The reproductive tissues of C. elegans influence how the whole animal ages. The two cell types of the reproductive system have opposite effects on longevity. The germ cells inhibit a long lifespan, and the somatic reproductive tissues (somatic gonad) promote a long lifespan in the absence of the germ cells. How the somatic gonad promotes the longevity of other tissues is not well understood.

Here, we find that the somatic gonad has two effects on other tissues. First, the somatic gonad activates DAF-12, a nuclear hormone receptor that extends lifespan, through its ligand, dafachronic acid. Next, the somatic gonad ensures the full transcriptional activity of the FOXO transcription factor, DAF-16. Interestingly, the somatic gonad does not affect the entry of DAF-16/FOXO into the nucleus where it acts to promote longevity. The germ cells, however, do affect where DAF-16/FOXO is located within a cell. This supports the idea that the somatic gonad and germ cells affect lifespan, at least in part, through distinct mechanisms.

To extend lifespan, the somatic gonad activates both DAF-12/NHR and DAF-16/FOXO. However, we find that the two transcription factors do not operate in a simple linear pathway, with one being fully required for the activity of the other. Instead, we find the two transcription factors regulate distinct sets of genes. Additionally, the two transcription factors have modest effects on the activity of the other. That is, DAF-12/NHR is partially required for the full activity of DAF-16/FOXO, and vice versa.

To summarize, the somatic gonad affects lifespan by modulation of the activities of two transcription factors, DAF-16/FOXO and DAF-12/NHR, which work in parallel to promote longevity.

To date, many genes and pathways regulating lifespan have been identified in the nematode Caenorhabditis elegans. Over 20 years of research have uncovered conserved roles for insulin signaling, metabolism, dietary restriction, and reproductive signaling in the control of lifespan. Yet, despite this work, we still lack a complete picture of aging. To identify new components of lifespan regulation, we undertook two RNAi-based screens. In the first, we searched for genes which, when knocked down, accelerate aging in C. elegans. Using lifespan analysis as well as analysis of tissue morphology, we identified five potential "progeria regulators". These five genes, four of which were independently identified by other researchers, negatively regulate lifespan. Further work will determine their exact role in aging regulation.

A separate RNAi screen for components of dietary restriction-mediated longevity also identified a negative regulator of lifespan. mrvl-1 encodes a MARVEL-domain containing membrane protein which is partially localized to the Golgi apparatus. RNAi inhibition of mrvl-1 significantly shortens lifespan and accelerates aging while overexpression extends lifespan in C. elegans. Using transcriptional profiling to identify genes involved in mrvl-1-regulation of aging, we found significant overlap with the lifespan-regulating daf-2/insulin/IGF-1 signaling pathway as well as with the innate immune response in C. elegans. Investigations into the immune activity of mrvl-1 revealed that whereas RNAi treatment causes increased sensitivity to the bacterial pathogen Pseudomonas aeruginosa, overexpression of mrvl-1 increases resistance. As bacterial food sources may potentially pose a late-life hazard to C. elegans, mrvl-1 likely helps the worm cope with the detrimental effects of proliferating and pathogenic bacteria. Indeed, mrvl-1 overexpression no longer extends the lifespan of animals fed non-proliferating, UV-killed bacteria. This work describes a novel lifespan gene that protects the worm from harmful bacteria, thus promoting survival and extending lifespan.

In both C. elegans and Drosophila, removing germline stem cells increases lifespan. In C. elegans, this lifespan extension requires the DAF-16/FOXO transcription factor and the DAF-12 nuclear hormone receptor, and the role of DAF-16/FOXO in this process may also be shared in flies. To better understand the regulatory relationships between

DAF-16 and DAF-12, we used microarray analysis to identify downstream genes. We found that these two transcription factors regulate distinct but overlapping sets of genes in response to loss of the germline. In addition, we identified several new genes that are required for loss of the germline to increase lifespan in C. elegans. One, phi-62, encodes

a conserved, predicted RNA binding protein. PHI-62 influences DAF-16-dependent transcription, possibly by collaborating with TCER-1, a putative transcription-elongation factor, and FTT-2, a 14-3-3- protein known to bind DAF-16. Three other genes encode proteins involved in lipid metabolism; one is a triacylglycerol lipase, and another is an

acyl CoA reductase. These genes do not affect bulk fat storage levels, but rather may influence production of a lifespan-extending signal or metabolite.

Genome-wide expression measurements remain difficult to interpret. Two major challenges lie in drawing firm conclusions from hundreds or even thousands of significantly changing genes, and in deriving hypotheses from the data that merit further testing. Identifying the degree to which each gene regulator acts to increase or decrease the expression of each gene, a concept I refer to as transcriptional influence, would greatly increase our ability to make sense of these data.

This work describes a new method to calculate the transcriptional influence that each regulatory motif in a de novo predicted set has on each gene represented in a gene expression measurement platform, using only a compendium of data from the platform and genome sequence information. The method uses independent component analysis (ICA) first to generate genetic regulatory modules, and then to predict DNA sequence motifs (putative regulatory sites) that are enriched in these modules. In a final step, the relative membership of each gene in each gene module and the enrichment of each sequence motif in each module are used to predict the relative influence of each sequence motif on each gene.

The power of these predictions is demonstrated in the analysis of microarray data for several C. elegans variants, including isp-1 and hif-1 mutants. isp-1 mutations extend lifespan through the HIF-1 transcription factor, but there is no meaningful overlap among significant genes in hif-1 and isp-1 microarray datasets. In contrast, our method reveals extensive similarity in gene expression at a deeper level. Moreover, a regulatory motif predicted to have a strong influence in both datasets matches the canonical HIF-1 binding site.

In nature, there is a strong correlation between physiological aging and tumor susceptibility. Understanding how youthful animals resist tumors may provide new insights into tumor biology, particularly if genes that regulate aging influence tumor susceptibility. Mutations in the C. elegans gene gld-1 produce a lethal germline tumor. We find that mutations that extend C. elegans' lifespan, by caloric restriction, mitochondrial dysfunction, or a reduction in daf-2/insulin/IGF-1 signaling, confer resistance to these tumors. Remarkably, mutations that reduce daf-2 function, which double the lifespan of worms, can completely prevent the shortened lifespan caused by these tumors. daf-2 mutations protect tumorous animals by triggering apoptosis through the FOXO transcription factor DAF-16 and the tumor-suppressor p53, and by activating daf-16 dependent and daf-16-independent processes that inhibit tumor cell division. Interestingly, the inhibition of mitosis is specific to tumor cells. Together these findings suggest a fundamental link between mechanisms that promote cell maintenance and animal longevity and mechanisms that inhibit tumor cell proliferation.

To identify downstream components of this process, we have screened through 734 potential daf-16/FOXO targets and found 16 genes that are necessary for daf-2 mutations to fully suppress tumor growth, and 14 genes that normally promote tumor growth. Approximately one quarter of these genes are known to have roles in human cancer, suggesting that the other genes may be involved in mammalian cancer as well.

Understanding the evolutionary links between an animal’s reproductive state and its rate of aging has been of interest to scientists since the field of aging research first began. Contrary to the “Disposable Soma Theory” on the evolution of aging, which proposes that the greedy germline poses an unavoidable cost to the rest of the body, studies exploring germline-less C. elegans have revealed that the longevity afforded these animals is due to complex and highly regulated molecular mechanisms, rather than just a change in resource allocation from progeny production to somatic tissue maintenance. The fact that lifespan benefits are seen when the germline tissues are disrupted in worms, flies, grasshoppers, rats and humans excitingly suggests an evolutionarily conserved pathway linking reproductive tissues to aging. This study aimed to uncover the upstream effectors of this longevity pathway, in order to determine how the germline functions to prevent life extension in normal intact C. elegans. Unbiased genetic and biochemical screens were performed to find genes and proteins involved in the KRI-1-mediated activation of DAF-16, which occurs upon germline-ablation and is integral to the resulting longevity. Many overlapping factors were found using these complimentary techniques, validating the combined genetic/biochemical approach. One particular gene of interest found to promote DAF-16 activity in germline-less worms was itr-1, a gene encoding the worm’s only IP3-responsive Ca2+ channel. However, RNAi knockdown of itr-1 not only decreased the lifespan of germline(-) worms, but did so in germline(+) worms as well, suggesting ITR-1 plays more broad role in lifespan regulation. Consistent with this, itr-1(RNAi) was found to impair DAF-16 activity in the intestine of two daf-2 mutants (e1370 and e1368), and also greatly inhibit the lifespan of these long-lived strains. Thus, ITR-1 appears to act as a more general longevity promoting factor in C. elegans, possibly working through regulation of intestinal DAF-16. Interestingly though, ITR-1 was also shown to regulate activity of several other components of germline-less longevity, specifically SKN-1, DAF-12 and the MitoUPR. Thus, ITR-1 may actually control lifespan through regulation of multiple downstream pathways. Understanding these pathways will be of great interest for future research.