Author Summary The reproductive status and longevity of animals are strongly interlinked. Increasing age influences the repro- ductive capacities of most animals. However, little is known about how reproductive status might affect lifespan. Experiments in worms and flies have shown that removing cells that give rise to gametes, the ‘‘germ cells’’, makes them live longer. We know very little about the genes and molecules that are involved in this process. In this study, we have identified a gene called tcer-1 that promotes the longevity of the roundworm Caenorhabditis elegans when its germ cells are removed. The gene tcer-1 codes for a protein, TCER-1, that is predicted to function as a ‘‘transcription elongation factor’’ (it allows the comple- tion of RNA synthesis during the process of gene expression). Our experiments imply that when the germ cells of worms are removed, TCER-1 collaborates with a transcription factor called DAF-16/FOXO to express genes that contribute to increased longevity. DAF-16/FOXO can extend lifespan in response to other physiological cues besides loss of germ cells. However, TCER-1 specifically helps this widely used longevity protein to respond to signals that reflect the reproductive status. Counterparts of DAF-16/FOXO are known to control aging in other organisms, including humans, so the identification of TCER-1 may lead to a better understanding of the relationship between reproduction and aging in other species, too. We find that TCER-1 plays a key regulatory role in transducing signals from the reproductive system to the somatic tissues. When the germline is removed, TCER-1 levels rise in the intestine. This up-regulation is functionally significant because overexpressing tcer-1 in normal, fertile animals bypasses the requirement for germ- cell loss and extends lifespan. Our findings indicate that the role of tcer-1 in the reproductive longevity pathway is to promote the transcription of a subset of DAF-16/FOXO-target genes that are up-regulated upon germline removal. Thus, TCER-1 appears to act in association with DAF-16/FOXO to extend lifespan. Interestingly, tcer-1 is not invariably required for DAF-16/ FOXO transcriptional activity, as it is not needed for DAF-16/ FOXO to up-regulate its target genes or to extend lifespan in daf-2 insulin/IGF-1 receptor mutants. At least some of the genes that are up-regulated by DAF-16/FOXO in a tcer-1-dependent fashion in long-lived, germline-defective animals do not require tcer-1 for their expression in long-lived insulin/IGF-1 mutants. Thus, this transcription elongation factor appears to link germline loss to a precise DAF-16/FOXO-dependent transcriptional program. Results tcer-1 is required for the longevity associated with germline ablation We identified tcer-1 in a Chromosome II RNAi screen for genes required to extend the lifespan of germline-depleted glp-1 mutants (see Materials and Methods). When grown at the non-permissive temperature (25uC), temperature-sensitive glp-1 mutants are sterile because the germline-stem cells fail to proliferate [34]. These mutants recapitulate the lifespan extension of animals whose germline-precursor cells have been eliminated by laser ablation [2]. Since lifespan can be shortened by conditions that simply compromise the animals’ health, we focused on RNAi clones that suppressed the extended lifespan of glp-1 mutants but had little or no effect on the lifespan of wild-type animals. From this screen, we TCER-1 Links Reproductive Signals to Longevity identified a clone of the gene tcer-1 (ZK1127.9), which encodes the C. elegans homolog of a human transcription elongation factor, TCERG1 (also known as CA150) [35–37]. tcer-1 RNAi strongly suppressed the extended lifespan of glp-1 mutants, but had no effect on the lifespan of wild-type animals (Figure 1A and 1B; Table S1A). Wild-type animals grown on bacteria expressing tcer-1 dsRNA appeared normal and healthy, had regular developmental rates and displayed normal reproduction. We obtained a tcer-1 mutant, tcer-1(tm1452), from the C. elegans National Bioresource Project, Japan. The tm1452 mutation is a 10 bp insertion coupled to a 392 bp deletion that eliminates parts of the WW domains of the protein and is likely to reduce gene function. We constructed a tcer-1; glp-1 double mutant and found that it had a much shorter lifespan than did glp-1 mutants; for example, in one experiment the 74% extension in lifespan produced by the glp-1 mutation was reduced by tcer-1(tm1452) to 15% (Figure 1C, Table S1B). There was no effect on the lifespan of wild-type worms (Figure 1C, Table S1C). This finding is significant, as it indicates that tcer-1 is unlikely to play a general role in transcription elongation, but instead has a more specific function in the animal. tcer-1 mutants displayed a delay in developmental timing, and a modest reduction in brood size that was not elicited by tcer-1 RNAi treatment (data not shown). Germline loss elevates TCER-1 levels in the intestine The observation that reducing tcer-1 activity had only mild effects on wild type but almost completely prevented germline loss from extending lifespan suggested that tcer-1 might play a regulatory role in this pathway. If so, it seemed possible that its level or location in the animal might change upon germline removal. To investigate this possibility, we constructed transgenic animals expressing a TCER-1::GFP fusion protein under the control of the endogenous tcer-1 promoter (see Materials and Methods). In the wild type, TCER-1::GFP was visible at all stages of embryonic and larval development (data not shown). In adults, we observed strong nuclear localization of TCER-1::GFP in intestinal cells, many head and body neurons, muscle and hypodermal cells (Figure 2A–2C). In some intestinal cells, weak expression was also observed in the cytoplasm. To test if this fusion protein was functional, we examined the ability of the construct to rescue the shortened lifespan of tcer-1; glp-1 double mutants. Two independent lines expressing the TCER-1::GFP fusion protein completely rescued the lifespan of tcer-1; glp-1 mutants in one trial (Figure 2D, Table S1C). In a second trial, the rescue was between 77–88% (Table S1C). Thus, TCER-1::GFP is a functional protein that likely reflects the endogenous expression of tcer-1. Importantly, these observations showed that a functional TCER-1 protein was present in intestinal nuclei in the adult, where DAF-16/FOXO has been shown to act to increase lifespan [11,12]. Next, we examined the effect of germline ablation on the pattern of TCER-1::GFP expression. We found that eliminating the germline increased the level of TCER-1 in somatic cells (Figure 3A–3C). This finding raised the possibility that TCER-1 might have an important regulatory role in the animal’s response to germline ablation. An independent microarray analysis designed to identify longevity genes whose expression is altered by germline ablation also found tcer-1 amongst the top 10% of germline- regulated genes (M. McCormick and C. Kenyon, unpublished data). Thus, tcer-1 is likely to be transcriptionally up-regulated following germline depletion. TCER-1 levels increased primarily in two tissues, the intestine and neurons, upon germline removal (Figure 3A and 3B). tcer-1 RNAi suppressed longevity without affecting neuronal expression (Figure S1). Because neurons are PLoS Genetics | www.plosgenetics.org 2 September 2009 | Volume 5 | Issue 9 | e1000639