuronal cell-types to non-autonomously regulate divergent components of the proteostatic network to enhance stress resistance, improve proteostasis and delay aging.

Discoveries made in the nematode Caenorhabditis elegans revealed that aging is under genetic control. Since these transformative initial studies, C. elegans has become a premier model system for aging research. Critically, the genes, pathways, and processes that have fundamental roles in organismal aging are deeply conserved throughout evolution. This conservation has led to a wealth of knowledge regarding both the processes that influence aging and the identification of molecular and cellular hallmarks that play a causative role in the physiological decline of organisms. One key feature of age-associated decline is the failure of mechanisms that maintain proper function of the proteome (proteostasis). Here we highlight components of the proteostatic network that act to maintain the proteome and how this network integrates into major longevity signaling pathways. We focus in depth on the heat shock transcription factor 1 (HSF1), the central regulator of gene expression for proteins that maintain the cytosolic and nuclear proteomes, and a key effector of longevity signals.

The advent of feeding based RNAi in Caenorhabditis elegans led to an era of gene discovery in aging research. Hundreds of gerogenes were discovered, and many are evolutionarily conserved, raising the exciting possibility that the underlying genetic basis for healthy aging in higher vertebrates could be quickly deciphered. Yet, the majority of putative gerogenes have still only been cursorily characterized, highlighting the need for high-throughput, quantitative assessments of changes in aging. A widely used surrogate measure of aging is lifespan. The traditional way to measure mortality in C. elegans tracks the deaths of individual animals over time within a relatively small population. This traditional method provides straightforward, direct measurements of median and maximum lifespan for the sampled population. However, this method is time consuming, often underpowered, and involves repeated handling of a set of animals over time, which in turn can introduce contamination or possibly damage increasingly fragile, aged animals. We have previously developed an alternative "Replica Set" methodology, which minimizes handling and increases throughput by at least an order of magnitude. The Replica Set method allows changes in lifespan to be measured for over one hundred feeding-based RNAi clones by one investigator in a single experiment- facilitating the generation of large quantitative phenotypic datasets, a prerequisite for development of biological models at a systems level. Here, we demonstrate through analysis of lifespan experiments simulated in silico that the Replica Set method is at least as precise and accurate as the traditional method in evaluating and estimating lifespan, and requires many fewer total animal observations across the course of an experiment. Furthermore, we show that the traditional approach to lifespan experiments is more vulnerable than the Replica Set method to experimental and measurement error. We find no compromise in statistical power for Replica Set experiments, even for moderate effect sizes, or when simulated experimental errors are introduced. We compare and contrast the statistical analysis of data generated by the two approaches, and highlight pitfalls common with the traditional methodology. Collectively, our analysis provides a standard of measure for each method across comparable parameters, which will be invaluable in both experimental design and evaluation of published data for lifespan studies.

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington's disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Lifespan is the most straightforward surrogate measure of aging, as it is easily quantifiable. A common approach to measure Caenorhabditis elegans lifespan is to follow a population of animals over time and score viability based on movement. We previously developed an alternative approach, called the Replica Set method, to quantitatively measure lifespan of C. elegans in a high-throughput manner. The replica set method allows a single investigator to screen more treatments or conditions in the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. Unlike traditional approaches, the Replica Set method centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with "traditional" longitudinal methods. The protocols provided here describe both the traditional experimental approach and the Replica Set method, as well as practical considerations for each.

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods. Scoring entails adding liquid to the wells of a multi-well plate, which stimulates C. elegans to move and facilitates quantifying changes in healthspan. Other major benefits of the Replica Set method include reduced exposure of agar surfaces to airborne contaminants (e.g. mold or fungus), minimal handling of animals, and robustness to sporadic mis-scoring (such as calling an animal as dead when it is still alive). To appropriately analyze and visualize the data from a Replica Set style experiment, a custom software tool was also developed. Current capabilities of the software include plotting of survival curves for both Replica Set and traditional (Kaplan-Meier) experiments, as well as statistical analysis for Replica Set. The protocols provided here describe the traditional experimental approach and the Replica Set method, as well as an overview of the corresponding data analysis.

An extensive proteostatic network comprised of molecular chaperones and protein clearance mechanisms functions collectively to preserve the integrity and resiliency of the proteome. The efficacy of this network deteriorates during aging, coinciding with many clinical manifestations, including protein aggregation diseases of the nervous system. A decline in proteostasis can be delayed through the activation of cytoprotective transcriptional responses, which are sensitive to environmental stress and internal metabolic and physiological cues. The homeodomain-interacting protein kinase (hipk) family members are conserved transcriptional co-factors that have been implicated in both genotoxic and metabolic stress responses from yeast to mammals. We demonstrate that constitutive expression of the sole Caenorhabditis elegans Hipk homolog, hpk-1, is sufficient to delay aging, preserve proteostasis, and promote stress resistance, while loss of hpk-1 is deleterious to these phenotypes. We show that HPK-1 preserves proteostasis and extends longevity through distinct but complementary genetic pathways defined by the heat shock transcription factor (HSF-1), and the target of rapamycin complex 1 (TORC1). We demonstrate that HPK-1 antagonizes sumoylation of HSF-1, a post-translational modification associated with reduced transcriptional activity in mammals. We show that inhibition of sumoylation by RNAi enhances HSF-1-dependent transcriptional induction of chaperones in response to heat shock. We find that hpk-1 is required for HSF-1 to induce molecular chaperones after thermal stress and enhances hormetic extension of longevity. We also show that HPK-1 is required in conjunction with HSF-1 for maintenance of proteostasis in the absence of thermal stress, protecting against the formation of polyglutamine (Q35::YFP) protein aggregates and associated locomotory toxicity. These functions of HPK-1/HSF-1 undergo rapid down-regulation once animals reach reproductive maturity. We show that HPK-1 fortifies proteostasis and extends longevity by an additional independent mechanism: induction of autophagy. HPK-1 is necessary for induction of autophagosome formation and autophagy gene expression in response to dietary restriction (DR) or inactivation of TORC1. The autophagy-stimulating transcription factors pha-4/FoxA and mxl-2/Mlx, but not hlh-30/TFEB or the nuclear hormone receptor nhr-62, are necessary for extended longevity resulting from HPK-1 overexpression. HPK-1 expression is itself induced by transcriptional mechanisms after nutritional stress, and post-transcriptional mechanisms in response to thermal stress. Collectively our results position HPK-1 at a central regulatory node upstream of the greater proteostatic network, acting at the transcriptional level by promoting protein folding via chaperone expression, and protein turnover via expression of autophagy genes. HPK-1 therefore provides a promising intervention point for pharmacological agents targeting the protein homeostasis system as a means of preserving robust longevity.

Spns1 (Spinster homolog 1 [Drosophila]) in vertebrates, as well as Spin (Spinster) in Drosophila, is a hypothetical lysosomal H+-carbohydrate transporter, which functions at a late stage of macroautophagy (hereafter autophagy). The Spin/Spns1 defect induces aberrant autolysosome formation that leads to developmental senescence in the embryonic stage and premature aging symptoms in adulthood. However, the molecular mechanism by which loss of Spin/Spns1 leads to the specific pathogenesis remains to be elucidated. Using chemical, genetic and CRISPR/Cas9-mediated genome-editing approaches in zebrafish, we investigated and determined a mechanism that suppresses embryonic senescence as well as autolysosomal impairment mediated by Spns1 deficiency. Unexpectedly, we found that a concurrent disruption of the vacuolar-type H+-ATPase (v-ATPase) subunit gene, atp6v0ca (ATPase, H+ transporting, lysosomal, V0 subunit ca) led to suppression of the senescence induced by the Spns1 defect, whereas the sole loss of Atp6v0ca led to senescent embryos similar to the single spns1 mutation. Moreover, we discovered that the combined stable defect seen in the presence of both the spns1 and atp6v0ca mutant genes still subsequently induced premature autophagosome-lysosome fusion marked by insufficient acidity, while extending developmental life span, compared with the solely mutated spns1 defect. Our data suggest that Spns1 and the v-ATPase orchestrate proper autolysosomal biogenesis with optimal acidification that is critically linked to developmental senescence and survival.

The Myc family of transcription factors regulates a variety of biological processes, including the cell cycle, growth, proliferation, metabolism, and apoptosis. In Caenorhabditis elegans, the "Myc interaction network" consists of two opposing heterodimeric complexes with antagonistic functions in transcriptional control: the Myc-Mondo:Mlx transcriptional activation complex and the Mad:Max transcriptional repression complex. In C. elegans, Mondo, Mlx, Mad, and Max are encoded by mml-1, mxl-2, mdl-1, and mxl-1, respectively. Here we show a similar antagonistic role for the C. elegans Myc-Mondo and Mad complexes in longevity control. Loss of mml-1 or mxl-2 shortens C. elegans lifespan. In contrast, loss of mdl-1 or mxl-1 increases longevity, dependent upon MML-1:MXL-2. The MML-1:MXL-2 and MDL-1:MXL-1 complexes function in both the insulin signaling and dietary restriction pathways. Furthermore, decreased insulin-like/IGF-1 signaling (ILS) or conditions of dietary restriction increase the accumulation of MML-1, consistent with the notion that the Myc family members function as sensors of metabolic status. Additionally, we find that Myc family members are regulated by distinct mechanisms, which would allow for integrated control of gene expression from diverse signals of metabolic status. We compared putative target genes based on ChIP-sequencing data in the modENCODE project and found significant overlap in genomic DNA binding between the major effectors of ILS (DAF-16/FoxO), DR (PHA-4/FoxA), and Myc family (MDL-1/Mad/Mxd) at common target genes, which suggests that diverse signals of metabolic status converge on overlapping transcriptional programs that influence aging. Consistent with this, there is over-enrichment at these common targets for genes that function in lifespan, stress response, and carbohydrate metabolism. Additionally, we find that Myc family members are also involved in stress response and the maintenance of protein homeostasis. Collectively, these findings indicate that Myc family members integrate diverse signals of metabolic status, to coordinate overlapping metabolic and cytoprotective transcriptional programs that determine the progression of aging.

Purpose of reviewThe vertebrate cap'n'collar family transcription factor Nrf2 and its invertebrate homologues SKN-1 (in worms) and CncC (in flies) function as master mediators of antioxidant and detoxification responses and regulators of the cellular redox state. Nrf2 controls gene expression programs that defend various tissues against diverse electrophilic stressors and oxidative insults, thus protecting the organism from disorders that are caused or exacerbated by such stresses. Moreover, studies on model organisms implicate the Nrf2 pathway in the prevention of aging-related diseases and suggest that SKN-1-regulated and CncC-regulated gene expression can promote longevity. These facets of Nrf2 signaling have been thoroughly reviewed. This article discusses another aspect of the Nrf2 pathway's function that has not yet received the same degree of attention, but emerges as a topic of increasing interest and potential clinical impact: its role in metabolic regulation and its interaction with central signaling systems that respond to nutritional inputs. Recent findingsRecent evidence identifies Nrf2 signaling as a mediator of the salutary effects of caloric restriction. Nrf2 signaling also crosstalks with metabolic signaling systems such as the insulin/Akt pathway as well as with the metabolism of lipids. Moreover, Nrf2 has a protective role in models of diabetic nephropathy. SummaryThe emerging role of Nrf2 as an effector of metabolic and longevity signals offers new therapeutic perspectives. The potential impact of pharmacological manipulation of Nrf2 signaling as a strategy for the prevention and treatment of metabolic disease can be envisioned.

The adenovirus E1A oncoprotein renders primary cells sensitive to the induction of apoptosis by diverse stimuli, including many anticancer agents. ElA-expressing cells accumulate p53 protein, and p53 potentiates druginduced apoptosis. To determine how E1A promotes chemosensitivity, a series of E1A mutants were introduced into primary human and mouse fibroblasts using high-titer recombinant retroviruses, allowing analysis of E1A in genetically normal cells outside the context of adenovirus infection. Mutations that disrupted apoptosis and chemosensitivity separated into two complementation groups, which correlated precisely with the ability of E1A to associate with either the p300/CBP or retinoblastoma protein families. Furthermore, ElA mutants incapable of binding RB, pl07, and pl30 conferred chemosensitivity to fibroblasts derived from RBdeficient mice, but not fibroblasts from mice lacking pl07 or pl30. Hence, inactivation of RB, but not pl07 or pl30, is required for chemosensitivity induced by E1A. Finally, the same E1A functions that promote drug-induced apoptosis also induce p53. Together, these data demonstrate that p53 accumulation and chemosensitivity are linked to ElA's oncogenic potential, and identify a strategy to selectively induce apoptosis in RB-deficient tumor cells. Despite the widespread use of cytotoxic agents to treat cancer, the molecular mechanisms underlying drug sensitivity and resistance remain poorly understood. Most anticancer agents induce apoptosis, suggesting that tumor-cell chemosensitivity is influenced by the efficiency with which anticancer agents activate apoptotic programs (1,2). This hypothesis implies that responsive tumors must be more susceptible to apoptosis than normal tissue, and that resistant tumors are unable to efficiently engage apoptotic programs. Tumorigenic mutations can have different effects on apoptosis. For example, activation of the c-myc oncogene enhances apoptosis (3) whereas inactivation of the p53 tumor suppressor gene suppresses cell death (reviewed in ref. 4). These diverse effects suggest that tumor-cell chemosensitivity is determined, in part, by the combined effects of oncogenic mutations on apoptosis (1, 5). Given the varied impact of oncogenic mutations on apoptosis, it is difficult to study the molecular determinants of chemosensitivity in the unknown genetic background of tumor cells. However, cells expressing the adenovirus early region 1A (ElA) oncogene provide a simple model for studying cellular processes that modulate chemosensitivity. E1A promotes apoptosis in nontumorigenic cells (6). As a consequence, ElAexpressing cells become extremely sensitive to toxic agents and readily undergo apoptosis following treatment with anticancer agents (1, 7). E1A can impinge on a variety of other cellular processes, including transcription, differentiation, and tumor necrosis The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424/97/9412094-6S2.00/0 PNAS is available online at http://www.pnas.org. factor cytolysis (reviewed in ref. 8). During adenovirus infection, E1A makes quiescent cells permissive for virus replication by promoting S phase entry (8). Consequently, ElA has oncogenic potential: ElA facilitates the immortalization of primary rodent cells and cooperates with viral (e.g., E1B) or cellular (e.g., oncogenic ras) genes to transform primary cells to a tumorigenic state (9). E1B prevents the apoptosis associated with ElA (6), whereas ElA prevents a senescent-like cell cycle arrest provoked by oncogenic Ras (10). Consequently, these transforming interactions illustrate the compensatory mechanisms normal cells possess to suppress transformation (reviewed in ref. 11). The ElA gene expresses several alternatively spliced transcripts, including the 12S and 13S messages encoding 243 (243R) and 289 (289R) amino acid oncoproteins, respectively (reviewed in ref. 12). The 289R protein contains three regions that are conserved between different adenovirus serotypes, designated conserved regions 1, 2, and 3 (CR1, CR2, CR3). CR3 encodes a domain required for transcriptional activation of other viral genes and is absent in the 243R protein, whereas CR1 and CR2 are present in both ElA proteins and are essential for many ElA activities, including oncogenic transformation (13, 14). ElA 243R associates with a series of cellular proteins, including the retinoblastoma gene product (RB), the RBrelated proteins pl07 and pl30, the p300 and CREB binding protein (CBP) transcriptional coactivators, cyclin A, and certain cyclin-dependent kinases (cdk) (refs. 13 and 14; reviewed in refs. 15 and 16). Because most of these interactions also require residues in CR1 and CR2, the ability of ElA to disrupt the function of these proteins may be crucial for its transforming activities. For example, ElA associates with RB (14, 17) and mutations in either CR1 or CR2 that disrupt this interaction also abolish oncogenicity (13). By binding RB, ElA disrupts RB-E2F heterodimers, thereby relieving repression and promoting transactivation of S phase genes (reviewed in refs. 15 and 16). Mutational inactivation of RB achieves a similar effect; consequently, ElA mimics mutational events that occur in familial retinoblastoma and many sporadic tumors (reviewed in ref. 18). In adenovirus-infected cells, ElA expression appears sufficient for apoptosis (19-21). However, cells tolerate ectopic ElA expression but become extremely prone to apoptosis (1, 7). ElA-expressing cells accumulate p53 protein, and both p53 and Bax—a pro-apoptotic member of the Bcl-2 family— contribute to apoptosis in this setting (1,7,22-25). p53 and Bax are inefficient at inducing apoptosis in normal cells lacking ElA (7); indeed, p53 functions to promote cell-cycle arrest (26). Furthermore, ElA-expressing cells possess a discrete factor, absent in normal cells, that is capable of activating the This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: CBP, CREB-binding protein; RB, retinoblastoma; CR1, -2, -3, conserved regions 1, 2, and 3; MEF, mouse embryonic fibroblast; puro, puromycin; hygro, hygromycin. To whom reprint requests should be addressed at: Cold Spring Harbor Laboratory, 1 Bungtown Road, P.O. Box 100, Cold Spring Harbor, NY 11724. e-mail: lowe@cshl.org.

Insulin-like growth factor signaling and other endocrine axes contribute to stress and longevity regulation in C. elegans and other animals. By genetic and functional genomic analysis, we have identified players in this pathway. Many of these genes are conserved and likely to mediate endocrine regulation of longevity across the animal kingdom.

Adenovirus E1A drives oncogenesis by targeting key regulatory pathways that are critical for cellular growth control. The interaction of E1A with p400 is essential for many E1A activities, but the downstream target of this interaction is unknown. Here, we present evidence that the oncoprotein transcription factor Myc is the target of this interaction. We show that E1A stabilizes Myc protein via p400 and promotes the coassociation of Myc and p400 at Myc target genes, leading to their transcriptional induction. We also show that E1A requires Myc for its ability to activate Myc-dependent gene expression and induce apoptosis, and that forced expression of Myc is sufficient to rescue the activity of an E1A-mutant defective in p400 binding. Together, these findings establish that Myc, via p400, is an essential downstream target of E1A.

Genetic and RNA interference (RNAi) screens for life span regulatory genes have revealed that the daf-2 insulin-like signaling pathway plays a major role in Caenorhabditis elegans longevity. This pathway converges on the DAF-16 transcription factor and may regulate life span by controlling the expression of a large number of genes, including free-radical detoxifying genes, stress resistance genes, and pathogen resistance genes. We conducted a genome-wide RNAi screen to identify genes necessary for the extended life span of daf-2 mutants and identified approximately 200 gene inactivations that shorten daf-2 life span. Some of these gene inactivations dramatically shorten daf-2 mutant life span but less dramatically shorten daf-2; daf-16 mutant or wild-type life span. Molecular and behavioral markers for normal aging and for extended life span in low insulin/IGF1 (insulin-like growth factor 1) signaling were assayed to distinguish accelerated aging from general sickness and to examine age-related phenotypes. Detailed demographic analysis, molecular markers of aging, and insulin signaling mutant test strains were used to filter progeric gene inactivations for specific acceleration of aging. Highly represented in the genes that mediate life span extension in the daf-2 mutant are components of endocytotic trafficking of membrane proteins to lysosomes. These gene inactivations disrupt the increased expression of the DAF-16 downstream gene superoxide dismutase sod-3 in a daf-2 mutant, suggesting trafficking between the insulin-like receptor and DAF-16. The activities of these genes may normally decline during aging.

A systematic genome-wide RNA interference screen was performed in the Caenorhabditis elegans lin-15b;eri-1 strain, which has an enhanced response to double-stranded RNA including the nervous system, to identify life-span regulatory factors. In total, 16,757 genes were examined, revealing 115 gene inactivations that extended life span. A more stringent longitudinal analysis revealed 18 gene inactivations that induced the greatest increase in life span (10-90%), all of which extended life span when inactivated either in eri-1 alone or in a second strain with an enhanced response to double-stranded RNA, eri-3. Most reduced the rate of aging, implying that animals aged more slowly. As was the case in previous studies, genes critical for metabolism caused the greatest extension of longevity. Extension of life span occurs through disparate mechanisms as increased resistance to thermal stress, oxidative damage, and decreased age pigment accumulation analysis of the 18 stronger positives failed to demonstrate a correlation between enhanced stress resistance and decreased lysosomal function. Consistently, aps-3 and lys-10, two genes annotated to have lysosomal functions, extended life span when inactivated without enhancing stress resistance. The results of this study reinforce the importance of metabolism, mitochondrial and lysosomal functions, genomic stability, and stress resistance on animal life-span determination.

The adenovirus E1A oncoprotein promotes proliferation and transformation by binding cellular proteins, including members of the retinoblastoma protein family, the p300/CREB-binding protein transcriptional coactivators, and the p400-TRRAP chromatin-remodeling complex. E1A also promotes apoptosis, in part, by engaging the ARF-p53 tumor suppressor pathway. We show that E1A induces ARF and p53 and promotes apoptosis in normal fibroblasts by physically associating with the retinoblastoma protein and a p400-TRRAP complex and that its interaction with p300 is largely dispensable for these effects. We further show that E1A increases p400 expression and, conversely, that suppression of p400 using stable RNA interference reduces the levels of ARF, p53, and apoptosis in E1A-expressing cells. Therefore, whereas E1A inactivates the retinoblastoma protein, it requires p400 to efficiently promote cell death. These results identify p400 as a regulator of the ARF-p53 pathway and a component of the cellular machinery that couples proliferation to cell death.