Sci. Aging Knowl. Environ., 30 April 2003
Vol. 2003, Issue 17, p. pe9
[DOI: 10.1126/sageke.2003.17.pe9]


Direct and Indirect Transcriptional Targets of DAF-16

Pamela L. Larsen

The author is in the Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, USA. E-mail: larsenp{at};2003/17/pe9

The free-living soil nematode Caenorhabditis elegans provides an excellent model system for addressing specific molecular, genetic, and biochemical questions pertaining to development and aging. Several genes involved in the determination of life span have been identified by mutation in C. elegans (1-4) (see Johnson Review ), and the increase in life span reported for these mutants is from ~30 to 400% relative to the wild type. The work by McElwee, Bubb, and Thomas (5) (see McElwee et al.) published in the current issue of Aging Cell focuses on the daf-2 pathway, for which a mechanism of efficient life maintenance has been inferred from the molecular identity of these genes. The daf-2 gene is homologous to the insulin receptor tyrosine kinase, insulin receptor-related receptor, and insulin-like growth factor 1 receptor family (6). Downstream genes age-1 and daf-16 encode a phosphatidylinositol-3-OH kinase and a protein that is homologous to the forkhead transcription factor, respectively (7-9). Like insulin signaling in mammalian systems, the C. elegans DAF-2 pathway controls multiple intracellular pathways that appear to include metabolism, development, stress resistance, and life span (4, 8, 10-13). The daf-16 gene is under the negative control of daf-2 signaling and is speculated to transcriptionally regulate downstream targets that are required for the longevity observed in daf-2 mutant adults. McElwee et al. used gene expression array analysis to search for genes that are differentially expressed between the long-lived daf-2(e1370) animals and the short-lived daf-16(m27);daf-2(e1370) animals. They also identified candidate direct targets of DAF-16. They then tested whether some of the candidate genes identified are necessary for the daf-2 mutant longevity and found that a putative protease is involved in this process.

The experimental design of McElwee et al. was to isolate mRNA from four separate synchronized samples of genetically sterilized 1-day-old adults, generate cDNA, and hybridize the labeled cDNA to DNA microarrays prepared by the Kim laboratory at Stanford. The microarrays contained fragments of genomic DNA from 17,871 C. elegans genes. Each genotype was labeled with one dye and the samples hybridized to the same chip for direct comparison, rather than one sample and a reference sample being hybridized, in an effort to increase measurement precision. It would have been even better if the direct comparisons would have included some chips with the samples labeled with the opposite dye. This swapping of the dyes would have controlled for dye incorporation and detection effects. The log of the average intensity for selected genes was analyzed from several perspectives to assess which genes were most likely to be important for the Age (long-lived) phenotype. First, groups of genes involved in functions previously associated with aging were considered, and the researchers asked whether expression of the particular gene sets was higher or lower than expected. No systematic difference was found for groups of genes involved in the general categories of protein synthesis, protein degradation, mitochondrial function, stress response, programmed cell death, and DNA modification or repair. However, some subgroups showed individual genes outside the average range. For nonproteasomal proteases and cytochrome P450 genes, transcriptional regulation was divergent, in that some of these genes were more highly expressed and others less highly expressed than the bulk of the group. Another subgroup pattern was the up-regulation of several heat shock protein transcripts in mRNA isolated from the long-lived [daf-2(e1370)] strain. An interesting difference is hsp-90, which they mark as more highly expressed in daf-2(e1370) than in daf-16;daf-2, but this was not observed in another report that used a different long-lived allele, daf-2(m41) (14). The availability of many alleles of daf-2 with a range of life span extensions (15) could be useful in sorting out the degree of changes relative to the severity of the Age phenotype. Another important aspect to remember is that microarrays measure steady-state concentrations of mRNA, and changes in these concentrations can be due to either altered transcription or altered message stability, and it is predominantly the latter that is responsible for high hsp-90 mRNA concentrations in dauer larvae (16).

The transcriptional program for the dauer larva has been analyzed previously by serial analysis of gene expression, and dauer-specific and non-dauer-specific genes were identified (17). McElwee et al. assessed the status of the genes classified as dauer-specific and non-dauer-specific in their data and concluded that the differentially expressed transcripts of long-lived daf-2 adults had more dauer-specific transcripts than would be expected to occur by chance. This supports the notion that the efficient life maintenance program of the dauer stage is heterochronically expressed and contributes to adult longevity in daf-2 mutants. In a separate analysis of transcriptional programs, mountains of genes that are thought to be co-regulated have been assembled from the compilation of chip data from a large variety of C. elegans genotypes, environmental conditions, and developmental ages (18). McElwee et al. compared their list of 1646 genes that were differentially expressed by at least 1.5-fold between their long-lived and short-lived strains on each of the four chips to the genes listed in the various mountains. For mounts 6, 15, 19, 22, and 27, differentially expressed genes in the daf-2(e1370) or daf-16;daf-2 strains were overrepresented. The clusters of genes in these mounts have been assigned to the following functions or tissues: dauer-enriched; intestine-enriched; lipid and fatty acid metabolism-enriched; amino acid metabolism- and energy generation-enriched; and unknown function-enriched, respectively. The authors concluded, therefore, that these classes of functions are regulated by DAF-16 and may be involved in the longevity phenotype of daf-2 mutant adults.

The genes highlighted from each of the sorting methods above could be regulated by DAF-16 either directly or indirectly. The final rationale used to whittle the number of genes considered to be important for the Age phenotype was to search the genome for DAF-16 consensus binding sites proximal to the transcriptional start site. The adjacent gene was considered to be a putative direct transcriptional target if there was at least a 1.5-fold difference in expression between the long-lived and short-lived samples for all four arrays. The authors relied on the repeatability of an increase observed on each chip, but it is not clear whether this fold difference is statistically significant for this data set. This approach of searching the genome for consensus binding sites has recently been taken by two other groups, with some variations in selection criteria (19, 20) (see "Foundations of Longevity"). Each of the three groups tested some of the genes identified in their analyses functionally by using RNA-mediated interference (RNAi) to knock down the amount of gene product (21). Not surprisingly, each of the groups identified different genes that can alter the life span of at least one C. elegans genotype. Ookuma et al. identified scl-1 (gene clone name F49E11.9) as being DAF-16-regulated and necessary for daf-2(e1370) longevity. The scl-1 gene encodes a putative secretory protein with an unknown function. McElwee et al. also found F49E11.9 to be differentially expressed in daf-2(e1370) versus daf-16;daf-2, but did not observe changes in the life span phenotype. What might account for this difference? The cultivation temperature and genotypes were the same in both sets of experiments, but each group made its own RNAi construct, and McElwee et al. raised the animals from eggs on the RNAi, whereas Ookuma et al. did not. McElwee et al. found two genes that slightly increased the life span of daf-2(e1370), and these genes shared homology with insulin-like peptides and a putative superoxide dismutase (SOD) that has some homology to previously identified Cu/Zn SOD genes from other organisms (see, for example, SOD1). RNAi of another gene that shares homology with aspartyl proteases, ZK384.3, shortened the life span of daf-2(e1370) and thus appears to be necessary for the longevity phenotype. However, a shortened life span phenotype is a difficult result to interpret, because it could result from the lack of a necessary longevity component or from general sickliness of the new strain. ZK384.3 did not shorten the wild-type life span, so this outcome might indeed result from the lack of a necessary longevity component. However, the extent of the decrease in gene expression by RNAi could differ between genotypes and thus contribute to the observed phenotypes. Further studies with genomic mutants of the ZK384.3 locus could clarify the in vivo role of this gene. It is not clear how ZK384.3 might function in regulating life span. Apartyl proteases participate in a variety of cellular functions ranging from digestion to cleavage of the amyloid precursor protein. Biochemical analyses may help to discriminate between the authors' proposed models of the protease functioning in (i) nonproteasomal protein degradation and/or (ii) proteolytic activation of proteins that subsequently play a role in life span determination.

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Not there yet. Solving the life span puzzle will require further dissection of biochemical pathways and integration of vast amounts of data. [Credit: Terry E. Smith]

Overall, the current work has provided new information on specific gene expression differences between long-lived and short-lived strains of C. elegans. However, the generality of the conclusion that metabolism and stress responses are involved in the longevity phenotype is somewhat frustrating, in that it supports the current beliefs without more enlightenment from the presumed unbiased approach of full genome profiling. So, the bad news is that the molecular basis of life span extension remains undetermined. The particular balance of expression of effector genes that produce intracellular changes that generate the daf-2 Age phenotype is complex and not solely a result of DAF-16 targets. It has been shown that at least two transcription factors affect aging, daf-16 and daf-12 (4, 22, 23). Multiple mRNAs are produced by differential splicing from the daf-12 locus and show homology to nuclear hormone receptors such as the vitamin D receptor and the ecdysone receptor. True understanding of the molecular basis of life span extension will require a more detailed approach with regard to biochemical pathways and probably additional data with different genotypes and manipulations. Sorting the information from vast data sets and integrating previous and current findings will be a computational and intellectual challenge. Thus, the bad news is also good news: The molecular mechanism for life span extension remains undetermined, and we all have much work to do to integrate the pieces and solve this puzzle.

April 30, 2003
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Citation: P. L. Larsen, Direct and Indirect Transcriptional Targets of DAF-16. Sci. SAGE KE 2003, pe9 (30 April 2003);2003/17/pe9

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