SMK-1/PPH-4.1-mediated silencing of the CHK-1 response to DNA damage in early C. elegans embryos

During early embryogenesis in Caenorhabditis elegans, the ATL-1-CHK-1 (ataxia telangiectasia mutated and Rad3 related-Chk1) checkpoint controls the timing of cell division in the future germ line, or P lineage, of the animal. Activation of the CHK-1 pathway by its canonical stimulus DNA damage is actively suppressed in early embryos so that P lineage cell divisions may occur on schedule. We recently found that the rad-2 mutation alleviates this checkpoint silent DNA damage response and, by doing so, causes damage-dependent delays in early embryonic cell cycle progression and subsequent lethality. In this study, we report that mutations in the smk-1 gene cause the rad-2 phenotype. SMK-1 is a regulatory subunit of the PPH-4.1 (protein phosphatase 4) protein phosphatase, and we show that SMK-1 recruits PPH-4.1 to replicating chromatin, where it silences the CHK-1 response to DNA damage. These results identify the SMK-1-PPH-4.1 complex as a critical regulator of the CHK-1 pathway in a developmentally relevant context.     

Inducible systemic RNA silencing in Caenorhabditis elegans

Introduction of double-stranded RNA (dsRNA) can elicit a gene-specific RNA interference response in a variety of organisms and cell types. In many cases, this response has a systemic character in that silencing of gene expression is observed in cells distal from the site of dsRNA delivery. The molecular mechanisms underlying the mobile nature of RNA silencing are unknown. For example, although cellular entry of dsRNA is possible, cellular exit of dsRNA from normal animal cells has not been directly observed. We provide evidence that transgenic strains of Caenorhabditis elegans transcribing dsRNA from a tissue-specific promoter do not exhibit comprehensive systemic RNA interference phenotypes. In these same animals, modifications of environmental conditions can result in more robust systemic RNA silencing. Additionally, we find that genetic mutations can influence the systemic character of RNA silencing in C. elegans and can separate mechanisms underlying systemic RNA silencing into tissue-specific components. These data suggest that trafficking of RNA silencing signals in C. elegans is regulated by specific physiological and genetic factors.     

Mutator phenotype of Caenorhabditis elegans DNA damage checkpoint mutants

DNA damage response proteins identify sites of DNA damage and signal to downstream effectors that orchestrate either apoptosis or arrest of the cell cycle and DNA repair. The C. elegans DNA damage response mutants mrt-2, hus-1, and clk-2(mn159) displayed 8- to 15-fold increases in the frequency of spontaneous mutation in their germlines. Many of these mutations were small- to medium-sized deletions, some of which had unusual sequences at their breakpoints such as purine-rich tracts or direct or inverted repeats. Although DNA-damage-induced apoptosis is abrogated in the mrt-2, hus-1, and clk-2 mutant backgrounds, lack of the apoptotic branch of the DNA damage response pathway in cep-1/p53, ced-3, and ced-4 mutants did not result in a Mutator phenotype. Thus, DNA damage checkpoint proteins suppress the frequency of mutation by ensuring that spontaneous DNA damage is accurately repaired in C. elegans germ cells. Although DNA damage response defects that predispose humans to cancer are known to result in large-scale chromosome aberrations, our results suggest that small- to medium-sized deletions may also play roles in the development of cancer.     

Altered DNA damage response in Caenorhabditis elegans with impaired poly(ADP-ribose) glycohydrolases genes expression

Poly(ADP-ribosyl)ation is one of the first cellular responses induced by DNA damage. Poly(ADP-ribose) is rapidly synthesized by nick-sensor poly(ADP-ribose) polymerases, which facilitate DNA repair enzymes to process DNA damage. ADP-ribose polymers are rapidly catabolized into free ADP-ribose units by poly(ADP-ribose) glycohydrolase (PARG). The metabolism of poly(ADP-ribose) is a well-defined biochemical process for which a physiological role in animals is just beginning to emerge. Two Caenorhabditis elegans PARGs, PME-3 and PME-4, have been cloned by our group. The pme-3 gene encodes an enzyme of 89kDa having less than 18% overall identity with human PARG but 42% identity with the PARG signature motif. The pme-4 gene codes for a PARG of 55kDa with approximately 22% overall identity with human PARG and 40% identity with the PARG signature motif. Two alternatively spliced forms of PME-3 were identified with an SL1 splice leader on both forms of the mRNA and were found to be expressed throughout the worm's life cycle. Similarly, pme-4 was shown to be expressed in all developmental stages of the worm. Recombinant enzymes that were expressed in bacteria displayed a PARG activity that may partly account for the PARG activity measured in the total worm extract. Reporter gene analysis of pme-3 and pme-4 using a GFP fusion construct showed that pme-3 and pme-4 are mainly expressed in nerve cells. PME-3 was shown to be nuclear while PME-4 localized to the cytoplasm. Worms with pme-3 and pme-4 expression knocked-down by RNAi showed a significant sensitivity toward ionizing radiations. Taken together, these data provide evidence for a physiological role for PARGs in DNA damage response and survival. It also shows that PARGs are evolutionarily conserved enzymes and that they are part of an ancient cellular response to DNA damage.     

Interactions between Caenorhabditis elegans individuals during chemotactic response

The chemotactic response of the nematode Caenorhabditis elegans is known to be affected by the population density on an assay plate, suggesting the existence of interactions between individual animals. To clarify the interactions between individuals during chemotaxis, we investigated the effect of population density at an attractant area on the chemotactic response to water-soluble sodium acetate and odorant diacetyl using wild-type N2 animals and daf-22 (m130) mutants, which have defective pheromone secretion but can sense pheromone. The chemotaxis index of N2 animals at 90 min of the assay negatively correlated with the number of animals on the assay plate regardless of the type of attractant used (p<0.01). On the other hand, there was no significant difference in the chemotaxis indices of daf-22 (m130) mutants for either of the attractants between the low-and high-population groups. When daf-22 (m130) mutants of a high population density were placed at the attractant location in advance, the chemotaxis index of N2 animals was almost the same as that in the control assay in which no animals were placed at the attractant location in advance. When N2 animals of a high population density were placed at the attractant location in advance, the chemotaxis indices of N2 animals and daf-22 (m130) mutants were significantly smaller than those obtained in the control assay (p<0.05). In the absence of an attractant, we observed a decline in the fraction of animals in the neighborhood of N2 animals of a high population density, although the nematodes were not influenced by daf-22 (m130) mutants of a high population density. These results suggest that the attraction of nematodes to chemicals is inhibited by an increase in the concentration of the pheromone generated by N2 animals at the attractant location.     

Gene silencing in Caenorhabditis elegans by transitive RNA interference

When a cell is exposed to double-stranded RNA (dsRNA), mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi). Here, we provide evidence that dsRNA is amplified in Caenorhabditis elegans to ensure a robust RNAi response. Our data suggest a model in which mRNA targeted by RNAi functions as a template for 5' to 3' synthesis of new dsRNA (termed transitive RNAi). Strikingly, the effect is nonautonomous: dsRNA targeted to a gene expressed in one cell type can lead to transitive RNAi-mediated silencing of a second gene expressed in a distinct cell type. These data suggest dsRNA synthesized in vivo can mediate systemic RNAi.     

Ribosomal DNA of Caenorhabditis elegans

We have characterized the organization of the genes coding for 18 S, 5·8 S and 26 S ribosomal RNAs in the nematode Caenorhabditis elegans. These ribosomal genes, present in about 55 copies per haploid genome, alternate in a repeating tandem array. The repeating unit is only 7000 base-pairs, containing a non-transcribed spacer of no more than 1000 base-pairs. Most of the repeating units have identical restriction maps, but one repeat contains a deletion of 2900 base-pairs, which eliminates all or part of the 18 S coding region. We have found no difference in the major ribosomal DNA restriction endonuclease cleavage patterns between two interbreeding strains of C. elegans, but found differences between C. elegans and the closely related Caenorhabditis briggsae.     

Roles for two partially redundant alpha-tubulins during mitosis in early Caenorhabditis elegans embryos

The Caenorhabditis elegans genome encodes multiple isotypes of alpha-tubulin and beta-tubulin. Roles for a number of these tubulins in neuronal development have been described, but less is known about the isoforms that function during early embryonic development. Microtubules are required for multiple events after fertilization produces a one-cell zygote in C. elegans, including pronuclear migration, mitotic spindle assembly and function, and proper spindle positioning. Here we describe a conditional and dominant mis-sense mutation in the C. elegans alpha-tubulin gene tba-1 that disrupts pronuclear migration and positioning of the first mitotic spindle, and results in a highly penetrant embryonic lethality, at the restrictive temperature of 26 degrees C. Our analysis of the dominant tba-1 (or346ts) allele suggests that TBA-1 assembles into microtubules in early embryonic cells. However, we also show that reduction of tba-1 function using RNA interference results in defects much less severe than those caused by the dominant or346ts mutation, due to partial redundancy of TBA-1 and another alpha-tubulin called TBA-2. Reducing the function of both TBA-1 and TBA-2 results in severe defects in microtubule-dependent processes. We conclude that microtubules in the early C. elegans embryo are composed of both TBA-1 and TBA-2, and that the dominant tba-1(or346ts) mutation disrupts MT assembly or stability. Cell Motil.     

Checkpoint and physiological apoptosis in germ cells proceeds normally in spaceflown Caenorhabditis elegans

It is important for human life in space to study the effects of environmental factors during spaceflight on a number of physiological phenomena. Apoptosis plays important roles in development and tissue homeostasis in metazoans. In this study, we have analyzed apoptotic activity in germ cells of the nematode C. elegans, following spacefight. Comparison of the number of cell corpses in wild type or ced-1 mutants, grown under either ground or spaceflight conditions, showed that both pachytene-checkpoint apoptosis and physiological apoptosis in germ cells occurred normally under spaceflight conditions. In addition, the expression levels of the checkpoint and apoptosis related genes are comparable between spaceflight and ground conditions. This is the first report documenting the occurrence of checkpoint apoptosis in the space environment and suggests that metazoans, including humans, would be able to eliminate cells that have failed to repair DNA lesions introduced by cosmic radiation during spaceflight.     

The Caenorhabditis elegans FancD2 ortholog is required for survival following DNA damage

Fanconi anemia (FA) is an autosomal recessive disease characterized by bone-marrow failure, congenital abnormalities, and cancer susceptibility. There are 11 FA complementation groups in human where 8 genes have been identified. We found that FancD2 is conserved in evolution and present in the genome of the nematode Caenorhabditis elegans. The gene Y41E3.9 (CeFancD2) encodes a structural ortholog of human FANCD2 and is composed of 10 predicted exons. Our analysis showed that exons 6 and 7 were absent from a CeFancD2 EST suggesting the presence of a splice variant. In an attempt to characterize its role in DNA damage, we depleted worms of CeFANCD2 using RNAi. When the CeFANCD2(RNAi) worms were treated with a crosslinking agent, a significant drop in the progeny survival was noted. These worms were also sensitive, although to a lesser extent, to ionizing radiation (IR). Therefore, these data support an important role for CeFANCD2 in DNA damage response as for its human counterpart. The data also support the usefulness of C. elegans to study the Fanconi anemia pathway, and emphasize the biological importance of FANCD2 in DNA damage response throughout evolution.     

The dynamics of replication licensing in live Caenorhabditis elegans embryos

Accurate DNA replication requires proper regulation of replication licensing, which entails loading MCM-2-7 onto replication origins. In this paper, we provide the first comprehensive view of replication licensing in vivo, using video microscopy of Caenorhabditis elegans embryos. As expected, MCM-2-7 loading in late M phase depended on the prereplicative complex (pre-RC) proteins: origin recognition complex (ORC), CDC-6, and CDT-1. However, many features we observed have not been described before: GFP-ORC-1 bound chromatin independently of ORC-2-5, and CDC-6 bound chromatin independently of ORC, whereas CDT-1 and MCM-2-7 DNA binding was interdependent. MCM-3 chromatin loading was irreversible, but CDC-6 and ORC turned over rapidly, consistent with ORC/CDC-6 loading multiple MCM-2-7 complexes. MCM-2-7 chromatin loading further reduced ORC and CDC-6 DNA binding. This dynamic behavior creates a feedback loop allowing ORC/CDC-6 to repeatedly load MCM-2-7 and distribute licensed origins along chromosomal DNA. During S phase, ORC and CDC-6 were excluded from nuclei, and DNA was overreplicated in export-defective cells. Thus, nucleocytoplasmic compartmentalization of licensing factors ensures that DNA replication occurs only once.     

Getting into shape: epidermal morphogenesis in Caenorhabditis elegans embryos

The change in shape of the C. elegans embryo from an ovoid ball of cells into a worm-shaped larva is driven by three events within the cells of the hypodermis (epidermis): (1) intercalation of two rows of dorsal cells, (2) enclosure of the ventral surface by hypodermis, and (3) elongation of the embryo. While the behavior of the hypodermal cells involved in each of these processes differs dramatically, it is clear that F-actin and microtubules have essential functions in each of these processes, whereas contraction of actomyosin structures appears to be involved specifically in elongation. Molecular analysis of these processes is revealing components specific to C. elegans as well as components found in other systems. Since C. elegans hypodermal cells demonstrate dramatically different behaviors during intercalation, enclosure and elongation, the study of cytoskeletal dynamics in these processes may reveal both unique and conserved activities during distinct epithelial morphogenetic movements. BioEssays 23:12-23, 2001.     

Control of cell-cycle timing in early embryos of Caenorhabditis elegans

A technique has been developed for extruding either substantial amounts of cytoplasm without nuclei or individual nuclei with small amounts of cytoplasm from early embryos of C. elegans after perforating the eggshell with a laser microbeam. This technique, in conjunction with laser-induced cell fusion, has allowed the altering of nuclear/cytoplasmic ratios and the exposing of the nucleus of one cell to cytoplasm from another. Using these approaches the roles of nuclei and cytoplasm in determining the different cell-cycle periods of the several blastomere lineages in early embryos have been examined. It was found that nuclei in a common cytoplasm divide synchronously; enucleated blastomeres retain a cycling period characteristic of their lineage; cycling period is not substantially affected by changes in the ratio of nuclear to cytoplasmic volumes or the DNA content per cell; the period of a cell from one lineage can be substantially altered by introduction of cytoplasm from a cell of another lineage with a different period; and short-term effects of foreign cytoplasm on the timing of the subsequent mitosis differ depending on position of the donor cell in the cell cycle. These results are discussed in connection with models for the action of cytoplasmic factors in controlling cell-cycle timing.