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The extracellular matrix (ECM) is important for normal development and disease states, including inflammation and fibrosis. To understand the complex regulation of ECM, we performed a suppressor screening using Caenorhabditis elegans expressing the mutant ROL-6 collagen protein. One cuticle mutant has a mutation in dpy-23 that encodes the μ2 adaptin (AP2M1) of clathrin-associated protein complex II (AP-2). The subsequent suppressor screening for dpy-23 revealed the lon-2 mutation. LON-2 functions to regulate body size through negative regulation of the tumor growth factor-beta (TGF-β) signaling pathway responsible for ECM production. RNA-seq analysis showed a dominant change in the expression of collagen genes and cuticle components. We noted an increase in the cav-1 gene encoding caveolin-1, which functions in clathrin-independent endocytosis. By knockdown of cav-1, the reduced TGF-β signal was significantly restored in the dpy-23 mutant. In conclusion, the dpy-23 mutation upregulated cav-1 expression in the hypodermis, and increased CAV-1 resulted in a decrease of TβRI. Finally, the reduction of collagen expression including rol-6 by the reduced TGF-β signal influenced the cuticle formation of the dpy-23 mutant. These findings could help us to understand the complex process of ECM regulation in organism development and disease conditions.

PHF8 is a JmjC domain-containing histone demethylase, defects in which are associated with X-linked mental retardation. In this study, we examined the roles of two PHF8 homologs, JMJD-1.1 and JMJD-1.2, in the model organism C. elegans in response to DNA damage. A deletion mutation in either of the genes led to hypersensitivity to interstrand DNA crosslinks (ICLs), while only mutation of jmjd-1.1 resulted in hypersensitivity to double-strand DNA breaks (DSBs). In response to ICLs, JMJD-1.1 did not affect the focus formation of FCD-2, a homolog of FANCD2, a key protein in the Fanconi anemia pathway. However, the dynamic behavior of RPA-1 and RAD-51 was affected by the mutation: the accumulations of both proteins at ICLs appeared normal, but their subsequent disappearance was retarded, suggesting that later steps of homologous recombination were defective. Similar changes in the dynamic behavior of RPA-1 and RAD-51 were seen in response to DSBs, supporting a role of JMJD-1.1 in homologous recombination. Such a role was also supported by our finding that the hypersensitivity of jmjd-1.1 worms to ICLs was rescued by knockdown of lig-4, a homolog of Ligase 4 active in nonhomologous end-joining. The hypersensitivity of jmjd-1.1 worms to ICLs was increased by rad-54 knockdown, suggesting that JMJD-1.1 acts in parallel with RAD-54 in modulating chromatin structure. Indeed, the level of histone H3 Lys9 tri-methylation, a marker of heterochromatin, was higher in jmjd-1.1 cells than in wild-type cells. We conclude that the histone demethylase JMJD-1.1 influences homologous recombination either by relaxing heterochromatin structure or by indirectly regulating the expression of multiple genes affecting DNA repair.

53BP1 contributes to activation of the G2/M checkpoint downstream of ATM and MDC1 in response to ionizing radiation and promotes nonhomologous end-joining (NHEJ) in mammalian cells. In order to determine whether the cellular activities of 53BP1 are conserved in the model organism C. elegans, we analyzed the function of its homolog, HSR-9 in response to DNA damage. Deletion or Mos1-insertion in hsr-9 did not affect the sensitivity of worms to double strand DNA breaks (DSBs), as reflected in embryonic survival and larval development. Nevertheless, the hsr-9 mutations, as well as a lig-4 deletion, reversed the hypersensitivity of rad-54-deficient worms to DSBs. In addition, oocyte chromosomal aberrations, which were increased by rad-54 knockdown in response to DSBs, were also reduced by the hsr-9 mutations. The hsr-9 mutations did not prevent the cell cycle arrest induced by DSBs in mitotically proliferating germ cells. However, they attenuated apoptosis induced by DSBs, but not when CEP-1 (a p53 ortholog) was absent, suggesting that HSR-9 functions in the same pathway as CEP-1. We concluded that the 53BP1 homolog in C. elegans is not directly involved in cell cycle arrest in response to DSBs, but that it promotes apoptosis and also a form of NHEJ that occurs only when rad-54 is deficient.

The Fanconi anemia (FA) pathway recognizes interstrand DNA crosslinks (ICLs) and contributes to their conversion into double-strand DNA breaks, which can be repaired by homologous recombination. Seven orthologs of the 15 proteins associated with Fanconi anemia are functionally conserved in the model organism C. elegans. Here we report that RNF-113, a ubiquitin ligase, is required for RAD-51 focus formation after inducing ICLs in C. elegans. However, the formation of foci of RPA-1 or FCD-2/FANCD2 in the FA pathway was not affected by depletion of RNF-113. Nevertheless, the RPA-1 foci formed did not disappear with time in the depleted worms, implying serious defects in ICL repair. As a result, RNF-113 depletion increased embryonic lethality after ICL treatment in wild-type worms, but it did not increase the ICL-induced lethality of rfs-1/rad51C mutants. In addition, the persistence of RPA-1 foci was suppressed in doubly-deficient rnf-113;rfs-1 worms, suggesting that there is an epistatic interaction between the two genes. These results lead us to suggest that RNF-113 and RFS-1 interact to promote the displacement of RPA-1 by RAD-51 on single-stranded DNA derived from ICLs.

WRN-1 is the Caenorhabditis elegans homolog of the human Werner syndrome protein, a RecQ helicase, mutations of which are associated with premature aging and increased genome instability. Relatively little is known as to how WRN-1 functions in DNA repair and DNA damage signaling. Here, we take advantage of the genetic and cytological approaches in C. elegans to dissect the epistatic relationship of WRN-1 in various DNA damage checkpoint pathways. We found that WRN-1 is required for CHK1 phosphorylation induced by DNA replication inhibition, but not by UV radiation. Furthermore, WRN-1 influences the RPA-1 focus formation, suggesting that WRN-1 functions in the same step or upstream of RPA-1 in the DNA replication checkpoint pathway. In response to ionizing radiation, RPA-1 focus formation and nuclear localization of ATM depend on WRN-1 and MRE-11. We conclude that C. elegans WRN-1 participates in the initial stages of checkpoint activation induced by DNA replication inhibition and ionizing radiation. These functions of WRN-1 in upstream DNA damage signaling are likely to be conserved, but might be cryptic in human systems due to functional redundancy.

Background: A strong association between stress resistance and longevity in multicellular organisms has been established as many mutations that extend lifespan also show increased resistance to stress. AAK-2, the C. elegans homolog of an alpha subunit of AMP-activated protein kinase (AMPK) is an intracellular fuel sensor that regulates cellular energy homeostasis and functions in stress resistance and lifespan extension. Findings: Here, we investigated global transcriptional responses of aak-2 mutants to oxidative stress and in turn identified potential downstream targets of AAK-2 involved in stress resistance in C. elegans. We employed massively parallel Illumina sequencing technology and performed comprehensive comparative transcriptome analysis. Specifically, we compared the transcriptomes of aak-2 and wild type animals under normal conditions and conditions of induced oxidative stress. This research has presented a snapshot of genome-wide transcriptional activities that take place in C. elegans in response to oxidative stress both in the presence and absence of AAK-2. Conclusions: The analysis presented in this study has enabled us to identify potential genes involved in stress resistance that may be either directly or indirectly under the control of AAK-2. Furthermore, we have extended our current knowledge of general defense responses of C. elegans against oxidative stress supporting the function for AAK-2 in inhibition of biosynthetic processes, especially lipid synthesis, under oxidative stress and transcriptional regulation of genes involved in reproductive processes. [ABSTRACT FROM AUTHOR]

Human mitofilin is a mitochondrial protein that controls cristae formation. Here, we investigated the role of the Caenorhabditis elegans mitofilin homologs, IMMT-1 and -2, in reproduction, physiology, and mitochondrial cristae formation. Mutation of either immt-1 or immt-2 produced defects in germline development and egg-laying. These defects were exacerbated by the double mutation, which greatly reduced motility, increased levels of reactive oxygen species, decreased mitochondrial mass, and imparted resistance to oxidative stress. Cryo-electron microscopy and electron tomography revealed that each of the single mutations resulted in curved and stacked mitochondrial crista tubules as well as a reduced number of crista junctions. The immt-2 mutation was also associated with the presence of outer mitochondrial membrane pores, which were larger in the double mutant. IMMT-1 and IMMT-2 proteins were localized to the inner mitochondrial membrane, as seen by immunoelectron microscopy, and they behaved as oligomers or large complexes with F1F0 ATP synthase in native polyacrylamide gel electrophoresis. These findings suggest that the two C. elegans mitofilin isoforms have non-overlapping functions in controlling mitochondrial cristae formation. J. Cell. Physiol. 224: 748–756, 2010. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Deficiency of the Caenorhabditis elegans protein, DIC-1, located in the inner membrane of mitochondria produces an abnormal mitochondrial morphology. The mechanism by which DIC-1 controls the topology of the inner membrane was investigated by transiently over-expressing DIC-1 in C. elegans. Cryo-electron microscopy showed that DIC-1 over-expression greatly increased the number and fractional area of mitochondrial cristae, suggesting that DIC-1 actively participates in cristae formation. These morphological changes were accompanied by increases in the oxygen consumption rate and ATP content of C. elegans worms, and decreases in reactive oxygen species (ROS) and sensitivity to paraquat. DIC-1 knockdown induced the opposite changes in ATP, ROS and paraquat-sensitivity. The ability of DIC-1 to increase cristae formation and secondarily, oxidative phosphorylation, suggests a potential use of this factor to control mitochondrial activity. [ABSTRACT FROM AUTHOR]

RNA interference (RNA&/) was performed on several essential genes in the pinewood nematode bursaphelenchus xylophilus, which causes pine wilt disease. Doublestranded RNA (dsRNA) was delivered to larvae or adult worms by soaking, electroporation, or microinjection. Soaking and electroporation of L2-L3 stage worms in solutions containing dsRNA for essential genes induced over 25% lethality after 5 days, and gene-specific phenotypes were observed. This lethality agreed with significant reductions of the targeted transcripts, as assayed by reversetranscription coupled with real time PCR. Microinjection was the most efficient route as measured by the hatching rate of F1 embryos, which was reduced by 46%. When adult worms were soaked in dsRNA, lethality was induced in the F1 larvae, revealing the persistence of knockdown phenotypes. The penetrance of the RNAá phenotypes for essential genes was relatively low but consistent, indicating that RNAá should be useful for studying the invivo functions of b. xylophilus gene products. [ABSTRACT FROM AUTHOR]

Caenorhabditis elegans him-6 mutants, which show a high incidence of males and partial embryonic lethality, are defective in the orthologue of human Bloom's syndrome protein (BLM). When strain him-6(e1104) containing a missense him-6 mutation was irradiated with γ-rays during germ cell development or embryogenesis, embryonic lethality was higher than in the wild type, suggesting a critical function of the wild type gene in mitotic and pachytene stage germ cells as well as in early embryos. Even in the absence of γ- irradiation, apoptosis was elevated in the germ cells of the him-6 strain and this increase was dependent on a functional p53 homologue (CEP-1), suggesting that spontaneous DNA damage accumulates due to him-6 deficiency. However, induction of germline apoptosis by ionizing radiation was not significantly affected by the deficiency, indicating that HIM-6 has no role in the induction of apoptosis by exogenous DNA damage. We conclude that the C. elegans BLM orthologue is involved in DNA repair in promeiotic cells undergoing homologous recombination, as well as in actively dividing germline and somatic cells. [ABSTRACT FROM AUTHOR]

A Werner syndrome protein homolog in C. elegans (WRN1) was immunolocalized to the nuclei of germ cells, embryonic cells, and many other cells of larval and adult worms. When wrn-1 expression was inhibited by RNA interference (RNAi), a slight reduction in C. elegans life span was observed, with accompanying signs of premature aging, such as earlier accumulation of lipofuscin and tissue deterioration in the head. In addition, various developmental defects, including small, dumpy, ruptured, transparent body, growth arrest and bag of worms, were induced by RNAi. The frequency of these defects was accentuated by γ-irradiation, implying that they were derived from spontaneous or induced DNA damage. wrn-1(RNAi) worms showed accelerated larval growth irrespective of γ-irradiation, and pre-meiotic germ cells had an abnormal checkpoint response to DNA replication blockage. These observations suggest that WRN-1 acts as a checkpoint protein for DNA damage and replication blockage. This idea is also supported by an accelerated S phase in wrn-1(RNAi) embryonic cells, wrn-1(RNAi) phenotypes similar to those of Werner syndrome, such as premature aging and short stature, suggest wrn-1-deficient C. elegans as a useful model organism for Werner syndrome. [ABSTRACT FROM AUTHOR]

Describes the Caenorhabditis elegans xeroderma pigmentosum complementation group A (XPA) homolog of human XPA. Nucleotide sequence of a complete C elegans XPA gene; Comparison of the human Drosophila and C. elegans XPA sequences; Effect of double-stranded RNA interference against ultraviolet radiation.

By screening Caenorhabditis elegans cDNA libraries, overlapping cDNA clones encoding DNA to-poisomerase I were obtained. An open reading frame of 751 amino acids was found in the 3.2-kb cDNA sequence. The open reading frame has 54% and 50% identities to the amino acid sequences of human and Drosophila melanogaster DNA topoisomerases I, respectively. Northern blot analysis showed the presence of an mRNA of 3.4 kb which suggests that the cDNA sequence is close to full length. The 72-kDa C-terminal polypeptide expressed in Escherichia coli cells showed catalytic DNA topoisomerase I activity. The DNA topoisomerase I gene was mapped to position 18 of chromosome I by screening polytene YAC plasmid DNAs. [ABSTRACT FROM AUTHOR]