[
International C. elegans Meeting,
2001]
Aging is a physiological phenomenon characteristic of metazoan life. As animals age, they suffer a broad functional decline and an exponentially increasing probability of death. Numerous gerontogene mutants have been identified in the nematode Caenorhabditis elegans. These mutants suggest several models by which aging may be slowed and life span prolonged. This study of global transcription during the chronological aging of the worm is a first step to testing several of these models including the stress response, metabolic rate and programmed aging models. DNA microarrays can be used to profile the expression levels of a large number of genes in parallel. Using our array, which contains a PCR product representing every C. elegans gene, we are profiling expression patterns during the normal aging process. We isolated RNA from synchronized cultures of sterile animals at various ages from young adult to old age. We hybridized labeled cDNA made from this RNA to DNA microarrays, and find that 212 (p<.001) genes show statistically significant changes with chronological age. This is a relatively small number of genes relative the number with significant changes during development, those specific for male and hermaphrodite developmental programs, and those involved in reproduction. We find that transposons show progressively increasing transcript levels as the worms age, and several gene classes including histone and mitochondrial genes exhibit progressive down-regulation as the worms age. Many oocyte genes maintain transcript levels past the reproductive period, declining only in old worms. This global transcript profile is the first detailed and complete molecular profile of aging in the worm.
[
Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI,
2010]
DAF-16, a forkhead-type transcription factor, function in downstream of an insulin/insulin-like growth factor-1 (Ins/IGF-1) signaling pathway, and is related to regulation of aging and oxidative stress resistance in the nematode Caenorhabditis elegans (C. elegans). In the latest report, we described that a consensus DAF-16 binding element (DBE), which binds the DAF-16 transcription factor, was discovered in the promoter region of
sod-5 gene encoding a Cu/Zn superoxide dismutase (SOD) in C. elegans. The DBE position was 192 base pairs (bp) upstream of the first exon of
sod-5 gene from the C. elegans genomic database. In a
daf-16 gene null mutant,
daf-16(mgDf50) strain, the
sod-5 gene expression as well as
sod-3 gene was obviously suppressed compared with it in wild-type N2. The phenotype of lifespan in
daf-16(mgDf50) animals was reduced when compared with N2. Therefore, it is suggested that
sod-5 gene expression is necessary to maintain the lifespan in wild-type with
sod-3 gene during normal aging. In addition, we could not find a functional compensation by
sod-1 gene in the
sod-5 deletion mutant such as the compensatory expression of
sod-5 gene via the Ins/IGF-1 signaling pathway in the
sod-1 deletion mutants (1).
sod-1 gene might have considerable capacity in the expression during the C. elegans normal aging. Thus, it is assumed that the Ins/IGF-1 signaling pathway plays more important role to epigenetic regulation of the target genes under aging and stressful conditions. Reference 1. Yanase S, Onodera A, Tedesco P, Johnson TE, Ishii N (2009) SOD-1 deletions in Caenorhabditis elegans alter localization of intracellular reactive oxygen species and show molecular compensation. J Geront 64A: 530-539.
[
West Coast Worm Meeting,
2004]
Despite the wealth of knowledge about conditions and mutations that cause worms to live longer, we still have an incomplete understanding of what varies from young to old worms -- especially at the molecular level. A long-term goal of our laboratory is to determine the molecular changes that occur as an organism ages in order to better understand the aging process and its regulation. Using the nematode Caenorhabditis elegans ( C. elegans ) as a genetic model for aging, we have used DNA microarrays to identify a common set of genes regulated throughout several age-related conditions: genes that change in old age (Lund et al. , 2002), genes that change in the exit from the dauer state (Wang and Kim, 2003), and genes that change expression in long-lived mutants of the insulin-like pathway such as the
age-1 and
daf-16 mutants (unpublished data). Analysis of the upstream regions of these age-regulated genes showed that they are heavily enriched for a GATA DNA consensus sequence suggesting that a GATA transcription factor may be involved in regulating the expression of these genes. This GATA motif may represent a novel regulatory pathway of the aging process that might act either together or separately from the
daf-2 /insulin-like pathway. Using GFP reporters of these genes, we have begun to determine how these genes are age-regulated and to identify the key tissues regulating lifespan. We have also used RNAi to abrogate the expression of these genes to determine their role in longevity. The results of these studies will define a set of molecular biomarkers that will allow a more accurate description of the aging process. In addition, these biomarkers will allow identification of new aging mutants perhaps leading to identification of mutants with accelerated aging. Lund, J., Tedesco, P., Duke, K., Wang, J., Kim, S. K., and Johnson, T. E. (2002). Transcriptional profile of aging in C. elegans. Curr Biol 12, 1566-1573. Wang, J., and Kim, S. K. (2003). Global analysis of dauer gene expression in Caenorhabditis elegans. Development 130, 1621-1634.
[
European Worm Meeting,
2006]
Peter I. Joyce and Patricia E. Kuwabara . Regulated proteolysis of receptors, cytoskeletal proteins and transcription factors is an important process that modulates cell growth and development. A family of calcium-regulated thiol proteases, known as calpains, has been shown to perform such processes in mammals, and other eukaryotes. Loss of function mutations in calpains are linked to certain pathological conditions including limb-girdle muscular dystrophy 2A (LGMD2A); whereas, gain of function mutations are associated with the formation of cataracts. . We are investigating the biochemical and functional role of atypical calpains in C. elegans. We have identified seven atypical calpains and eight calpain-like sequences within the C. elegans genome. To gain an understanding of their possible roles in C. elegans development we have created calpain promoter transcriptional expression constructs for the atypical calpains 1 to 7 using monomeric RFP. The transcriptional expression patterns were analysed by co-localisation with the following tissue specific GFP markers:
unc-119::GFP (all neurons),
unc-47::GFP (GABAergic neurons within the ventral nerve cord),
myo-3::GFP (body wall muscle), exc::GFP (excretory cell) and seam::GFP (seam cells). Five out of the seven atypical calpains show promoter transcriptional expression activity;
clp-1,
clp-2,
clp-4,
tra-3 and
clp-7. Two of the atypical calpain promoter constructs fail to show detectable transcriptional expression activity;
clp-3 (lacks all catalytic residues) and
clp-6 (possible pseudogene).
clp-1 shows the highest expression throughout the animal, specifically in neurons and body wall muscle. . In parallel with expression studies we have taken a global approach into studying calpain function using RNAi, calpain mutant analysis and ectopic over-expression. Preliminary studies have revealed no obvious effects. Because calpains require calcium for proteolytic activity, we are investigating ways in which calcium levels can be perturbed within C. elegans, as a way to activate calpains ectopically. We are also undertaking biochemical studies to characterise the proteolytic properties of the atypical calpains.
[
International Worm Meeting,
2005]
Mutations in genes of the
daf-2 insulin/insulin-like growth factor signaling (IIS) pathway have been found to extend adult lifespan in C. elegans1. This increase in lifespan resulting from decreased IIS is supressed by a loss of function of the
daf-16 gene which encodes a forkhead transcription factor2,3. A microarray analysis was carried out at the Sanger Institute on two temperature sensitive (ts) long-lived strains of C. elegans:
daf-2(
sa193) and
daf-2 (
m41), and compared their transcription profiles to those of wild type N2 and
daf-7 (
e1372) ts adult worms. From this study, 1,700 genes were identified as being 2-fold up- or down-regulated in the long-lived nematodes. These 1,700 genes were compared to the gene expression profiles obtained from other microarray ageing studies4,5,6. Based on these comparisons we selected 20 up- and 20 down-regulated genes for gene knockout analysis by RNAi. An RNAi feeding protocol was designed using the ts sterile C. elegans mutant
glp-4 (
bn2 ts).
glp-4 nematodes were fed on the E. coli dsRNA feeding strains and lifespan was measured by counting the number of live nematodes every second day. A long-lived control consisting of a C. elegans
glp-4 fed on
daf-2 dsRNA expressing E. coli (
daf-2 knockout) and a normal lifespan control consisting of C. elegans
glp-4 fed on E. coli transformed with the empty vector (no gene knockout) were also used. These experiments are ongoing. Our results to date show that RNAi of 4 down-regulated genes increases lifespan and RNAi of 3 up-regulated genes decreases lifespan. We will report on our progress on these RNAi experiments. We are grateful to Dr. Patricia Kuwabara in whose laboratory the microarry experiment was carried out, to Dr. David Gems and Dr. Joshua McElwee for helpful discussions and to Enterprise Ireland for financial support. 1. Kenyon, C. et al. (1993). Nature 366, 461-464. 2. Lin K. et al. (1997). Science 278, 1319-1322. 3. Ogg. S. et al. (1997). Nature 389, 994-999. 4. Murphy, C.T. et al. (2003). Nature, 424, 277-284. 5. Kimura, K.D. et al. (1997). Nature, 277, 942-946. 6. McElwee J.J. et al. (2004). J. Biol. Chem. 279, 44533-44543.
[
West Coast Worm Meeting,
2004]
Regulatory motifs are short sequences of DNA that regulate the level, timing, and location of gene expression. Identifying these motifs and their functions is crucial in our understanding of gene regulation and disease processes. We developed CompareProspector, a motif-finding program that takes advantage of cross-species sequence comparison to identify putative regulatory motifs from sets of co-regulated genes [1] . We applied CompareProspector to 30 sets of genes with very similar patterns of expression, identified from the C. elegans topomap [2] and individual DNA microarray experiments. The statistical significance of each candidate motif identified was evaluated using criteria such as motif enrichment-the ratio of prevalence of the motif in a given set of promoters to its prevalence elsewhere in the genome, and the expression coherence of genes with the motif. We identified twelve significant regulatory motifs, three of which have literature evidence confirming they are true regulatory motifs. Overall, these twelve motifs are found in the upstream regulatory regions of 2970 different genes, and may be involved in gene regulation in 24 clusters of co-expressed genes. The first known motif, with the consensus TGATAA, matches the consensus of known binding sites for GATA factors. As GATA factors are known to be involved in worm intestine development [3] and hyperdermis development, it is not surprising that the GATA motif is identified from a set intestine-specific genes (F. Pauli, unpublished), mount08 of the topomap, which is enriched in genes from the intestine, and several collagen-related datasets (mount14, 17, and 35 of the topomap). We correctly identified GATA sites in the promoters of genes known to be regulatory by GATA factors. Interestingly, the GATA motif is also identified from several data sets involved in the aging process. This result parallels that of Murphy and colleagues, who independently identified this motif from their data set of DAF-16 target genes [4] . Both our result and the result from Murphy suggest that GATA factors may be involved in worm aging. Motif 2, which is identified in the two heat shock-related data sets, matches the consensus of known binding sites for heat shock factors [5] . Motif 3 matches the consensus of heat shock associated sites (HSAS), a motif that was first predicted computationally to be involved in the heat shock process [6] and later experimentally validated to be involved in ethanol stress response (14 th International C. elegans Conference abstract 1113C). We are currently in the process of validating the rest of the motifs and their individual binding sites using mutagenesis studies of promoters with predicted motifs. 1. Liu, Y., Liu, X.S., Wei, L., Altman, R.B. and Batzoglou, S. (2004) Eukaryotic regulatory element conservation analysis and identification using comparative genomics . Genome Res. 14 , 451-8. 2. Kim, S.K., Lund, J., Kiraly, M., Duke, K., Jiang, M., Stuart, J.M., Eizinger, A., Wylie, B.N. and Davidson, G.S. (2001) A gene expression map for Caenorhabditis elegans . Science. 293 , 2087-92. 3. Maduro, M.F. and Rothman, J.H. (2002) Making worm guts: the gene regulatory network of the Caenorhabditis elegans endoderm . Dev Biol. 246 , 68-85. 4. Murphy, C.T., McCarroll, S.A., Bargmann, C.I., Fraser, A., Kamath, R.S., Ahringer, J., Li, H. and Kenyon, C. (2003) Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans . Nature. 424 , 277-83. 5. Amin, J., Ananthan, J. and Voellmy, R. (1988) Key features of heat shock regulatory elements . Mol Cell Biol. 8 , 3761-9. 6. GuhaThakurta, D., Palomar, L., Stormo, G.D., Tedesco, P., Johnson, T.E., Walker, D.W., Lithgow, G., Kim, S. and Link, C.D. (2002) Identification of a novel cis-regulatory element involved in the heat shock response in Caenorhabditis elegans using microarray gene expression and computational methods . Genome Res. 12 , 701-12 .