Model for TCER-1 function We can suggest hypotheses about the molecular function of TCER-1 from studies of its human homolog, the transcription elongation factor TCERG1 (also known as CA150) [35,36]. TCERG1 is known to associate with elongation-competent RNA Polymerase II (RNAPII) complexes and alter the elongation efficiency of nascent mRNA transcripts [36,49,50]. It has also been implicated in splicing [51–53]. TCERG1 (like TCER-1) has a modular structure, with FF repeats that associate with the C Terminal Domain (CTD) of RNAPII, and WW domains that can be used for association with other regulatory proteins [37,50,51,53], consistent with its role in regulating gene expres- sion. The known function of TCERG1 suggests the model that TCER-1 influences longevity by promoting transcript elongation of DAF-16/FOXO-target genes. Specifically, we propose that following germline ablation, DAF-16/FOXO accumulates in nuclei and initiates transcription of a set of downstream targets, many of which require TCER-1 for transcript elongation. In germline-ablated tcer-1 mutants, the transcription of such genes is stalled, so lifespan extension is prevented. This hypothesis is supported by recent discoveries of transcription elongation factors that selectively influence transcription initiated by particular transcription initiation (activation) factors [54,55]. FOXO proteins are known to be regulated by many types of covalent modifications, including phosphorylation on multiple sites, dea- cetlyation, and in addition by regulated proteolysis [56,57]. To our knowledge, this is the first suggestion of DAF-16/FOXO regulation exerted at the level of transcription elongation. However, it is important to note that our experiments, which utilized GFP fusions and RT-PCR (that report on the efficiency of transcription in general and not the elongation step in particular), do not rule out the possibility that tcer-1 acts at a different stage of transcription, such as transcription initiation. Unexpectedly, we noticed that the expression of some DAF-16/ FOXO target genes, such as sod-3 and F52H3.5, was further increased in tcer-1(2); glp-1(2) mutants (Figure 4U–4X and Figure S4). We also found that the cytoplasmic levels of DAF-16::GFP were elevated in these animals (Figure S3). One explanation for these observations is that in germline-depleted tcer-1 mutants, the silence of many DAF-16/FOXO targets triggers a compensatory response that elevates DAF-16/FOXO levels in the cytoplasm (and possibly to some extent in the nucleus as well). For tcer-1- dependent genes (such as nnt-1), this elevated DAF-16/FOXO cannot compensate for the absence of TCER-1, and their transcription remains stalled. However, for tcer-1-independent genes (such as sod-3), elevated DAF-16/FOXO levels increase transcription. DAF-16/FOXO activates different patterns of gene expression in response to insulin/IGF-1-pathway inhibition and germline loss Since DAF-16/FOXO increases lifespan in response to germ- line loss and reduced insulin/IGF-1 signaling, a simple model would be that it activates the same downstream lifespan-extending genes in both situations. However, we found that at least some of the genes that are up-regulated in germline-defective mutants in a tcer-1 dependent manner are not up-regulated in daf-2 mutants (for example T21D12.9; see Table S3). This finding suggests that the differential deployment of tcer-1 activity allows reproductive cues and reduced insulin/IGF-1 levels to trigger different patterns of DAF-16/FOXO-dependent gene expression. Thus, this situation illustrates how the activities of broadly-deployed transcription TCER-1 Links Reproductive Signals to Longevity factors can be tailored to precise transcriptional outputs in response to specific signals. Interestingly, some genes that are up-regulated in germline-defective mutants in a tcer-1-dependent way are also up-regulated in daf-2 mutants, but independently of tcer-1 (for example, dod-8). Perhaps a different transcription- elongation factor, one that can substitute for TCER-1, is activated by reduction of insulin/IGF-1 activity. Activation of the reproductive longevity pathway in animals that have germ cells A major question in the field of aging is whether a mode of lifespan extension that is normally triggered by one specific condition, such as caloric restriction, can be triggered instead by intervention at a downstream regulatory step of the pathway. As described above, we found that the longevity pathway activated by loss of the germ cells could potentially operate independently of signals related to reproduction. The finding that germline removal triggers the transcriptional up-regulation of tcer-1 suggested that this regulatory step might function as a germline-pathway specific switch that activates downstream longevity processes. In fact this appears to be the case: when tcer-1 is overexpressed in intact animals that have germ cells, lifespan is extended. This lifespan extension has no detrimental effects on progeny production (data not shown) and is accompanied by several hallmarks of the germline-longevity pathway, including a requirement for KRI-1 and DAF-16/FOXO, and the stimulation of a pattern of gene expression similar to that produced by germline removal. The lifespan increment obtained by overexpressing tcer-1 was not as large as that produced by germ-cell loss. Nevertheless, these data, and other recent observations [58], suggest that at least some of the beneficial longevity effects produced by loss of the germline can be replicated without perturbing reproduction itself. Perspective The last few decades have transformed our view of aging from a haphazard, unregulated phenomenon to a process influenced by conserved genetic pathways. The insulin/IGF-1 pathway and DAF-16/FOXO orthologs have been implicated in lifespan regulation in worms, flies, mice, dogs and several human populations [16,26–33,59,60]. Studies in flies and mice indicate that pathways linking reproduction and aging maybe widespread in nature too. Given this context, the identification of a regulator, TCER-1, that selectively responds to reproductive signals to influence the transcriptional response of a conserved longevity- determinant, DAF-16/FOXO, is exciting. It will be interesting to learn whether TCER-1’s function in aging has also been conserved during evolution. Materials and Methods Strains Strains were maintained as described earlier [61]. The tcer- 1(tm1452) mutant was provided by the National Bioresource Project (Japan) and outcrossed three times to the Kenyon Lab N2 stock. Transgenic strains expressing GFP under control of promoters of DAF-16/FOXO-target genes were obtained from the CGC, or from Prof. David Baillie’s laboratory (Caenorhabditis Gene Expression Consortium). The details of these transgenic animals, strains generated using them and other strains used in this study are described in Table S6 and Table S3. Molecular biology The Ptcer-1::tcer-1::gfp fusion construct was generated as described previously [62]. The complete coding sequence of tcer- PLoS Genetics | www.plosgenetics.org 10 September 2009 | Volume 5 | Issue 9 | e1000639