[
International Worm Meeting,
2007]
The insulin/insulin-like growth factor-I signaling (IIS) pathway regulates larval diapause and adult lifespan in Caenorhabditis elegans. To date, many of the 38 insulin-like genes have been identified1), and a few of the genes have been investigated to identify their physiological function by RNAi knockdown. For instance, Murphy and co-workers reported that
ins-7 RNAi knockdown induces larva diapause and an extended lifespan.2) In our previous study, we disrupted Ceinsulin-1 (
ins-18) and Ceinsulin-2 (
ins-17), and elucidated their function on larval diapause and adult lifespan. Disruption of
ins-17 and/or
ins-18 reduced dauer larva formation caused by a crude extract of dauer-inducing pheromone, while the disruption showed no effect on adult lifespan. To investigate redundant function of the insulin-like genes, we disrupted the
ins-7, and then established multiple-gene-disrupted animals. Disruption of
ins-7 induced an extended lifespan as expected. Disruption of
ins-7 and
ins-17 also revealed lifespan extension, suggesting that
ins-17 is not relevant to lifespan regulation. On the other hand, disruption of
ins-7 and
ins-18 revealed no lifespan extension, indicating that
ins-18 is necessary for lifespan extension induced by the gene-disruption of
ins-7. Now we are measuring lifespan of each gene-disrupted animal under
daf-2(-) conditions. 1) Pierce, S. B. et al. (2001) Genes Dev. 15:672. 2) Murphy, C. T. et al. (2003) Nature 424:277.
[
International C. elegans Meeting,
1991]
mig-l O III mutants exhibit partially defective embryonic migration of CAN, ALM and HSN neurons and have shortened posterior excretory canals (pec's). Genetic mosaic analysis suggests that mig-lO(+) activity within the excretory cell is neither necessary nor sufficient to effect normal pec outgrowth. Thus,
mig-10 may encode a cell- extrinsic component involved in cell movement. Patterns of mosaic Dp loss are consistent with a hypodermal focus, although certain other tissues have not been ruled out. Because the cells whose movements are affected in
mig-10 mutants develop in contact with the body wall, a hypodermal focus would not be surprising. mig-l O has been cloned by transformation rescue. Germline microiniection of either of two overlapping cosmid clones, FlOE9 or F49A3, can completely rescue mig-l O
(ct41 ) phenotypes. Both cosmids have been used as probes to isolate a set of at least 22 genomic lambda phage clones containing sequences from the overlap. The lambdas are currently being used in transformation rescue experiments designed to narrow
mig-10 to a physical interval of manageable size for molecular analysis.
[
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 .
Merlet, Jorge, Gomes, Jose-Eduardo, Tavernier, Nicolas, Pintard, Lionel, Burger, Julien, Richaudeau, Benedicte
[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
In early C. elegans embryos, the ATR (ataxia telangectasia mutated and
rad3 related) checkpoint pathway controls the timing of cell division in the P lineage, or future germ line of the animal. However, the mechanisms regulating the ATR checkpoint pathway are still poorly understood. Here, we establish that the Leucine Rich Repeats protein, LRR-1, promotes cell cycle progression during development in C. elegans , both in the adult germ line and in the early embryo. Our results indicate that LRR-1 is a substrate-recruitment factor of a cullin 2-RING E3-ligase (CRL2), which down regulates the ATR signaling pathway in C. elegans . LRR-1 contains a typical BC/Cul2-box through which it binds CRL2 components in vitro and in vivo . Lo ss of
lrr-1 function causes a cell-cycle arrest in the mitotic region of the germ line resulting in sterility owing to depletion of germ cells. Inactivation of the DNA replication checkpoint signaling components ATR/ATL-1 and Chk1/CHK-1 suppresses this cell cycle arrest and fully restores
lrr-1 mutant fertility. Likewise, in early embryos,
lrr-1 inactivation leads to hyper-activation of the ATL-1/Chk1 pathway, which delays mitotic entry and causes embryonic lethality. Our findings suggest a model in which the CRL2 LRR-1 E3 ligase negatively regulates the DNA replication checkpoint and thereby coordinates cell cycle progression and development.
[
International Worm Meeting,
2005]
Studies in our and other labs have suggested that critical events during the mid-life of the nematode can influence the aging and lifespan of this organism. In an effort to better understand the biology of aging with an emphasis on mid-life changes that influence healthspan, we performed a DNA microarray analysis of global gene expression profiles over time using Affymetrix gene chip arrays. Our experiment includes time points covering the reproductive and post-reproductive periods, with a series of consecutive mid-life time points. For our analyses, we used unsupervised methods, including a clustering method called Two-Way SPC (see. Domany et al.,Phys. Rev.Lett 76,3521). The method is actually a new approach to clustering, based on rules which can be described with help of statistical mechanics. Interestingly, we do find a sharp change in the transcriptional levels of numerous genes at about 10 days post egg-lay. This abrupt change in gene expression on day 10 of adulthood is consistent with a transition point at which potentially harmful autofluorescent biomarkers accumulate at accelerated rates (see abstract by Gerstbrein et al.) and at which age-related muscle decline may also accelerate. Since similar microarray experiments have been published (Lund et al., 2001, Curr Biol. 12(18): 1566-73; Murphy et al., 2003, Nature, 424(6946):277-83), we attempted a detailed cross-comparison between data from all three experiments, using again unsupervised, as well as supervised techniques. From the supervised technique we found approximately 100 genes that change expression during adult lifespan that are common to all three studies. We consider that these expression changes might be relevant to rapid end-stage deterioration in old nematodes.
[
International Worm Meeting,
2007]
Enteropathogenic E. coli (EPEC) is a gastrointestinal pathogen that kills infants in developing countries through diarrhea and dehydration. To better understand the mechanisms of EPEC virulence and host response, we developed an EPEC/C. elegans model and showed that EPEC paralyzes and kills C. elegans via a secreted toxin. However,
daf-2 and
age-1 mutant worms proved to be more resistant to EPEC virulence.
daf-2 via
age-1 prevents the translocation of
daf-16 to the nucleus to up-regulate survival genes and antimicrobial factors. Thus, EPEC killing of C. elegans appears to involve inactivation of factors that permit the worm to accommodate to environmental or other stresses associated with aging. Using microarray analysis, Murphy et. al., (2003) demonstrated that 263 and 251 genes were up-regulated to promote longevity, or down-regulated to prevent life shortening by
daf-16 respectively. To determine which genes are important for mediation against EPEC toxins, we inhibited in
daf-2(
e1370) mutants, the up-regulated genes by RNA interference and exposed these worms to EPEC. We report that RNAi against
abl-1 and
hsf-1 renders
daf-2(
e1370) mutants as sensitive to EPEC virulence as wild type. Other genes such as
spp-1,
aqp-1, and
dod-6 rendered the
daf-2 mutants partially sensitive. We are currently studying the mechanisms underlying the sensitivity of
abl-1 and
hsf-1 mutants. We further determined whether specific tissues act as signaling centers by expressing DAF-16 under tissue specific promoters in the
daf-2;
daf-16 mutants and exposing the worms to EPEC virulence. Our analyses show that expression of DAF-16 under a neuronal promoter elicited the strongest resistance to EPEC. Together, our findings suggest that contact with EPEC initiates a protective neuronal response that may decrease
daf-2 activity leading to the transcription of antimicrobial genes to facilitate survival.