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BRCA2 is a multifunctional tumor suppressor involved in homologous recombination ( HR), mitotic checkpoint regulation, and telomere homeostasis. Absence of Brca2 in mice results in progressive shortening of telomeres and senescence, yet cells are prone to neoplastic transformation with elongated telomeres, suggesting that BRCA2 has positive and negative effects on telomere length regulation along the path to tumorigenesis. Using Caenorhabditis elegans as a model, we show here that depletion of BRC-2, an ortholog of BRCA2, paradoxically delays senescence in telomerase-deficient mutant worms. Telomerase-deficient worms ( trt-1) exhibit early replication senescence due to short telomeres. It should be noted that worms mutated in brc-2 are not viable as well due to massive genotoxic insults. However, when BRC-2 is depleted by RNA interference in trt-1 mutant worms, the number of generations is unexpectedly increased with telomere length maintained, compared to telomerase mutants. Interestingly, depletion of other HR genes such as rad-51 and rad-54 exhibited similar effects. In worms doubly deficient of telomerase and brc-2, rad-51, or rad-54, extra telomeric C-circles were generated, suggesting that abrogation of HR induces an alteration in telomere environment favorable to illegitimate telomere maintenance when telomerase is absent. Collectively, absence of BRC-2 in telomerase-deficient background first leads to telomere shortening, followed by an induction of an as-yet-unknown telomere maintenance pathway, resulting in delay of senescence. The results have implications in the understanding of dysfunctional BRCA2-associated tumorigenesis. [ABSTRACT FROM AUTHOR]

Cilia are important for the interaction with environments and the proper function of tissues. While the basic structure of cilia is well conserved, ciliated cells have various functions. To understand the distinctive identities of ciliated cells, the identification of cell-specific proteins and its regulation is essential. Here, we report the mechanism that confers a specific identity on IL2 neurons in Caenorhabditis elegans , neurons important for the dauer larva-specific nictation behavior. We show that DAF-19M, an isoform of the sole C. elegans RFX transcription factor DAF-19, heads a regulatory subroutine, regulating target genes through an X-box motif variant under the control of terminal selector proteins UNC-86 and CFI-1 in IL2 neurons. Considering the conservation of DAF-19M module in IL2 neurons for nictation and in male-specific neurons for mating behavior, we propose the existence of an evolutionarily adaptable, hard-wired genetic module for distinct behaviors that share the feature "recognizing the environment." [Display omitted] • DAF-19M heads a regulatory subroutine for target genes via an X-box motif variant • This DAF-19M regulatory subroutine is active in ciliated IL2 neurons • DAF-19M in IL2 in turn is regulated by terminal selector proteins UNC-86 and CFI-1 • We propose a genetic module for distinct ciliary and behavioral specializations In Caenorhabditis elegans , IL2 ciliated neurons are required for nictation behavior. What makes IL2 neurons different from other ciliated neurons? Ahn et al. demonstrate that DAF-19M, an isoform of the sole C. elegans RFX transcription factor DAF-19, regulates IL2-specific, cilia-associated genes through an X-box motif variant. [ABSTRACT FROM AUTHOR]

Abstract: Caenorhabditis elegans microRNAs (miRNAs) bind to partially complementary sequences in the 3′ untranslated region of target mRNAs, resulting in translational repression through mRNA destabilization. High-throughput sequencing of RNA cleavage fragments was performed to directly detect miRNA-directed cleavage targets in adult stage C. elegans. From this analysis, we found that miR-249 directed the cleavage of the ZK637.6 transcript with extensive and evolutionarily conserved complementarity in nematode. In addition, expression of the ZK637.6 transcript was strongly dependent on the expression of miR-249. These findings may lead to a better understanding of miRNA-mediated gene regulation in nematodes. [Copyright &y& Elsevier]

Abstract: During development of Caenorhabditis elegans, expression of the RUNX homolog, rnt-1, is tightly regulated both spatially and temporally. In this study, we investigated the mechanism underlying the temporal regulation of rnt-1. We found that rnt-1 contained evolutionarily conserved consensus RUNX binding sequences within one of its introns, and that RNT-1 bound to these intronic sequences both in vitro and in vivo in the presence of BRO-1, suggesting that RNT-1 together with BRO-1 represses its own transcription. Fine deletion and substitution experiments revealed a binding site within the intron that was critical for rnt-1 regulation. Importantly, we found that the TGFβ homolog, DBL-1, was required for counteracting the repressive activity of BRO-1 at postembryonic stages. Accordingly, ectopic expression of DBL-1 induced transcription of rnt-1 in the lateral hypodermis and other tissues even at the postembryonic stages. Taken together, our data suggest that rnt-1 expression is regulated by the balance between DBL-1-mediated activation and BRO-1-mediated repression at the postembryonic stages. [Copyright &y& Elsevier]

Many protein components of telomeres, the multifunctional DNA–protein complexes at the ends of eukaryotic chromosomes, have been identified in diverse species ranging from yeast to humans. In Caenorhabditis elegans, CEH-37 has been identified by a yeast one hybrid screen to be a double-stranded telomere-binding protein. However, the role of CEH-37 in telomere function is unclear because a deletion mutation in this gene does not cause severe telomere defects. This observation raises the possibility of the presence of genetic redundancy. To identify additional double-stranded telomere-binding proteins in C. elegans, we used a different approach, namely, a proteomic approach. Affinity chromatography followed by Finnigan LCQ ion trap mass spectrometer analysis allowed us to identify several candidate proteins. We further characterized one of these, HMG-5, which is encoded by F45E4.9. HMG-5 bound to double-stranded telomere in vitro as shown by competition assays. At least two telomeric DNA repeats were needed for this binding. HMG-5 was expressed in the nuclei of the oocytes and all embryonic cells, but not in the hatched larvae or adults. HMG-5 mainly localized to the chromosomal ends, indicating that HMG-5 also binds to telomeres in vivo. These observations suggest that HMG-5 may participate, together with CEH-37, in early embryogenesis by acting at the telomeres. [Copyright &y& Elsevier]

In order to characterize the mechanisms regulating neuronal expression of the nematode SNAP-25 gene, we identified the SNAP-25 genes of Caenorhabditis elegans and Caenorhabditis briggsae. Comparative sequence analysis and reporter assays revealed two putative 5′ regulatory elements, P1 and P2, and four elements, I1h, I1m, I2h, and I2m, in the first intron. Nuclear extracts contained activities that bound the P2 and I1h elements. Different elements were required for SNAP-25 expression in different neuronal subsets; P1 was required in DA and DD motor neurons, and I1m and I2m were required in DB and DA neurons, respectively. P2 was active in amphid and phasmid neurons, I1h in pharyngeal neurons, and I2h in touch receptor neurons. The I2h element contained a putative binding site for transcription factor UNC-86. Both UNC-86 and MEC-3 were required for I2h activity in the mechanosensory neurons: in these neurons, GFP expression driven by I2h was abolished in animals bearing either an unc-86 null or a mec-3 null mutation, or an unc-86 mutation that leads to defective interaction with MEC-3. Deletion of the MEC-3 binding site also abolished the GFP expression. Gel mobility-shift assay results suggest that transcriptional regulation of SNAP-25 may involve multiple transcription factors. [Copyright &y& Elsevier]

Abstract: Acute exposure to ethanol causes paralysis at high concentrations in the nematode Caenorhabditis elegans. We set out to elucidate the mechanism of the anesthetic action of ethanol by genetic approaches. We identified nine mutations that conferred reduced sensitivity to ethanol after chemical, irradiation, or transposon insertion mutagenesis. Of these nine, we further characterized five mutations that defined four genes, jud-1–jud-4. Analysis of the phenotypes of the animals heterozygous for two unlinked genes revealed that jud-1 and jud-3 act synergistically in a gene dose-dependent manner. We cloned jud-4 and found that it encodes a protein with limited homology to human Homer proteins. jud-4 was expressed in the hypodermis and vulva muscles, suggesting that this gene acts in tissues directly exposed to the external environment. Characterization of the other mutations identified in this study will facilitate the elucidation of the molecular mechanism for the anesthetic action of ethanol. [Copyright &y& Elsevier]

To study the interplay of rare outcrossing and metapopulation structure, we focus on the nematode Caenorhabditis elegans. Its remarkably low outcrossing rate is at the extreme end of the spectrum for facultative selfing organisms. At the demographic level, C. elegans natural populations undergo boom and bust dynamics on ephemeral resources, with the dauer diapause larva acting as the dispersal form. Here we investigate the small-scale genetic structure of C. elegans populations in two localities over several years, using 2b restriction-associated DNA sequencing of nearly 1000 individuals. We find a remarkably small number of genome-wide haplotypes, almost exclusively in the homozygous state, confirming the low effective outcrossing rate. Most strikingly, the major haplotypes in a locality remain intact and do not effectively recombine over several years. From the spatial pattern of diversity, we estimate that each subpopulation or deme is seeded by a mean of 3--10 immigrating individuals. Populations are thus formed by clones that compete at two levels, within a subpopulation and at the metapopulation level. We test for the presence of local phenotypic variation in pathogen resistance and dauer larva nictation, which could possibly explain the maintenance of different genotypes by heterogeneous selection in different local environments or lifecycles. This study is the first to address the local spatiotemporal genetic structure of C. elegans on feeding substrates. We conclude that these animals coexist as competing homozygous clones at the smallest population scale as well as in the metapopulation. [ABSTRACT FROM AUTHOR]

Free-living nematode Caenorhabditis elegans exhibits various behaviors to adapt to the fluctuating environment. When early larvae of C. elegans experience the harsh environmental condition, they develop to an alternative developmental stage called dauer, which shows nictation, a stage-specific waving behavior. Nictation enables dauers to attach to more mobile animals, which helps them disperse to other habitats beyond physical barriers. However, underlying molecular mechanisms that regulate nictation behavior are largely unknown. In this study, we show that insulin signaling and transforming growth beta (TGF-β) signaling, the two major parallel signaling pathways that mediate dauer development, are involved in the regulation of dauer-specific nictation behavior. Genetic analysis revealed that downregulation of insulin signaling enhanced nictation behavior. Heat-shock induced rescue experiments showed that the action period of the insulin signaling is before dauer formation. Surprisingly, lowering of TGF-β signaling inhibited the normal performance of nictation, suggesting that TGF-β signaling acts in an opposite way from that for dauer formation. Cell-specific rescue experiments revealed that two signaling pathways act in the nervous system and an epistasis experiment showed that TGF-β signaling is epistatic to insulin signaling. Taken together, we propose that the neuroendocrinal insulin signaling and TGF-β signaling regulate nictation behavior during development in response to environmental conditions. [ABSTRACT FROM AUTHOR]

Abstract: SNARE domain proteins are key molecules mediating intracellular fusion events. SNAP25 family proteins are unique target-SNAREs possessing two SNARE domains. Here we report the genetic, molecular, and cell biological characterization of C. elegans SNAP-29. We found that snap-29 is an essential gene required throughout the life-cycle. Depletion of snap-29 by RNAi in adults results in sterility associated with endomitotic oocytes and pre-meiotic maturation of the oocytes. Many of the embryos that are produced are multinucleated, indicating a defect in embryonic cytokinesis. A profound defect in secretion by oocytes and early embryos in animals lacking SNAP-29 appears to be the underlying defect connecting these phenotypes. Further analysis revealed defects in basolateral and apical secretion by intestinal epithelial cells in animals lacking SNAP-29, indicating a broad requirement for this protein in the secretory pathway. A SNAP-29-GFP fusion protein was enriched on recycling endosomes, and loss of SNAP-29 disrupted recycling endosome morphology. Taken together these results suggest a requirement for SNAP-29 in the fusion of post-Golgi vesicles with the recycling endosome for cargo to reach the cell surface. [Copyright &y& Elsevier]

Abstract: Telomeres are multifunctional elements that protect chromosomal ends. Telomere length affects lifespan and stress resistance. In our previous report, we found that the increase of telomere length extended organismic lifespan in Caenorhabditis elegans. To investigate the effect of the telomere length on organismic lifespan at the global gene expression level, we performed microarray experiments using mRNA from long telomere-containing worms. We identified genes that showed significant difference in transcript levels from long telomere-containing worms.We examined the expression patterns of these genes and measured lifespan of the worms over-expressing each of the up-regulated genes. Most up-regulated genes, when over-expressed, extended lifespan of C. elegans. In particular, over-expression of rnh-1.3 in the intestine extended lifespan in the daf-16-dependent manner and increased stress resistance. Interestingly, genes up-regulated in long telomeric worms partially overlapped with the genes whose transcription was increased by the rde-3 mutations, and the over-expression of each of these genes also extended the longevity of worms, raising the possibility that the telomere effect may involve molecular players shared with the RNAi machinery. [Copyright &y& Elsevier]

The nematode shows responses to acute ethanol exposure that are similar to those observed in humans, mice, and Drosophila, namely hyperactivity followed by uncoordination and sedation. We used in this report the nematode Caenorhabditis elegans as a model system to identify and characterize the genes that are affected by ethanol exposure and to link those genes functionally into an ethanol-induced gene network. By analyzing the expression profiles of all C. elegans ORFs using microarrays, we identified 230 genes affected by ethanol. While the ethanol response of some of the identified genes was significant at early time points, that of the majority was at late time points, indicating that the genes in the latter case might represent the physiological consequence of the ethanol exposure. We further characterized the early response genes that may represent those involved directly in the ethanol response. These genes included many heat shock protein genes, indicating that high concentration of ethanol acts as a strong stress to the animal. Interestingly, we identified two non-heat-shock protein genes that were specifically responsive to ethanol. glr-2 was the only glutamate receptor gene to be induced by ethanol. T28C12.4, which encodes a protein with limited homology to human neuroligin, was also specific to ethanol stress. Finally, by analyzing the promoter regions of the early response genes, we identified a regulatory element, TCTGCGTCTCT, that was necessary for the expression of subsets of ethanol response genes. [Copyright &y& Elsevier]