A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity

We report a systematic RNA interference (RNAi) screen of 5,690 Caenorhabditis elegans genes for gene inactivations that increase lifespan. We found that genes important for mitochondrial function stand out as a principal group of genes affecting C. elegans lifespan. A classical genetic screen identified a mutation in the mitochondrial leucyl-tRNA synthetase gene (lrs-2) that impaired mitochondrial function and was associated with longer-lifespan. The long-lived worms with impaired mitochondria had lower ATP content and oxygen consumption, but differential responses to free-radical and other stresses. These data suggest that the longer lifespan of C. elegans with compromised mitochrondria cannot simply be assigned to lower free radical production and suggest a more complex coupling of metabolism and longevity.     

Conservation and diversification of Wnt signaling function during the evolution of nematode vulva development

Cell-fate specification and cell-cell signaling have been well studied during vulva development in Caenorhabditis elegans and provide a paradigm in evolutionary developmental biology. Pristionchus pacificus has been developed as a 'satellite' organism with an integrated physical and genetic map that allows detailed comparisons to C. elegans. A common aspect of vulva formation in both species is the polarization of the P7.p lineage, which is responsible for vulval symmetry. In C. elegans, Wnt signaling is crucial for P7.p cell-fate patterning; nothing is known about vulval symmetry in P. pacificus. We isolated mutations that disrupt polarization of the P7.p lineage in P. pacificus and found that the corresponding gene encodes a Frizzled-like molecule. In addition, mutations in Ppa-lin-17 (encoding Frizzled) and morpholino knock-down of Ppa-lin-44 (encoding Wnt), Ppa-egl-20 (encoding Wnt), Ppa-mig-5 (encoding Dsh), Ppa-apr-1 (encoding APC) and Ppa-bar-1 (encoding beta-catenin) results in gonad-independent vulva differentiation, indicating that these genes have a role in a negative signaling process. In contrast, in C. elegans, Wnt signaling has a positive role in vulva induction, and mutations in bar-1 result in a hypoinduced phenotype. Therefore, whereas the molecular mechanisms that generate vulval symmetry are conserved, the genetic control of vulva induction diversified during evolution.     

The complete family of genes encoding G proteins of Caenorhabditis elegans

Caenorhabditis elegans is the first animal whose genomic sequence has been determined. One of the new possibilities in post-sequence genetics is the analysis of complete gene families at once. We studied the family of heterotrimeric G proteins. C. elegans has 20 Galpha, 2 Gbeta and 2 Ggamma genes. There is 1 homologue of each of the 4 mammalian classes of Galpha genes, G(i)/G(o)alpha, G(s)alpha , G(q)alpha and G12alpha, and there are 16 new alpha genes. Although the conserved Galpha subunits are expressed in many neurons and muscle cells, GFP fusions indicate that 14 new Galpha genes are expressed almost exclusively in a small subset of the chemosensory neurons of C. elegans. We generated loss-of-function alleles using target-selected gene inactivation. None of the amphid-expressed genes are essential for viability, and only four show any detectable phenotype (chemotaxis defects), suggesting extensive functional redundancy. On the basis of functional analysis, the 20 genes encoding Galpha proteins can be divided into two groups: those that encode subunits affecting muscle activity (homologues of G(i)/G(o)alpha, G(s)alpha and G(q)), and those (14 new genes) that encode proteins most likely involved in perception.     

Caenorhabditis elegans ABL-1 antagonizes p53-mediated germline apoptosis after ionizing irradiation

c-Abl, a conserved nonreceptor tyrosine kinase, integrates genotoxic stress responses, acting as a transducer of both pro- and antiapoptotic effector pathways. Nuclear c-Abl seems to interact with the p53 homolog p73 to elicit apoptosis. Although several observations suggest that cytoplasmic localization of c-Abl is required for antiapoptotic function, the signals that mediate its antiapoptotic effect are largely unknown. Here we show that worms carrying an abl-1 deletion allele, abl-1(ok171), are specifically hypersensitive to radiation-induced apoptosis in the Caenorhabditis elegans germ line. Our findings delineate an apoptotic pathway antagonized by ABL-1, which requires sequentially the cell cycle checkpoint genes clk-2, hus-1 and mrt-2; the C. elegans p53 homolog, cep-1; and the genes encoding the components of the conserved apoptotic machinery, ced-3, ced-9 and egl-1. ABL-1 does not antagonize germline apoptosis induced by the DNA-alkylating agent ethylnitrosourea. Furthermore, worms treated with the c-Abl inhibitor STI-571 (Gleevec; used in human cancer therapy), two newly synthesized STI-571 variants or PD166326 had a phenotype similar to that generated by abl-1(ok171). These studies indicate that ABL-1 distinguishes proapoptotic signals triggered by two different DNA-damaging agents and suggest that C. elegans might provide tissue models for development of anticancer drugs.     

Mutations in a member of the Ras superfamily of small GTP-binding proteins causes Bardet-Biedl syndrome

RAB, ADP-ribosylation factors (ARFs) and ARF-like (ARL) proteins belong to the Ras superfamily of small GTP-binding proteins and are essential for various membrane-associated intracellular trafficking processes. None of the approximately 50 known members of this family are linked to human disease. Using a bioinformatic screen for ciliary genes in combination with mutational analyses, we identified ARL6 as the gene underlying Bardet-Biedl syndrome type 3, a multisystemic disorder characterized by obesity, blindness, polydactyly, renal abnormalities and cognitive impairment. We uncovered four different homozygous substitutions in ARL6 in four unrelated families affected with Bardet-Biedl syndrome, two of which disrupt a threonine residue important for GTP binding and function of several related small GTP-binding proteins. Analysis of the Caenorhabditis elegans ARL6 homolog indicates that it is specifically expressed in ciliated cells, and that, in addition to the postulated cytoplasmic functions of ARL proteins, it undergoes intraflagellar transport. These findings implicate a small GTP-binding protein in ciliary transport and the pathogenesis of a pleiotropic disorder.     

Caenorhabditis elegans expressed sequence tags identify gene families and potential disease gene homologues.

A database containing mapped partial cDNA sequences from Caenorhabditis elegans will provide a ready starting point for identifying nematode homologues of important human genes and determining their functions in C. elegans. A total of 720 expressed sequence tags (ESTs) have been generated from 585 clones randomly selected from a mixed-stage C. elegans cDNA library. Comparison of these ESTs with sequence databases identified 422 new C. elegans genes, of which 317 are not similar to any sequences in the database. Twenty-six new genes have been mapped by YAC clone hybridization. Members of several gene families, including cuticle collagens, GTP-binding proteins, and RNA helicases were discovered. Many of the new genes are similar to known or potential human disease genes, including CFTR and the LDL receptor.     

Chromosome-scale selective sweeps shape Caenorhabditis elegans genomic diversity

The nematode Caenorhabditis elegans is central to research in molecular, cell and developmental biology, but nearly all of this research has been conducted on a single strain of C. elegans. Little is known about the population genomic and evolutionary history of this species. We characterized C. elegans genetic variation using high-throughput selective sequencing of a worldwide collection of 200 wild strains and identified 41,188 SNPs. Notably, C. elegans genome variation is dominated by a set of commonly shared haplotypes on four of its six chromosomes, each spanning many megabases. Population genetic modeling showed that this pattern was generated by chromosome-scale selective sweeps that have reduced variation worldwide; at least one of these sweeps probably occurred in the last few hundred years. These sweeps, which we hypothesize to be a result of human activity, have drastically reshaped the global C. elegans population in the recent past.     

Self-fertilization sweeps up variation in the worm genome

A new study reports a comprehensive survey of genetic diversity in natural populations of the nematode Caenorhabditis elegans. Their analyses suggest that recent chromosome-scale selective sweeps have reduced C. elegans genetic diversity worldwide and strongly structured genetic variation across its genome.     

Senescence bypass screen identifies TBX2, which represses Cdkn2a (p19(ARF)) and is amplified in a subset of human breast cancers

To identify new immortalizing genes with potential roles in tumorigenesis, we performed a genetic screen aimed to bypass the rapid and tight senescence arrest of primary fibroblasts deficient for the oncogene Bmi1. We identified the T-box member TBX2 as a potent immortalizing gene that acts by downregulating Cdkn2a (p19(ARF)). TBX2 represses the Cdkn2a (p19(ARF)) promoter and attenuates E2F1, Myc or HRAS-mediated induction of Cdkn2a (p19(ARF)). We found TBX2 to be amplified in a subset of primary human breast cancers, indicating that it might contribute to breast cancer development.     

Open-reading-frame sequence tags (OSTs) support the existence of at least 17,300 genes in C. elegans

The genome sequences of Caenorhabditis elegans, Drosophila melanogaster and Arabidopsis thaliana have been predicted to contain 19,000, 13,600 and 25,500 genes, respectively. Before this information can be fully used for evolutionary and functional studies, several issues need to be addressed. First, the gene number estimates obtained in silico and not yet supported by any experimental data need to be verified. For example, it seems biologically paradoxical that C. elegans would have 50% more genes than Drosophilia. Second, intron/exon predictions need to be tested experimentally. Third, complete sets of open reading frames (ORFs), or "ORFeomes," need to be cloned into various expression vectors. To address these issues simultaneously, we have designed and applied to C. elegans the following strategy. Predicted ORFs are amplified by PCR from a highly representative cDNA library using ORF-specific primers, cloned by Gateway recombination cloning and then sequenced to generate ORF sequence tags (OSTs) as a way to verify identity and splicing. In a sample (n=1,222) of the nearly 10,000 genes predicted ab initio (that is, for which no expressed sequence tag (EST) is available so far), at least 70% were verified by OSTs. We also observed that 27% of these experimentally confirmed genes have a structure different from that predicted by GeneFinder. We now have experimental evidence that supports the existence of at least 17,300 genes in C. elegans. Hence we suggest that gene counts based primarily on ESTs may underestimate the number of genes in human and in other organisms.     

Regulation of endocytosis by CUP-5, the Caenorhabditis elegans mucolipin-1 homolog

Loss of the human mucolipin-1 gene underlies mucolipidosis type IV (MLIV), a lysosomal storage disease that results in severe developmental neuropathology. Unlike other lysosomal storage diseases, MLIV is not associated with a lack of lysosomal hydrolases; instead, MLIV cells display abnormal endocytosis of lipids and accumulate large vesicles, indicating that a defect in endocytosis may underlie the disease. Here we report the identification of a loss-of-function mutation in the Caenorhabditis elegans mucolipin-1 homolog, cup-5, and show that this mutation results in an enhanced rate of uptake of fluid-phase markers, decreased degradation of endocytosed protein and accumulation of large vacuoles. Overexpression of cup-5(+) causes the opposite phenotype, indicating that cup-5 activity controls aspects of endocytosis. Studies in model organisms such as C. elegans have helped illuminate fundamental mechanisms involved in normal cellular function and human disease; thus the C. elegans cup-5 mutant may be a useful model for studying conserved aspects of mucolipin-1 structure and function and for assessing the effects of potential therapeutic compounds.     

A functional SNP in CILP, encoding cartilage intermediate layer protein, is associated with susceptibility to lumbar disc disease

Lumbar disc disease (LDD) is caused by degeneration of intervertebral discs of the lumbar spine. One of the most common musculoskeletal disorders, LDD has strong genetic determinants. Using a case-control association study, we identified a functional SNP (1184T --> C, resulting in the amino acid substitution I395T) in CILP, which encodes the cartilage intermediate layer protein, that acts as a modulator of LDD susceptibility. CILP was expressed abundantly in intervertebral discs, and its expression increased as disc degeneration progressed. CILP colocalized with TGF-beta1 in clustering chondrocytes and their territorial matrices in intervertebral discs. CILP inhibited TGF-beta1-mediated induction of cartilage matrix genes through direct interaction with TGF-beta1 and inhibition of TGF-beta1 signaling. The susceptibility-associated 1184C allele showed increased binding and inhibition of TGF-beta1. Therefore, we conclude that the extracellular matrix protein CILP regulates TGF-beta signaling and that this regulation has a crucial role in the etiology and pathogenesis of LDD. Our study also adds to the list of connective tissue diseases that are associated with TGF-beta.     

Systematic mapping of genetic interactions in Caenorhabditis elegans identifies common modifiers of diverse signaling pathways

Most heritable traits, including disease susceptibility, are affected by interactions between multiple genes. However, we understand little about how genes interact because very few possible genetic interactions have been explored experimentally. We have used RNA interference in Caenorhabditis elegans to systematically test approximately 65,000 pairs of genes for their ability to interact genetically. We identify approximately 350 genetic interactions between genes functioning in signaling pathways that are mutated in human diseases, including components of the EGF/Ras, Notch and Wnt pathways. Most notably, we identify a class of highly connected 'hub' genes: inactivation of these genes can enhance the phenotypic consequences of mutation of many different genes. These hub genes all encode chromatin regulators, and their activity as genetic hubs seems to be conserved across animals. We propose that these genes function as general buffers of genetic variation and that these hub genes may act as modifier genes in multiple, mechanistically unrelated genetic diseases in humans.     

A single gene network accurately predicts phenotypic effects of gene perturbation in Caenorhabditis elegans

The fundamental aim of genetics is to understand how an organism's phenotype is determined by its genotype, and implicit in this is predicting how changes in DNA sequence alter phenotypes. A single network covering all the genes of an organism might guide such predictions down to the level of individual cells and tissues. To validate this approach, we computationally generated a network covering most C. elegans genes and tested its predictive capacity. Connectivity within this network predicts essentiality, identifying this relationship as an evolutionarily conserved biological principle. Critically, the network makes tissue-specific predictions-we accurately identify genes for most systematically assayed loss-of-function phenotypes, which span diverse cellular and developmental processes. Using the network, we identify 16 genes whose inactivation suppresses defects in the retinoblastoma tumor suppressor pathway, and we successfully predict that the dystrophin complex modulates EGF signaling. We conclude that an analogous network for human genes might be similarly predictive and thus facilitate identification of disease genes and rational therapeutic targets.     

C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression

To verify the genome annotation and to create a resource to functionally characterize the proteome, we attempted to Gateway-clone all predicted protein-encoding open reading frames (ORFs), or the 'ORFeome,' of Caenorhabditis elegans. We successfully cloned approximately 12,000 ORFs (ORFeome 1.1), of which roughly 4,000 correspond to genes that are untouched by any cDNA or expressed-sequence tag (EST). More than 50% of predicted genes needed corrections in their intron-exon structures. Notably, approximately 11,000 C. elegans proteins can now be expressed under many conditions and characterized using various high-throughput strategies, including large-scale interactome mapping. We suggest that similar ORFeome projects will be valuable for other organisms, including humans.     

iASPP oncoprotein is a key inhibitor of p53 conserved from worm to human

We have previously shown that ASPP1 and ASPP2 are specific activators of p53; one mechanism by which wild-type p53 is tolerated in human breast carcinomas is through loss of ASPP activity. We have further shown that 53BP2, which corresponds to a C-terminal fragment of ASPP2, acts as a dominant negative inhibitor of p53 (ref. 1). Hence, an inhibitory form of ASPP resembling 53BP2 could allow cells to bypass the tumor-suppressor functions of p53 and the ASPP proteins. Here, we characterize such a protein, iASPP (inhibitory member of the ASPP family), encoded by PPP1R13L in humans and ape-1 in Caenorhabditis elegans. iASPP is an evolutionarily conserved inhibitor of p53; inhibition of iASPP by RNA-mediated interference or antisense RNA in C. elegans or human cells, respectively, induces p53-dependent apoptosis. Moreover, iASPP is an oncoprotein that cooperates with Ras, E1A and E7, but not mutant p53, to transform cells in vitro. Increased expression of iASPP also confers resistance to ultraviolet radiation and to cisplatin-induced apoptosis. iASPP expression is upregulated in human breast carcinomas expressing wild-type p53 and normal levels of ASPP. Inhibition of iASPP could provide an important new strategy for treating tumors expressing wild-type p53.