Search

SET-26, HCF-1, and HDA-1 are highly conserved chromatin factors with key roles in development and aging. Here we present mechanistic insights into how these factors regulate gene expression and modulate longevity inC. elegans. We show that SET-26 and HCF-1 cooperate to regulate a common set of genes, and both antagonize the histone deacetylase HDA-1 to limit longevity. We propose a model in which SET-26 recruits HCF-1 to chromatin in somatic cells, where they stabilize each other at the promoters of a subset of genes, particularly mitochondrial function genes, and regulate their expression. HDA-1 opposes SET-26 and HCF-1 on the regulation of a subset of their common target genes and in longevity. Our findings suggest that SET-26, HCF-1, and HDA-1 comprise a mechanism to fine-tune gene expression and longevity and likely have important implications for the mechanistic understanding of how these factors function in diverse organisms, particularly in aging biology.

Epigenetic alterations occur as organisms age, and lead to chromatin deterioration, loss of transcriptional silencing and genomic instability. Dysregulated epigenome has been linked to increased susceptibility to age-related disorders. We aim to characterize the age-dependent changes of the epigenome and, in turn, to understand epigenetic processes that drive aging phenotypes. In this study, we focused on the aging-associated changes in the repressive histone marks H3K9me3 and H3K27me3 in C. elegans. We observed redistribution of of both histone marks, but the changes are more significant for H3K9me3. We further found alteration of heterochromatic boundaries in aged somatic tissues. Interestingly, we discovered that the most significant changes reflected H3K9me3-marked regions that are formed during aging, and are absent in developing worms, which we termed “aging-associated repressive domains” (AARDs). These AARDs preferentially occur in genic regions that are marked by high levels of H3K9me2 and H3K36me2 in larval stages. Interestingly, maintenance of high H3K9me2 levels in these regions have been shown to correlate with longer lifespan. Next, we examined whether the changes in repressive histone marks lead to de-silencing of repetitive DNA elements, as reported for several other organisms. We observed increased expression of active repetitive DNA elements but not global re-activation of silent repeats in old worms, likely due to the distributed nature of repetitive elements in the C. elegans genome. Intriguingly, CELE45, a putative short interspersed nuclear elements (SINE), was greatly overexpressed at old age and upon heat stress. SINEs have been suggested to regulate transcription in response to various cellular stresses in mammals, it is likely that CELE45 RNAs also play roles in stress response and aging in C. elegans. Taken together, our study revealed significant and specific age-dependent changes in repressive histone modifications and repetitive elements, providing important insights into aging biology.Author summaryHeterochromatin refers to the portion of the genome that is tightly packed where genes stay silent. Heterochromatin is typically decorated by particular chemical groups called histone modifications, such as trimethylation of lysine 9 or lysine 27 on histone 3 (H3K9me3 or H3K27me3). To understand how the heterochromatin landscape may change from a “youthful” to an “aged” state, we monitored the genome-wide patterns of H3K9me3 and H3K27me3 during aging using the genetic model soil worm C. elegans. We found that while H3K27me3 remained relatively stable with age, H3K9me3 showed profound genome-wide redistribution in aged worms. We observed that new H3K9me3-marked heterochromatin preferentially formed in specific gene-rich regions in aged worms. Interestingly, these particular regions were marked by high levels of three other histone modifications when worms were young. This result suggested that H3K9me3 gain during aging is influenced by the gene-specific landscape of histone modifications established at young age rather than occurs in a stochastic manner. In summary, our study discovered reproducible and gene-specific changes in histone modifications that likely contribute to the aging phenotypes.

Chromatin Immunoprecipitation is a regularly used method to detect DNA–protein interaction in diverse biological samples. Here we describe the application of ChIP for histone modifications in adult-stage Caenorhabditis elegans somatic cells.

Objective More than one-third of U.S. adults have obesity, causing an alarming increase in obesity-related comorbidities such as type 2 diabetes. The functional role of mitochondrial carrier homolog 2 (MTCH2), a human obesity-associated gene, in lipid homeostasis was investigated in Caenorhabditis elegans, cell culture, and mice. Methods In C. elegans, MTCH2/MTCH-1 was depleted, using RNAi and a genetic mutant, and overexpressed to assess its effect on lipid accumulation. In cells and mice, shRNAs against MTCH2 were used for knockdown and MTCH2 overexpression vectors were used for overexpression to study the role of this gene in fat accumulation. Results MTCH2 knockdown reduced lipid accumulation in adipocyte-like cells in vitro and in C. elegans and mice in vivo. MTCH2 overexpression increased fat accumulation in cell culture, C. elegans, and mice. Acute MTCH2 inhibition reduced fat accumulation in animals subjected to a high-fat diet. Finally, MTCH2 influenced estrogen receptor 1 (ESR1) activity. Conclusions MTCH2 is a conserved regulator of lipid homeostasis. MTCH2 was found to be both required and sufficient for lipid homeostasis shifts, suggesting that pharmacological inhibition of MTCH2 could be therapeutic for treatment of obesity and related disorders. MTCH2 could influence lipid homeostasis through inhibition of ESR1 activity.

Tri-methylation on histone H3 lysine 4 (H3K4me3) is associated with active gene expression but its regulatory role in transcriptional activation is unclear. Here we used Caenorhabditis elegans to investigate the connection between H3K4me3 and gene expression regulation during aging. We uncovered around 30% of H3K4me3 enriched regions to show significant and reproducible changes with age. We further showed that these age-dynamic H3K4me3 regions largely mark gene-bodies and are acquired during adult stages. We found that these adult-specific age-dynamic H3K4me3 regions are correlated with gene expression changes with age. In contrast, H3K4me3 marking established during developmental stages remained largely stable with age, even when the H3K4me3 associated genes exhibited RNA expression changes during aging. Importantly, the genes associated with changes in H3K4me3 and RNA levels with age are enriched for functional groups commonly implicated in aging biology. Therefore, our findings suggested divergent roles of H3K4me3 in gene expression regulation during aging, with important implications on aging-dependent pathophysiologies., Author summary Histone modifications, the specific chemical modifications on histone proteins, are key for regulating the packing of DNA, and thus have important influence on diverse biological processes. An intensely studied function of histone modifications is their contribution to regulating gene expression. Recent studies in diverse model organisms demonstrated that the global alterations of particular histone modifications, for instance H3K4me3, extend the lifespan of the organism. However, the underlying molecular mechanisms remain largely unclear. In this study, we monitored whether and how the genome-wide pattern of the histone modification H3K4me3 changes during aging in the somatic cells of the model organism C. elegans. We identified interesting and non-conventional patterns of H3K4me3, which span gene-bodies and are acquired during adulthood, that are particularly prone to changes with aging. This is contrasted to the well-studied H3K4me3 patterns that span transcriptional start sites and 5’ promoter regions and are established early during development, which remain stable with age. Consistent with the close association between H3K4me3 marking and active transcription, we observed that the age-dynamic H3K4me3 markings are highly correlated with corresponding RNA expression changes. Importantly, the genes that are associated with both H3K4me3 and RNA expression changes with age are over-represented for functional groups commonly implicated in aging biology. In summary, our findings revealed a lesser known pattern of H3K4me3 modification that can have important biological roles in aging.

Summary A decline in mitochondrial electron transport chain (ETC) function has long been implicated in aging and various diseases. Recently, moderate mitochondrial ETC dysfunction has been found to prolong lifespan in diverse organisms, suggesting a conserved and complex role of mitochondria in longevity determination. Several nuclear transcription factors have been demonstrated to mediate the lifespan extension effect associated with partial impairment of the ETC, suggesting that compensatory transcriptional response to be crucial. In this study, we showed that the transcription factors CEP‐1/p53 and CEH‐23 act through a similar mechanism to modulate longevity in response to defective ETC in Caenorhabditis elegans. Genomewide gene expression profiling comparison revealed a new link between these two transcription factors and AAK‐2/AMP kinase (AMPK) signaling. Further functional analyses suggested that CEP‐1/p53 and CEH‐23 act downstream of AAK‐2/AMPK signaling and CRTC‐1 transcriptional coactivator to promote stress resistance and lifespan. As AAK‐2, CEP‐1, and CEH‐23 are all highly conserved, our findings likely provide important insights for understanding the organismal adaptive response to mitochondrial dysfunction in diverse organisms and will be relevant to aging and pathologies with a mitochondrial etiology in human.

Regular consumption of fruits and vegetables is associated with reduced risk of age-related functional decline and chronic diseases such as cancer and cardiovascular disease. These effects are primarily attributed to phytochemicals, plant compounds with a wide range of biological activities and health benefits. Apples, the top contributor of fruit phenolics in American diets, have high antioxidant, antiproliferative and chemopreventive activity in vitro and in vivo. However, little is known about their effects on aging. The objectives of this study were to determine the effects of whole apple phytochemical extracts on lifespan, healthspan and resistance to various stresses in vivo using Caenorhabditis elegans as a model. The mean and maximum lifespan of animals treated with 2.5, 5 and 10 mg/ml whole apple extracts increased significantly in a dose-dependent manner by up to 39% and 25%, respectively. Healthspan also significantly improved as indicated by improved motility and reduced lipofuscin accumulation. Animals pre-treated with whole apple extracts were more resistant to stresses such as heat, UV radiation, paraquat-induced oxidative stress, and pathogenic infection, suggesting that cellular defense and immune system functions also improved. Our findings indicate that, in C. elegans, whole apple extracts slow aging, extend lifespan, improve healthspan, and enhance resistance to stress.

Summary Host cell factor-1 (HCF-1) is a conserved regulator of the longevity and stress response functions of DAF-16/FOXO. SKN-1 transcription factor is an evolutionarily conserved xenobiotic stress regulator and a pro-longevity factor. Here, we demonstrate that SKN-1 contributes to the enhanced oxidative stress resistance incurred by hcf-1 mutation in C. elegans. HCF-1 prevents the nuclear accumulation of SKN-1 and represses the transcriptional activation of SKN-1 specifically at target genes involved in cellular detoxification pathways. Our findings reveal a novel and context-specific regulatory relationship between two highly conserved longevity and stress response factors HCF-1 and SKN-1.

Changes in epigenetic status and chromatin structure have been shown to associate with aging in many organisms. Here, we report an RNAi screen of putative histone methyltransferases and demethylases in wild-type Caenorhabditis elegans using reproduction inhibitor. We identified six genes that when inactivated by RNAi, consistently extend lifespan. Five of these genes do not require germline proliferation to affect lifespan. We further characterized two of these genes, the highly homologous SET domain containing genes, set-9 and set-26. They share redundant functions in maintaining normal lifespan, while exhibiting differential tissue expression patterns. Furthermore, we found that set-9 and set-26 partially act through the Forkhead box O (FOXO) transcription factor, DAF-16, to modulate lifespan. Interestingly, inactivation of somatic SET-26 alone results in a robust lifespan extension and alters the levels of histone H3 protein and the repressive histone marks, H3K9me3 and H3K27me3, in an age-dependent manner. We hypothesize that inactivation of SET-26 triggers compensation mechanisms to restore repressive chromatin structure and hence affects chromatin stability to promote longevity.

Mitochondria are dynamic organelles able to undergo frequent morphological and numeral changes. A delicate balance between mitochondrial fusion and fission is critical for broad aspects of animal physiology, including apoptosis and control of mitochondrial inheritance and quality (Seo et al. 2010). From yeast to human, deregulations of mitochondrial network equilibrium as evident by disrupted mitochondrial morphology and accumulation of abnormally shaped mitochondria have been associated with senescence, aging and aging-related diseases (Sohal 1975; Yasuda et al. 2006; Lee et al. 2007). However, no direct evidence implicates mitochondrial dynamics in longevity determination in animals. The only evidence that mitochondrial plasticity positively impacts lifespan was shown in fungal models, in which reduced mitochondrial fission led to increased lifespan (Scheckhuber et al. 2007; Palermo et al. 2010). Mitochondrial dynamics are governed by molecular machineries that are highly conserved (Okamoto & Shaw 2005). The dynamin-related protein DRP-1 is the only profission protein identified in C. elegans and is demonstrated to control the scission of the mitochondrial outer membrane (Labrousse et al. 1999; Westermann 2010). To explore how mitochondrial fission can impact animal lifespan, we monitored the lifespan of worms either treated with drp-1 RNAi or bearing a putative null-mutation in the drp-1 gene (Breckenridge et al. 2008). The mean lifespan of wild-type worms treated with drp-1 RNAi or drp-1 mutant worms was indistinguishable from that of control animals (Fig.1A-B, Table S1), even though their mitochondrial morphology are greatly disrupted (Labrousse et al. 1999, Fig.2B-C and S1), indicating that reduced mitochondrial fission does not affect C. elegans lifespan under normal culturing condition Figure 1 Loss of drp-1 enhances the longevity phenotype of IIS mutant worms in a daf-16-dependent manner Figure 2 Loss of drp-1 affects mitochondrial morphology and stress resistance without affecting DAF-16 sub-cellular localization The insulin/IGF-1 signaling (IIS) pathway is a key longevity pathway and C. elegans mutants with reduced IIS, such as the phosphatidyl-inositol 3-kinase age-1 mutant (Friedman & Johnson 1988; Morris et al. 1996) and the tyrosine kinase insulin/IGF receptor daf-2 mutant (Kenyon et al. 1993), are long-lived (Fig.1A,C-D). Given that mitochondrial fission plays a critical role in insulin secretion in mammals (Yoon et al. 2010), we next tested how inactivating drp-1 might affect the longevity of IIS mutants. Strikingly, both age-1 and daf-2 mutants treated with drp-1 RNAi showed substantial further increase in mean lifespan (>65% compared to age-1 and daf-2 single mutant on control RNAi; Fig.1A, Table S1). Similarly, age-1;drp-1 and daf-2;drp-1 mutants exhibited a >75% increase in mean lifespan and an extension by up to 30 days in maximum lifespan when compared to their single mutant counterparts (Fig.1C-D, Table S1). Importantly, the enhancement of both the mean and maximum lifespans of age-1 mutants by the drp-1 mutation could be rescued by a transgene expressing drp-1 (Fig.S2). We also tested whether drp-1 inactivation will impact the lifespan of other long-lived strains, including worms undergoing the bacteria deprivation paradigm of dietary restriction (Kaeberlein et al. 2006), and worms with mild impairment of the mitochondrial respiratory chain (Felkai et al. 1999; Feng et al. 2001). We detected no or marginal effects (Table S2). Altogether, we demonstrated that inactivating drp-1 specifically and robustly synergizes with reduced IIS to prolong longevity. The longevity effect of reduced IIS is often associated with increased resistance to stress (Johnson et al. 2001). Surprisingly, wild-type and insulin mutant worms with drp-1 deletion were more sensitive to the oxidizing agent paraquat and to heat stress than their counterparts with wild-type drp-1 (Fig.2A, Table S3). The vulnerability of the age-1;drp-1 mutant to heat stress is due to drp-1 loss as it can be rescued by a transgene overexpressing drp-1 (Fig.S2). Therefore, inactivating drp-1 specifically prolongs longevity, but not stress resistance, of worms with reduced IIS, suggesting that manipulation of mitochondrial fission uncouples the function of IIS in aging and stress resistance. However, we cannot exclude that inactivating drp-1 causes an early stress in young worms but will confer stress resistance later in life. We next examined how inactivation of drp-1 affects mitochondrial morphology by comparing the indices of circularity (a mathematical estimation of circular shapes) of individual mitochondrion (see Supplementary Materials and Methods). We found that mitochondrial morphology was similarly disrupted in single drp-1 mutant and in IIS;drp-1 mutants (Fig.2C), suggesting DRP-1 does not cooperate with IIS to regulate mitochondrial fission. Interestingly, our data also revealed that mitochondria of daf-2 and age-1 single mutants presented lower indices of circularity than those of wild-type animals (Fig.2B-C). Signaling from DAF-2 and AGE-1 results in cytoplasmic retention of the FOXO transcription factor DAF-16, and DAF-16 translocation into the nucleus in age-1 and daf-2 mutants enables DAF-16-mediated regulation of target genes that contribute to diverse functional outcomes (Hekimi et al. 2001; Lee et al. 2001). Because the prolonged lifespan of age-1 and daf-2 mutants are fully dependent on daf-16 (Dorman et al. 1995), we next tested whether the extraordinary longevity of the IIS;drp-1 mutants also requires daf-16. We found that daf-16 RNAi completely abolished the synergistic effect of drp-1 and daf-2/age-1 mutations (Fig.1C-D, Table S1). Interestingly, we did not detect any changes in DAF-16∷GFP sub-cellular localization upon drp-1 inactivation (Fig.2D). Thus, the mechanism allowing DRP-1 to cooperate with IIS in modulating lifespan fully depends on DAF-16 but is unlikely to be mediated via DAF-16’s nuclear translocation. In considering a mechanism that enables drp-1 inactivation to specifically synergize with IIS mutants to extend lifespan, several possibilities come to mind. In mammals, mitochondrial fission is critical for insulin secretion (Zorzano et al. 2009; Yoon et al. 2010). Thus, drp-1 inactivation in worms may similarly interfere with insulin secretion and further reduce IIS in age-1/daf-2 mutants. This possibility is unlikely however as DAF-16 nuclear translocation was not enhanced in IIS;drp-1 double mutants. Although we found that age-1 and daf-2 single mutants show slight alterations in mitochondrial morphology, our data do not support the possibility that mitochondrial dynamics was further perturbed in IIS;drp-1 double mutants, as mitochondrial morphology in those mutants were similar to drp-1 single mutant. On the other hand, because the drp-1 single mutant has a brood size defect (Breckenridge et al. 2008), it is possible that germline proliferation is inhibited when drp-1 is inactivated, and defective germline proliferation is known to synergize with IIS mutant to increase lifespan (Hsin & Kenyon 1999; Spanier et al.). Alternatively, similar to what was shown with mitochondrial prohibitins (Artal-Sanz & Tavernarakis 2009), inactivation of drp-1 may perturb lipid metabolism, a process highly dependent on mitochondrial activity, to synergize with IIS mutants to extend lifespan. Lastly, C. elegans DRP-1 is shown to regulate apoptosis under sensitized conditions (Breckenridge et al. 2008). Intriguingly, daf-2 mutants exhibit enhanced germline apoptosis (Pinkston et al. 2006). It is possible that the apoptotic function of DRP-1 is induced in IIS mutants, and loss of drp-1 may block apoptosis in IIS mutants and further promote longevity. Interestingly, in fungal models, mitochondrial fission modulates wild-type lifespan by interfering with apoptosis (Scheckhuber et al. 2007; Palermo et al. 2010). It is possible that fungal aging is more closely connected to apoptosis (Rockenfeller & Madeo 2008) than C. elegans aging, providing an explanation for why reduced mitochondrial fission affects wild-type lifespan in fungi but not in worms. This is the first report of a manipulation of mitochondrial dynamics that positively impacts lifespan in an animal. As components of the mitochondrial fission/fusion machinery and IIS are highly conserved, our observations are likely relevant to mitochondrial biology and longevity in mammals.

Summary Background Aging is under genetic control in C. elegans , but the mechanisms of life-span regulation are not completely known. MicroRNAs (miRNAs) regulate various aspects of development and metabolism, and one miRNA has been previously implicated in life span. Results Here we show that multiple miRNAs change expression in C. elegans aging, including novel miRNAs, and that mutations in several of the most upregulated miRNAs lead to life-span defects. Some act to promote normal life span and stress resistance, whereas others inhibit these phenomena. We find that these miRNAs genetically interact with genes in the DNA damage checkpoint response pathway and in the insulin signaling pathway. Conclusions Our findings reveal that miRNAs both positively and negatively influence life span. Because several miRNAs upregulated during aging regulate genes in conserved pathways of aging and thereby influence life span in C. elegans , we propose that miRNAs may play important roles in stress response and aging of more complex organisms.

The Snail family of transcription factors are core inducers of epithelial-to-mesenchymal transition (EMT). Here we show that the F-box protein FBXO11 recognizes and promotes ubiquitin-mediated degradation of multiple Snail family members including Scratch. The association between FBXO11 and Snai1 in vitro is independent of Snai1 phosphorylation. Overexpression of FBXO11 in mesenchymal cells reduces Snail protein abundance and cellular invasiveness. Conversely, depletion of endogenous FBXO11 in epithelial cancer cells causes Snail protein accumulation, EMT, and tumor invasion, as well as loss of estrogen receptor expression in breast cancer cells. Expression of FBXO11 is downregulated by EMT-inducing signals TGFβ and nickel. In human cancer, high FBXO11 levels correlate with expression of epithelial markers and favorable prognosis. The results suggest that FBXO11 sustains the epithelial state and inhibits cancer progression. Inactivation of FBXO11 in mice leads to neonatal lethality, epidermal thickening, and increased Snail protein levels in epidermis, validating that FBXO11 is a physiological ubiquitin ligase of Snail. Moreover, in C. elegans, the FBXO11 mutant phenotype is attributed to the Snail factors as it is suppressed by inactivation/depletion of Snail homologs. Collectively, these findings suggest that the FBXO11-Snail regulatory axis is evolutionarily conserved and critically governs carcinoma progression and mammalian epidermal development.

We report here the first genome-wide functional genomic screen for longevity genes. We systematically surveyed Caenorhabditis elegans genes using large-scale RNA interference (RNAi), and found that RNAi inactivation of 89 genes extend C. elegans lifespan. Components of the daf-2/insulin-like signaling pathway are recovered, as well as genes that regulate metabolism, signal transduction, protein turnover, and gene expression. Many of these candidate longevity genes are conserved across animal phylogeny. Genetic interaction analyses with the new longevity genes indicate that some act upstream of the daf-16/FOXO transcription factor or the sir2.1 protein deacetylase, and others function independently of daf-16/FOXO and sir2.1, and might define new pathways to regulate lifespan.

Signaling from the DAF-2/insulin receptor to the DAF-16/FOXO transcription factor controls longevity, metabolism, and development in disparate phyla. To identify genes that mediate the conserved biological outputs of daf-2 /insulin-like signaling, we usedcomparative genomics to identify 17 orthologous genes from Caenorhabditis and Drosophila , each of which bears a DAF-16 binding site in the promoter region. One-third of these DAF-16 downstream candidate genes were regulatedby daf-2 /insulin-like signaling in C. elegans , and RNA interference inactivation of the candidates showed that many of these genes mediate distinct aspects of daf-16 function, including longevity, metabolism, anddevelopment.

Adenovirus type 9 (Ad9) is distinct among human adenoviruses because it elicits solely mammary tumors in animals and its primary oncogenic determinant is the E4 region-encoded ORF1 (E4-ORF1) protein. We report here that the PDZ domain-containing protein ZO-2, which is a candidate tumor suppressor protein, is a cellular target for tumorigenic Ad9 E4-ORF1 but not for non-tumorigenic wild-type E4-ORF1 proteins encoded by adenovirus types 5 and 12. Complex formation was mediated by the C-terminal PDZ domain-binding motif of Ad9 E4- ORF1 and the first PDZ domain of ZO-2, and in cells this interaction resulted in aberrant sequestration of ZO-2 within the cytoplasm. Furthermore, transformation-defective Ad9 E4-ORF1 mutants exhibited impaired binding to and sequestration of ZO-2 in cells, and overexpression of wild-type ZO-2, but not mutant ZO-2 lacking the second and third PDZ domains, interfered with Ad9 E4-ORF1-induced focus formation. Our results suggest that the select capacity to complex with the candidate tumor suppressor protein ZO-2 is key to defining the unique transforming and tumorigenic properties of the Ad9 E4-ORF1 oncoprotein.

A yeast two-hybrid screen was employed to identify ligands for the cytoplasmic domain of the NG2 chondroitin sulfate proteoglycan. Two overlapping cDNA clones selected in the screen are identical in sequence to a DNA segment coding for the most amino-terminal of the 13 PDZ domains found in the multi-PDZ-protein MUPP1. Antibodies made against recombinant polypeptides representing these two clones (NIP-2 and NIP-7) are reactive with the same 250-kDa molecule recognized by anti-MUPP1 antibodies, confirming the presence of the NIP-2 and NIP-7 sequences in the MUPP1 protein. NIP-2 and NIP-7 GST fusion proteins effectively recognize NG2 in pull-down assays, demonstrating the ability of these polypeptide segments to interact with the intact proteoglycan. The fusion proteins fail to bind NG2 missing the C-terminal half of the cytoplasmic domain, emphasizing the role of the NG2 C-terminus in the interaction with MUPP1. The existence of an NG2/MUPP1 interaction in situ is demonstrated by the ability of NG2 antibodies to co-immunoprecipitate both NG2 and MUPP1 from detergent extracts of cells expressing the two molecules. MUPP1 may serve as a multivalent scaffold that provides a means of linking NG2 with key structural and/or signaling components in the cytoplasm.

Previous studies have shown that the oncogenic HPV E6 proteins form a complex with the human homologue of the Drosophila tumour suppressor protein, discs large (Dlg). This is mediated by the carboxy terminus of the E6 proteins and involves recognition of at least one PDZ domain of Dlg. This region of E6 is not conserved amongst E6 proteins from the low risk papillomavirus types and, hence, binding of HPV E6 proteins to Dlg correlates with the oncogenic potential of these viruses. We have performed studies to investigate the consequences of the interaction between E6 and Dlg. Mutational analysis of both the HPV18 E6 and Dlg proteins has further defined the regions of E6 and Dlg necessary for complex formation. Strikingly, co-expression of wild type HPV18 E6 with Dlg in vitro or in vivo results in a dramatic decrease in the amount of Dlg protein, whereas mutants of E6 which fail to complex with Dlg have minimal effect on Dlg protein levels. The oncogenic HPV16 E6 also decreased the Dlg levels, but this was not observed with the low risk HPV11 E6 protein. Moreover, a region within the first 544 amino acids of Dlg containing the three PDZ domains confers susceptibility to E6 mediated degradation. Finally, treatment of cells with a proteasome inhibitor overrides the capacity of E6 to degrade Dlg. These results demonstrate that Dlg is targeted by high risk HPV E6 proteins for proteasome mediated degradation.

Recent findings in diverse organisms strongly support a conserved role for mitochondrial electron transport chain dysfunction in longevity modulation, but the underlying mechanisms are not well understood. One way cells cope with mitochondrial dysfunction is through a retrograde transcriptional reprogramming response. In this review, we primarily focus on the work that has been performed in Caenorhabditis elegans to elucidate these mechanisms. We describe several transcription factors that participate in mitochondria-to-nucleus signaling and discuss how they mediate the relationship between mitochondrial dysfunction and life span.

The 9ORF1 gene encodes an adenovirus E4 region oncoprotein that requires a C-terminal region for transforming activity. Screening a λgt11 cDNA expression library with a 9ORF1 protein probe yielded a novel cellular PDZ domain-containing protein, 9BP-1, which binds to wild-type, but not a transformation-defective, C-terminal, mutant 9ORF1 protein. The fact that PDZ domains complex with specific sequences at the free C-terminal end of some proteins led to the recognition that the 9ORF1 C-terminal region contained such a consensus-binding motif. This discovery prompted investigations into whether the 9ORF1 protein associates with additional cellular proteins having PDZ domains. It was found that the 9ORF1 protein interacts directly, in vitro and in vivo , with the PDZ domain-containing protein hDlg/SAP97 (DLG), which is a mammalian homolog of the Drosophila discs large tumor suppressor protein and which also binds the adenomatous polyposis coli tumor suppressor protein. Of interest, in forming complexes, the 9ORF1 protein preferentially associated with the second PDZ domain of DLG, similar to adenomatous polyposis coli protein. Human T cell leukemia virus type 1 Tax and most oncogenic human papillomavirus E6 oncoproteins also possessed PDZ domain-binding motifs at their C termini and, significantly, human T cell leukemia virus type 1 Tax and human papillomavirus 18 E6 proteins bound DLG in vitro . Considering the requirement of the 9ORF1 C-terminal region in transformation, these findings suggest that interactions with the cellular factor DLG may contribute to the tumorigenic potentials of several different human virus oncoproteins.

Caenorhabditis elegans CEP-1 and its mammalian homolog p53 are critical for responding to diverse stress signals. In this study, we found that cep-1 inactivation suppressed the prolonged lifespan of electron transport chain (ETC) mutants, such as isp-1 and nuo-6, but rescued the shortened lifespan of other ETC mutants, such as mev-1 and gas-1. We compared the CEP-1-regulated transcriptional profiles of the long-lived isp-1 and the short-lived mev-1 mutants and, to our surprise, found that CEP-1 regulated largely similar sets of target genes in the two mutants despite exerting opposing effects on their longevity. Further analyses identified a small subset of CEP-1-regulated genes that displayed distinct expression changes between the isp-1 and mev-1 mutants. Interestingly, this small group of differentially regulated genes are enriched for the “aging” Gene Ontology term, consistent with the hypothesis that they might be particularly important for mediating the distinct longevity effects of CEP-1 in isp-1 and mev-1 mutants. We further focused on one of these differentially regulated genes, ftn-1, which encodes ferritin in C. elegans, and demonstrated that it specifically contributed to the extended lifespan of isp-1 mutant worms but did not affect the mev-1 mutant lifespan. We propose that CEP-1 responds to different mitochondrial ETC stress by mounting distinct compensatory responses accordingly to modulate animal physiology and longevity. Our findings provide insights into how mammalian p53 might respond to distinct mitochondrial stressors to influence cellular and organismal responses., Author Summary Perturbing different components of the mitochondrial electron transport chain (ETC) can affect the lifespan of Caenorhabditis elegans in divergent ways. ETC dysfunction can either attenuate or extend C. elegans lifespan. Here we demonstrate that the C. elegans homolog of mammalian p53, CEP-1, is key in mediating differential survival of distinct ETC mutants. Inactivating cep-1 in two long-lived ETC mutants shortened their lifespans, whereas cep-1 deletion in two short-lived ETC mutants restored their lifespans. Because how CEP-1 does this is unknown, we compared the genome-wide expression profiles of the long-lived isp-1 and short-lived mev-1 ETC mutants upon cep-1 removal. We found that CEP-1 is able to differentially regulate a small subset of genes depending on the specific ETC mutations. We validated one of the genes, the iron transporter ferritin (ftn-1), to be an important target through which CEP-1 mediates distinct lifespan outcome in specific ETC mutants. We also identified numerous other candidate CEP-1 targets that merit future analysis, which will further elucidate how CEP-1 differentially responds to the distinct ETC dysfunction of various mitochondrial mutants. Our study provides new insights that will inform how mammalian p53, a critical tumor suppressor, might respond to mitochondrial and metabolic stresses.

The transcriptional co-regulator host cell factor-1 (HCF-1) plays critical roles in promoting cell cycle progression in diverse cell types, and in maintaining self-renewal of embryonic stem cells, but its role in pancreatic β-cell function has not been investigated. Immunhistochemistry of mouse pancreas revealed nuclear expression of HCF-1 in pancreatic islets. Reducing HCF-1 expression in the INS-1 pancreatic β-cell line resulted in reduced cell proliferation, reduced glucose-stimulated insulin secretion, and reduced expression of the critical β-cell transcription factor Pdx1. HCF-1 is a known co-activator of the E2F1 transcription factor, and loss of E2F1 results in pancreatic β-cell dysfunction and reduced expression of Pdx1. Therefore we wondered whether HCF-1 might be required for E2F1 regulation of Pdx1. Chromatin immunoprecipitation experiments revealed that HCF-1 and E2F1 co-localize to the Pdx1 promoter. These results indicate that HCF-1 represents a novel transcriptional regulator required for maintaining pancreatic β-cell function.

Larvae of the nematode Caenorhabditis elegans must choose between reproductive development and dauer diapause. This decision is based on sensing of environmental inputs and dauer pheromone, a small molecule signal that serves to monitor population density. These signals are integrated via conserved neuroendocrine pathways that converge on steroidal ligands of the nuclear receptor DAF-12, a homolog of the mammalian vitamin D receptor and liver X receptor. DAF-12 acts as the main switch between gene expression programs that drive either reproductive development or dauer entry. Extensive studies in the past two decades demonstrated that biosynthesis of two bile acid-like DAF-12 ligands, named dafachronic acids (DA), controls developmental fate. In this issue of PLoS Biology, Wollam et al. showed that a conserved steroid-modifying enzyme, DHS-16, introduces a key feature in the structures of the DAF-12 ligands, closing a major gap in the DA biosynthesis pathway. The emerging picture of DA biosynthesis in C. elegans enables us to address a key question in the field: how are complex environmental signals integrated to enforce binary, organism-wide decisions on developmental fate? Schaedel et al. demonstrated that pheromone and DA serve as competing signals, and that a positive feedback loop based on regulation of DA biosynthesis ensures organism-wide commitment to reproductive development. Considering that many components of DA signaling are highly conserved, ongoing studies in C. elegans may reveal new aspects of bile acid function and lifespan regulation in mammals.

Changes in epigenetic status and chromatin structure have been shown to associate with aging in many organisms. Here, we report an RNAi screen of putative histone methyltransferases and demethylases in wild-type Caenorhabditis elegans using reproduction inhibitor. We identified six genes that when inactivated by RNAi, consistently extend lifespan. Five of these genes do not require germline proliferation to affect lifespan. We further characterized two of these genes, the highly homologous SET domain containing genes, set-9 and set-26. They share redundant functions in maintaining normal lifespan, while exhibiting differential tissue expression patterns. Furthermore, we found that set-9 and set-26 partially act through the Forkhead box O (FOXO) transcription factor, DAF-16, to modulate lifespan. Interestingly, inactivation of somatic SET-26 alone results in a robust lifespan extension and alters the levels of histone H3 protein and the repressive histone marks, H3K9me3 and H3K27me3, in an age-dependent manner. We hypothesize that inactivation of SET-26 triggers compensation mechanisms to restore repressive chromatin structure and hence affects chromatin stability to promote longevity.

Larvae of the nematode Caenorhabditis elegans must choose between reproductive development and dauer diapause. This decision is based on sensing of environmental inputs and dauer pheromone, a small molecule signal that serves to monitor population density. These signals are integrated via conserved neuroendocrine pathways that converge on steroidal ligands of the nuclear receptor DAF-12, a homolog of the mammalian vitamin D receptor and liver X receptor. DAF-12 acts as the main switch between gene expression programs that drive either reproductive development or dauer entry. Extensive studies in the past two decades demonstrated that biosynthesis of two bile acid-like DAF-12 ligands, named dafachronic acids (DA), controls developmental fate. In this issue of PLoS Biology, Wollam et al. showed that a conserved steroid-modifying enzyme, DHS-16, introduces a key feature in the structures of the DAF-12 ligands, closing a major gap in the DA biosynthesis pathway. The emerging picture of DA biosynthesis in C. elegans enables us to address a key question in the field: how are complex environmental signals integrated to enforce binary, organism-wide decisions on developmental fate? Schaedel et al. demonstrated that pheromone and DA serve as competing signals, and that a positive feedback loop based on regulation of DA biosynthesis ensures organism-wide commitment to reproductive development. Considering that many components of DA signaling are highly conserved, ongoing studies in C. elegans may reveal new aspects of bile acid function and lifespan regulation in mammals.

Our understanding of the genetic mechanisms of organismal aging has advanced dramatically during the past two decades. With the development of large-scale RNAi screens, the last few years saw the remarkable identifications of hundreds of new longevity genes in the roundworm Caenorhabditis elegans. The various RNAi screens revealed many biological pathways previously unknown to be related to aging. In this review, we focus on findings from the recent large-scale RNAi longevity screens, and discuss insights they have provided into the complex biological process of aging and considerations of the RNAi technology will continue to have on the future development of the aging field.

For a long time, mitochondria have been considered as primary targets of the aging process, notably through accumulation of oxidative damage and mitochondrial deoxyribonucleic acid (DNA) mutations that lead to gradual decline of mitochondrial function. Recent investigations in various model organisms have revealed conserved molecular pathways that can impact longevity. Intriguingly, some of these longevity pathways involve proteins with key functions in the mitochondria. Furthermore, a growing number of studies indicate that signals emanated from mitochondria can directly impact the longevity of the whole organism. Thus, the mitochondrion organelle likely plays an active and causal role in longevity determination. This renders mitochondria a key determinant in the complex network of physiological signals that modulates longevity. Keywords: aging; signal transduction; mitochondria; ROS; dietary restriction

Insulin/IGF-signaling (IIS) affects longevity, stress resistance and metabolism in worms, flies, and mammals. The Forkhead transcription factor DAF-16/FOXO is the major downstream effector of IIS and is responsible for the activation and repression of genes that mediate the diverse effects of IIS. We surveyed a set of informatically predicted conserved DAF-16/FOXO target genes and identified the novel DAF-16 direct target hlh-13. hlh-13 is the predicted homolog of the mammalian transcription factor Ptf1a, a critical determinant of pancreatic development. We found that an hlh-13 mutant exits L1 arrest and IIS-dependent dauer diapause faster than control worms, but is not involved in lifespan or resistance to a variety of stresses. Our results have identified a novel DAF-16 target gene and linked its function to known outputs of IIS. Considering the high conservation of IIS in diverse species, our results also hint at an intriguing connection of IIS and Ptf1a in mammals.

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., Synopsis 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.

This article discusses the pros and cons of using RNAi screening to identify longevity genes in Caenorhabditis elegans. The discussion will focus on the results of two large-scale longevity RNAi screens that were recently published. The two screens revealed largely non-overlapping sets of candidate longevity genes. The possible reasons for such differences and their implications will be discussed.

Several recent studies have highlighted how RNA interference has advanced aging-related research in Caenorhabditis elegans . Two new genome-wide RNAi-based screens together identified more than 100 new candidate longevity genes. RNAi has also greatly facilitated the functional validation of several large-scale gene expression profiling studies. Furthermore, RNAi is flexible and can be administered at different times throughout life; this feature has been exploited to analyze the temporal requirement of genes in life-span determination.

Aging is a complex process that involves the gradual functional decline of many different tissues and cells. Gene expression microarray analysis provides a comprehensive view of the gene expression signature associated with age and is particularly valuable for understanding the molecular mechanisms that contribute to the aging process. However, because of the stochastic nature of the aging process, animals of the same chronological age often manifest great physiological differences. Therefore, profiling the gene expression pattern of a large population of aging animals risks either exaggerating or masking the changes in gene expression that correspond to physiological aging. In a recent paper, Golden and Melov surveyed the gene expression profiles of individual aging Caenorhabditis elegans , hoping to circumvent the problem of variability among worms of the same chronological age. This initial analysis of age-dependent gene expression in individual aging worms is an important step toward deciphering the molecular basis of physiological aging.