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Abstract:
The lifecycle of the nematode Caenorhabditis elegans depends greatly on environmental conditions. Under favorable conditions, worms develop from egg to adult in three days, passing 4 larval stages (Li-L4). In unfavorable conditions, they arrest development at larval stage 1 or 3 and enter diapause. The L3 diapause form, the dauer larva, is stress resistant, longlived and specialized for survival and dispersal. The nuclear hormone receptor DAF-12 regulates L3 developmental decision between reproductive growth and the dauer diapause. DAF-12 integrates signals from several endocrine pathways including serotonergic, insulin/IGF, TGF-ß and cGMP signaling (the dauer pathways) via a postulated DAF-12 regulating hormone. In addition, DAF-12 has a role in specifying developmental stage programs in the heterochronic pathway, and in concert with genes from insulin/IGF signaling, it influences the life span of C. elegans.
In order to identify components of postulated DAF-12 regulator complexes, we screened for DAF-12 interacting factors using the yeast-two hybrid method. In total, we identified 22 candidates, seven of which were selected for further studies. For functional characterization of the putative DAF-12 interactors, we performed RNAi feeding assays. One of the candidates, F07A11.6 = din-1 suppressed dauer constitutive (Daf-c) phenotypes of reduced insulin receptor (daf-2), TGF-ß (daf-7), guanyl cyclase (daf-11), cytochrome P450 (daf-9) and nuclear receptor daf-12 signaling. Moreover, din-1RNAi suppressed daf-9 and daf-12 heterochronic gonadal migration phenotypes (Mig), as well as daf-12 heterochronic phenotypes in the epidermis. These results place DIN-1 downstream or parallel to the known dauer pathways, as well as downstream or at the same point as nuclear hormone receptor signal transduction. Protein. BLAST alignments revealed DIN-1 homologous, the nuclear repressor proteins SHARP and MINT in mammals, the Split ends protein SPEN in Drosophila melanogaster, and the C. elegans protein F29C4.7.
Gene finder analyses predict that the largest din-1 product consists of 2784 aa which is organized in 21 exons. In contrast to gene finder prediction, analyses of din-1 EST-cDNA clones and cDNA clones obtained by RT-PCR revealed that most of the din-1 clones lacked exon 18 and harbored an additional exon 3. We found several lines of evidence for the presence of a short din-1 isoform, comprised of exons 18-22 (568 aa), suggesting that din-1 isoforms originate from two transcriptional units.
EMS-mutagenesis screens for revertants of the gonadal Mig phenotype of daf-12 ligand binding domain mutants revealed five din-1 alleles. Representative alleles suppressed dauer pathway mutant Daf-c and heterochronic phenotypes, thus confirming our findings for din-1 RNAi. din-1 mutants cluster in the exon 18, which corresponds to the DIN-1-DAF-12 interaction domain, suggesting that the DIN-1-DAF-12 association is disrupted, but also indicating that none of them is a null allele.
Aging experiments indicated that in daf-2(e1370);din-1(dh127) double mutants mean life span is increased by 35% compared to already long-lived daf-2(e1370) alone. din-1 mutants alone lived as long as wild type. Expression studies with green fluorescence protein (gfp) tagged DIN-1 constructs uncovered wide spread expression in nuclei of different cell types throughout development. In particular, DIN-1 is seen in hypodermis, gonad, muscle, intestine, pharynx and nervous system, tissues remodeled during dauer formation. The nuclear localization of din-1 is consistent with a function in transcriptional regulation.
We suggest that in unfavorable conditions where the production of a dauer diapause suppressing hormone is blocked, DIN-1 binds to DAF-12, assembling a corepressor complex that subsequently arrests reproductive development and promotes dauer diapause. In concordance with the homologues SHARP and MINT in mammals, SPEN in Drosophila and F29C4.7 in C. elegans, DIN-1 forms a class of large nuclear proteins involved in the coordination of basic developmental pathways.
