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[
Curr Biol,
2006]
The DAF-7/TGF-beta pathway in C. elegans interprets environmental signals relayed through amphid neurons and actively inhibits dauer formation during reproductive developmental growth . In metazoans, the STAT pathway interprets external stimuli through regulated tyrosine phosphorylation, nuclear translocation, and gene expression , but its importance for developmental commitment, particularly in conjuction with TGF-beta, remains largely unknown. Here, we report that the nematode STAT ortholog STA-1 accumulated in the nuclei of five head neuron pairs, three of which are amphid neurons involved in dauer formation . Moreover,
sta-1 mutants showed a synthetic dauer phenotype with selected TGF-beta mutations.
sta-1 deficiency was complemented by reconstitution with wild-type protein, but not with a tyrosine mutant. Canonical TGF-beta signaling involves the DAF-7/TGF-beta ligand activating the DAF-1/DAF-4 receptor pair to regulate the DAF-8/DAF-14 Smads . Interestingly, STA-1 functioned in the absence of DAF-7, DAF-4, and DAF-14, but it required DAF-1 and DAF-8. Additionally, STA-1 expression was induced by TGF-beta in a DAF-3-dependent manner, demonstrating a homeostatic negative feedback loop. These results highlight a role for activated STAT proteins in repression of dauer formation. They also raise the possibility of an unexpected function for DAF-1 and DAF-8 that is independent of their normal upstream activator, DAF-7.
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[
FASEB J,
2006]
STAT transcription factors have been implicated in many biological processes, particularly host immune defense and development. Here we characterize a STAT orthologue from the nematode, C. elegans. We show that this protein, termed STA-1, is structurally and functionally related to other vertebrate and invertebrate STAT proteins, recognizing a cis DNA element conserved through phylogeny. Unexpectedly, STA-1 lacks the conserved amino-terminal oligomerization domain found in vertebrate and other invertebrate STAT proteins, a feature also lacking in orthologues from a distantly related nematode species and from the slime mold, Dictyostelium discoideum. This absence suggests that a primordial STAT protein lacked this domain, which was accreted later in evolution to provide further regulatory control of STAT signaling. Derivation of null mutants demonstrated that STA-1 is not required for nematode viability, despite its widespread expression in multiple tissues of the worm. However, mutant STA-1 proteins that lack functional coiled-coil and DNA binding domains could still be activated and accumulated in the nucleus, suggesting that DNA binding is not a necessary prerequisite for nuclear retention of activated STAT proteins. Our results shed new light on the evolution and function of the STAT signaling pathway and on the structural requirements for STAT activation.
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[
Vet Parasitol,
2008]
Strongyloides sp. (Nematoda) are very wide spread small intestinal parasites of vertebrates that can form a facultative free-living generation. Most authors considered all Strongyloides of farm ruminants to belong to the same species, namely Strongyloides papillosus (Wedl, 1856). Here we show that, at least in southern Germany, the predominant Strongyloides found in cattle and the Strongyloides found in sheep belong to separate, genetically isolated populations. While we did find mixed infections in cattle, one form clearly dominated. This variety, in turn, was never found in sheep, indicating that the two forms have different host preferences. We also present molecular tools for distinguishing the two varieties, and an analysis of their phylogenetic relationship with the human parasite Strongyloides stercoralis and the major laboratory model species Strongyloides ratti. Based on our findings we propose that Strongyloides from sheep and the predominant Strongyloides from cattle should be considered separate species as it had already been proposed by [Brumpt, E., 1921. Recherches sur le determinisme des sexes et de l''evolution des Anguillules parasites (Strongyloides). Comptes rendu hebdomadaires des seances et memoires de la Societe de Biologie et de ses filiales 85, 149-152], but was largely ignored by later authors. For nomenclature, we follow [Brumpt, E., 1921. Recherches sur le determinisme des sexes et de l''evolution des Anguillules parasites (Strongyloides). Comptes rendu hebdomadaires des seances et memoires de la Societe de Biologie et de ses filiales 85, 149-152] and use the name S. papillosus for the Strongyloides of sheep and the name Strongyloides vituli for the predominant Strongyloides of cattle.
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Cho MT, Kok F, Mahida S, Wilson C, Demmer LA, Smith-Hicks C, Stumpel CTRM, Falik-Zaccai T, Angione KM, Brasington C, Sticht H, Shinde DN, Mandel H, Edwards SL, McWalter K, Di Donato N, Marquardt T, Allen W, Stegmann APA, Bonati MT, Hellenbroich Y, Miller KG, Jamra R, Pinz H, Gamble CN, Lemke JR, Iascone M, Panis B, Ramos L, Stobe P, Pepler A, Platzer K
[
Am J Hum Genet,
2018]
Using exome sequencing, we have identified de novo variants in MAPK8IP3 in 13 unrelated individuals presenting with an overlapping phenotype of mild to severe intellectual disability. The de novo variants comprise six missense variants, three of which are recurrent, and three truncating variants. Brain anomalies such as perisylvian polymicrogyria, cerebral or cerebellar atrophy, and hypoplasia of the corpus callosum were consistent among individuals harboring recurrent de novo missense variants. MAPK8IP3 has been shown to be involved in the retrograde axonal-transport machinery, but many of its specific functions are yet to be elucidated. Using the CRISPR-Cas9 system to target six conserved amino acid positions in Caenorhabditis elegans, we found that two of the six investigated human alterations led to a significantly elevated density of axonal lysosomes, and five variants were associated with adverse locomotion. Reverse-engineering normalized the observed adverse effects back to wild-type levels. Combining genetic, phenotypic, and functional findings, as well as the significant enrichment of de novo variants in MAPK8IP3 within our total cohort of 27,232 individuals who underwent exome sequencing, we implicate de novo variants in MAPK8IP3 as a cause of a neurodevelopmental disorder with intellectual disability and variable brain anomalies.
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[
J Am Soc Mass Spectrom,
2015]
De novo sequencing software has been widely used in proteomics to sequence new peptides from tandem mass spectrometry data. This study presents a new software tool, Novor, to greatly improve both the speed and accuracy of today's peptide de novo sequencing analyses. To improve the accuracy, Novor's scoring functions are based on two large decision trees built from a peptide spectral library with more than 300,000 spectra with machine learning. Important knowledge about peptide fragmentation is extracted automatically from the library and incorporated into the scoring functions. The decision tree model also enables efficient score calculation and contributes to the speed improvement. To further improve the speed, a two-stage algorithmic approach, namely dynamic programming and refinement, is used. The software program was also carefully optimized. On the testing datasets, Novor sequenced 7%-37% more correct residues than the state-of-the-art de novo sequencing tool, PEAKS, while being an order of magnitude faster. Novor can de novo sequence more than 300 MS/MS spectra per second on a laptop computer. The speed surpasses the acquisition speed of today's mass spectrometer and, therefore, opens a new possibility to de novo sequence in real time while the spectrometer is acquiring the spectral data. Graphical Abstract .
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[
Curr Biol,
2011]
DNA injected into the Caenorhabditis elegans germline forms extrachromosomal arrays that segregate during cell division [1, 2]. The mechanisms underlying array formation and segregation are not known. Here, we show that extrachromosomal arrays form de novo centromeres at high frequency, providing unique access to a process that occurs with extremely low frequency in other systems [3-8]. De novo centromerized arrays recruit centromeric chromatin and kinetochore proteins and autonomously segregate on the spindle. Live imaging following DNA injection revealed that arrays form after oocyte fertilization via homologous recombination and nonhomologous end-joining. Individual arrays gradually transition from passive inheritance to active segregation during the early embryonic divisions. The heterochromatin protein 1 (HP1) family proteins HPL-1 and HPL-2 are dispensable for de novo centromerization even though arrays become strongly enriched for the heterochromatin-associated H3K9me3 modification over time. Partial inhibition of HP1 family proteins accelerates the acquisition of segregation competence. In addition to reporting the first direct visualization of new centromere formation in living cells, these findings reveal that naked DNA rapidly builds de novo centromeres in C. elegans embryos in an HP1-independent manner and suggest that, rather than being a prerequisite, HP1-dependent heterochromatin antagonizes de novo centromerization.
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[
PLoS Genet,
2017]
Density-Enhanced Phosphatase-1 (DEP-1) de-phosphorylates various growth factor receptors and adhesion proteins to regulate cell proliferation, adhesion and migration. Moreover,
dep-1/scc1 mutations have been detected in various types of human cancers, indicating a broad tumor suppressor activity. During C. elegans development, DEP-1 mediates binary cell fate decisions by negatively regulating EGFR signaling. Using a substrate-trapping DEP-1 mutant in a proteomics approach, we have identified the C. elegans -integrin subunit PAT-3 as a specific DEP-1 substrate. DEP-1 selectively de-phosphorylates tyrosine 792 in the membrane-proximal NPXY motif to promote integrin activation via talin recruitment. The non-phosphorylatable -integrin mutant
pat-3(Y792F) partially suppresses the hyperactive EGFR signaling phenotype caused by loss of
dep-1 function. Thus, DEP-1 attenuates EGFR signaling in part by de-phosphorylating Y792 in the -integrin cytoplasmic tail, besides the direct de-phosphorylation of the EGFR. Furthermore, in vivo FRAP analysis indicates that the -integrin/talin complex attenuates EGFR signaling by restricting receptor mobility on the basolateral plasma membrane. We propose that DEP-1 regulates EGFR signaling via two parallel mechanisms, by direct receptor de-phosphorylation and by restricting receptor mobility through -integrin activation.
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[
Sci China Life Sci,
2019]
Orphan genes that lack detectable homologues in other lineages could contribute to a variety of biological functions. However, their origination and function mechanisms remain largely unknown. Herein, through a comprehensive and systematic computational pipeline, we identified 893 orphan genes in the lineage of C. elegans, of which only a low fraction (0.9%) were derived from transposon elements. Six new protein-coding genes that de novo originated from non-coding DNA sequences in the genome of C. elegans were also identified. The authenticity and functionality of these orphan genes and de novo genes are supported by three lines of evidences, consisting of transcriptional data, and in silico proteomic data, and the fixation status data in wild populations. Orphan genes and de novo genes exhibited simple gene structures, such as, short in protein length, of fewer exons, and are frequently X-linked. RNA-seq data analysis showed these orphan genes are enriched with expression in embryo development and gonad, and their potential function in early development was further supported by gene ontology enrichment analysis results. Meanwhile, de novo genes were found to be with significant expression in gonad, and functional enrichment analysis of the co-expression genes of these de novo genes suggested they may be functionally involved in signaling transduction pathway and metabolism process. Our results presented the first systematic evidence on the evolution of orphan genes and de novo origin of genes in nematodes and their impacts on the functional and phenotypic evolution, and thus could shed new light on our appreciation of the importance of these new genes.
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[
PLoS One,
2015]
The nematode Caenorhabditis elegans provides a unique opportunity to interrogate the neural basis of behavior at single neuron resolution. In C. elegans, neural circuits that control behaviors can be formulated based on its complete neural connection map, and easily assessed by applying advanced genetic tools that allow for modulation in the activity of specific neurons. Importantly, C. elegans exhibits several elaborate behaviors that can be empirically quantified and analyzed, thus providing a means to assess the contribution of specific neural circuits to behavioral output. Particularly, locomotory behavior can be recorded and analyzed with computational and mathematical tools. Here, we describe a robust single worm-tracking system, which is based on the open-source Python programming language, and an analysis system, which implements path-related algorithms. Our tracking system was designed to accommodate worms that explore a large area with frequent turns and reversals at high speeds. As a proof of principle, we used our tracker to record the movements of wild-type animals that were freshly removed from abundant bacterial food, and determined how wild-type animals change locomotory behavior over a long period of time. Consistent with previous findings, we observed that wild-type animals show a transition from area-restricted local search to global search over time. Intriguingly, we found that wild-type animals initially exhibit short, random movements interrupted by infrequent long trajectories. This movement pattern often coincides with local/global search behavior, and visually resembles Levy flight search, a search behavior conserved across species. Our mathematical analysis showed that while most of the animals exhibited Brownian walks, approximately 20% of the animals exhibited Levy flights, indicating that C. elegans can use Levy flights for efficient food search. In summary, our tracker and analysis software will help analyze the neural basis of the alteration and transition of C. elegans locomotory behavior in a food-deprived condition.
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Toshimoto K, Hayashi T, Nagayasu E, Kajitani R, Itoh T, Toyoda A, Okuno M, Fujiyama A, Yabana M, Noguchi H, Kohara Y, Maruyama H, Ogura Y, Harada M
[
Genome Res,
2014]
Although many de novo genome assembly projects have recently been conducted using high-throughput sequencers, assembling highly heterozygous diploid genomes is a substantial challenge due to the increased complexity of the de Bruijn graph structure predominantly used. To address the increasing demand for sequencing of nonmodel and/or wild-type samples, in most cases inbred lines or fosmid-based hierarchical sequencing methods are used to overcome such problems. However, these methods are costly and time consuming, forfeiting the advantages of massive parallel sequencing. Here, we describe a novel de novo assembler, Platanus, that can effectively manage high-throughput data from heterozygous samples. Platanus assembles DNA fragments (reads) into contigs by constructing de Bruijn graphs with automatically optimized k-mer sizes followed by the scaffolding of contigs based on paired-end information. The complicated graph structures that result from the heterozygosity are simplified during not only the contig assembly step but also the scaffolding step. We evaluated the assembly results on eukaryotic samples with various levels of heterozygosity. Compared with other assemblers, Platanus yields assembly results that have a larger scaffold NG50 length without any accompanying loss of accuracy in both simulated and real data. In addition, Platanus recorded the largest scaffold NG50 values for two of the three low-heterozygosity species used in the de novo assembly contest, Assemblathon 2. Platanus therefore provides a novel and efficient approach for the assembly of gigabase-sized highly heterozygous genomes and is an attractive alternative to the existing assemblers designed for genomes of lower heterozygosity.