Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 11 de 11
Filter
Add more filters










Publication year range
1.
Mol Cell ; 70(4): 639-649.e6, 2018 05 17.
Article in English | MEDLINE | ID: mdl-29775580

ABSTRACT

Animal cells have a remarkable capacity to adopt durable and heritable gene expression programs or epigenetic states that define the physical properties and diversity of somatic cell types. The maintenance of epigenetic programs depends on poorly understood pathways that prevent gain or loss of inherited signals. In the germline, epigenetic factors are enriched in liquid-like perinuclear condensates called nuage. Here, we identify the deeply conserved helicase-domain protein, ZNFX-1, as an epigenetic regulator and component of nuage that interacts with Argonaute systems to balance epigenetic inheritance. Our findings suggest that ZNFX-1 promotes the 3' recruitment of machinery that propagates the small RNA epigenetic signal and thus counteracts a tendency for Argonaute targeting to shift 5' along the mRNA. These functional insights support the idea that recently identified subdomains of nuage, including ZNFX-1 granules or "Z-granules," may define spatial and temporal zones of molecular activity during epigenetic regulation.


Subject(s)
Argonaute Proteins/metabolism , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/genetics , Cell Nucleus/genetics , Epigenesis, Genetic , Germ Cells/metabolism , RNA Helicases/metabolism , RNA, Small Interfering/genetics , Animals , Argonaute Proteins/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Organelles , RNA Helicases/genetics , RNA, Small Interfering/metabolism , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism
2.
Cell Rep ; 22(9): 2254-2264, 2018 02 27.
Article in English | MEDLINE | ID: mdl-29456100

ABSTRACT

Protein-coding genes undergo a wide array of regulatory interactions with factors that engage non-coding regions. Open reading frames (ORFs), in contrast, are thought to be constrained by coding function, precluding a major role in gene regulation. Here, we explore Piwi-interacting (pi)RNA-mediated transgene silencing in C. elegans and show that marked differences in the sensitivity to piRNA silencing map to the endogenous sequences within transgene ORFs. Artificially increasing piRNA targeting within the ORF of a resistant transgene can lead to a partial yet stable reduction in expression, revealing that piRNAs not only silence but can also "tune" gene expression. Our findings support a model that involves a temporal element to mRNA regulation by germline Argonautes, likely prior to translation, and suggest that piRNAs afford incremental control of germline mRNA expression by targeting the body of the mRNA, including the coding region.


Subject(s)
Argonaute Proteins/genetics , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/genetics , Gene Expression Regulation , Germ Cells/metabolism , Open Reading Frames/genetics , Animals , Argonaute Proteins/metabolism , Base Sequence , Caenorhabditis elegans Proteins/metabolism , Codon, Nonsense/genetics , Gene Silencing , Models, Biological , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Signal Transduction , Transgenes
3.
Dev Cell ; 44(6): 762-770.e3, 2018 03 26.
Article in English | MEDLINE | ID: mdl-29456136

ABSTRACT

In metazoans, Piwi-related Argonaute proteins engage piRNAs (Piwi-interacting small RNAs) to defend the genome against invasive nucleic acids, such as transposable elements. Yet many organisms-including worms and humans-express thousands of piRNAs that do not target transposons, suggesting that piRNA function extends beyond genome defense. Here, we show that the X chromosome-derived piRNA 21ux-1 downregulates XOL-1 (XO Lethal), a master regulator of X chromosome dosage compensation and sex determination in Caenorhabditis elegans. Mutations in 21ux-1 and several Piwi-pathway components sensitize hermaphrodites to dosage compensation and sex determination defects. We show that the piRNA pathway also targets xol-1 in C. briggsae, a nematode species related to C. elegans. Our findings reveal physiologically important piRNA-mRNA interactions, raising the possibility that piRNAs function broadly to ensure robust gene expression and germline development.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/genetics , Dosage Compensation, Genetic , Gene Expression Regulation , RNA, Small Interfering/genetics , Sex Chromosomes , Sex Determination Analysis , Animals , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Phenotype
4.
Genetics ; 197(4): 1069-80, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24879462

ABSTRACT

Genome editing based on CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease (Cas9) has been successfully applied in dozens of diverse plant and animal species, including the nematode Caenorhabditis elegans. The rapid life cycle and easy access to the ovary by micro-injection make C. elegans an ideal organism both for applying CRISPR-Cas9 genome editing technology and for optimizing genome-editing protocols. Here we report efficient and straightforward CRISPR-Cas9 genome-editing methods for C. elegans, including a Co-CRISPR strategy that facilitates detection of genome-editing events. We describe methods for detecting homologous recombination (HR) events, including direct screening methods as well as new selection/counterselection strategies. Our findings reveal a surprisingly high frequency of HR-mediated gene conversion, making it possible to rapidly and precisely edit the C. elegans genome both with and without the use of co-inserted marker genes.


Subject(s)
CRISPR-Associated Proteins/genetics , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Deoxyribonucleases/genetics , Genome, Helminth , Animals , Base Sequence , Genetic Markers , Homologous Recombination/genetics , Molecular Sequence Data , Mutagenesis, Insertional , Plasmids/genetics , Sequence Analysis, DNA
5.
Curr Biol ; 24(8): 845-51, 2014 Apr 14.
Article in English | MEDLINE | ID: mdl-24684931

ABSTRACT

Argonaute (AGO) proteins are key nuclease effectors of RNAi. Although purified AGOs can mediate a single round of target RNA cleavage in vitro, accessory factors are required for small interfering RNA (siRNA) loading and to achieve multiple-target turnover. To identify AGO cofactors, we immunoprecipitated the C. elegans AGO WAGO-1, which engages amplified small RNAs during RNAi. These studies identified a robust association between WAGO-1 and a conserved Vasa ATPase-related protein RDE-12. rde-12 mutants are deficient in RNAi, including viral suppression, and fail to produce amplified secondary siRNAs and certain endogenous siRNAs (endo-siRNAs). RDE-12 colocalizes with WAGO-1 in germline P granules and in cytoplasmic and perinuclear foci in somatic cells. These findings and our genetic studies suggest that RDE-12 is first recruited to target mRNA by upstream AGOs (RDE-1 and ERGO-1), where it promotes small RNA amplification and/or WAGO-1 loading. Downstream of these events, RDE-12 forms an RNase-resistant (target mRNA-independent) complex with WAGO-1 and may thus have additional functions in target mRNA surveillance and silencing.


Subject(s)
Argonaute Proteins/metabolism , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/physiology , DEAD-box RNA Helicases/metabolism , RNA Interference/physiology , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Amino Acid Sequence , Animals , Blotting, Northern , Caenorhabditis elegans Proteins/genetics , DEAD-box RNA Helicases/genetics , Immunoblotting , Immunoprecipitation , Microscopy, Fluorescence , Molecular Sequence Data , RNA-Binding Proteins/genetics
6.
J Clin Microbiol ; 52(2): 536-43, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24478485

ABSTRACT

Bovine tuberculosis remains one of the most damaging diseases to agriculture, and there is also a concern for human spillover. A critical need exists for rapid, thorough, and inexpensive diagnostic methods capable of detecting and differentiating Mycobacterium bovis infection from other pathogenic and environmental mycobacteria at multiple surveillance levels. In a previous study, Seth et al. (PLoS One 4:e5478, 2009, doi:10.1371/journal.pone.0005478) identified 32 host peptides that specifically increased in the blood serum of M. bovis-infected animals). In the current study, 16 M. bovis proteins were discovered in the blood serum proteomics data sets. A large-scale validation analysis was undertaken for selected host and M. bovis proteins using a cattle serum repository containing M. bovis (n = 128), Mycobacterium kansasii (n = 10), and Mycobacterium avium subsp. paratuberculosis (n = 10), cases exposed to M. bovis (n = 424), and negative controls (n = 38). Of the host biomarkers, vitamin D binding protein (VDBP) showed the greatest sensitivity and specificity for M. bovis detection. Circulating M. bovis proteins, specifically polyketide synthetase 5, detected M. bovis-infected cattle with little to no seroreactivity against M. kansasii- and M. avium subsp. paratuberculosis-infected animals. These data indicate that host and pathogen serum proteins can serve as reliable biomarkers for tracking M. bovis infection in animal populations.


Subject(s)
Biomarkers/blood , Clinical Laboratory Techniques/methods , Latent Tuberculosis/veterinary , Mycobacterium bovis/chemistry , Peptides/blood , Tuberculosis, Bovine/diagnosis , Veterinary Medicine/methods , Animals , Bacterial Proteins/blood , Blood Chemical Analysis , Cattle , Latent Tuberculosis/diagnosis , Proteome/analysis , Sensitivity and Specificity , Vitamin D-Binding Protein/blood
7.
Dev Cell ; 27(6): 656-63, 2013 Dec 23.
Article in English | MEDLINE | ID: mdl-24360782

ABSTRACT

Organisms can develop adaptive sequence-specific immunity by reexpressing pathogen-specific small RNAs that guide gene silencing. For example, the C. elegans PIWI-Argonaute/piwi-interacting RNA (piRNA) pathway recruits RNA-dependent RNA polymerase (RdRP) to foreign sequences to amplify a transgenerational small-RNA-induced epigenetic silencing signal (termed RNAe). Here, we provide evidence that, in addition to an adaptive memory of silenced sequences, C. elegans can also develop an opposing adaptive memory of expressed/self-mRNAs. We refer to this mechanism, which can prevent or reverse RNAe, as RNA-induced epigenetic gene activation (RNAa). We show that CSR-1, which engages RdRP-amplified small RNAs complementary to germline-expressed mRNAs, is required for RNAa. We show that a transgene with RNAa activity also exhibits accumulation of cognate CSR-1 small RNAs. Our findings suggest that C. elegans adaptively acquires and maintains a transgenerational CSR-1 memory that recognizes and protects self-mRNAs, allowing piRNAs to recognize foreign sequences innately, without the need for prior exposure


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/genetics , Epigenesis, Genetic , Gene Silencing , Germ Cells/metabolism , RNA, Helminth/genetics , RNA, Small Interfering/metabolism , Animals , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , Caenorhabditis elegans/growth & development , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , RNA, Helminth/metabolism , RNA, Small Interfering/genetics , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , Signal Transduction
8.
Cell ; 150(1): 65-77, 2012 Jul 06.
Article in English | MEDLINE | ID: mdl-22738726

ABSTRACT

Organisms employ a fascinating array of strategies to silence invasive nucleic acids such as transposons and viruses. Although evidence exists for several pathways that detect foreign sequences, including pathways that sense copy number, unpaired DNA, or aberrant RNA (e.g., dsRNA), in many cases, the mechanisms used to distinguish "self" from "nonself" nucleic acids remain mysterious. Here, we describe an RNA-induced epigenetic silencing pathway that permanently silences single-copy transgenes. We show that the Piwi Argonaute PRG-1 and its genomically encoded piRNA cofactors initiate permanent silencing, and maintenance depends on chromatin factors and the WAGO Argonaute pathway. Our findings support a model in which PRG-1 scans for foreign sequences and two other Argonaute pathways serve as epigenetic memories of "self" and "nonself" RNAs. These findings suggest how organisms can utilize RNAi-related mechanisms to detect foreign sequences not by any molecular signature, but by comparing the foreign sequence to a memory of previous gene expression.


Subject(s)
Caenorhabditis elegans/genetics , Epigenomics , RNA, Helminth/metabolism , RNA, Small Interfering/metabolism , Animals , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Gene Silencing , Germ Cells/metabolism , RNA Interference
9.
Am J Pathol ; 181(2): 548-59, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22688055

ABSTRACT

Neuropilins (NRPs) are transmembrane receptors that bind class 3 semaphorins and VEGF family members to regulate axon guidance and angiogenesis. Although expression of NRP1 by vascular smooth muscle cells (SMCs) has been reported, NRP function in smooth muscle (SM) in vivo is unexplored. Using Nrp2(+/LacZ) and Nrp2(+/gfp) transgenic mice, we observed robust and sustained expression of Nrp2 in the SM compartments of the bladder and gut, but no expression in vascular SM, skeletal muscle, or cardiac muscle. This expression pattern was recapitulated in vitro using primary human SM cell lines. Alterations in cell morphology after treatment of primary visceral SMCs with the NRP2 ligand semaphorin-3F (SEMA3F) were accompanied by inhibition of RhoA activity and myosin light chain phosphorylation, as well as decreased cytoskeletal stiffness. Ex vivo contractility testing of bladder muscle strips exposed to electrical stimulation or soluble agonists revealed enhanced tension generation of tissues from mice with constitutive or SM-specific knockout of Nrp2, compared with controls. Mice lacking Nrp2 also displayed increased bladder filling pressures, as assessed by cystometry in conscious mice. Together, these findings identify Nrp2 as a mediator of prorelaxant stimuli in SMCs and suggest a novel function for Nrp2 as a regulator of visceral SM contractility.


Subject(s)
Muscle Contraction/physiology , Muscle, Smooth/physiology , Neuropilin-2/deficiency , Neuropilin-2/metabolism , Animals , Cell Shape/drug effects , Cytoskeleton/drug effects , Cytoskeleton/metabolism , Down-Regulation/drug effects , Female , Gene Deletion , Heparin-binding EGF-like Growth Factor , Humans , In Vitro Techniques , Intercellular Signaling Peptides and Proteins/pharmacology , Intestinal Mucosa/metabolism , Intestines/cytology , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Muscle Contraction/drug effects , Muscle, Smooth/drug effects , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Nerve Tissue Proteins/metabolism , Organ Specificity/drug effects , Sus scrofa , Urinary Bladder/cytology , Urinary Bladder/metabolism , rho-Associated Kinases/metabolism , rhoA GTP-Binding Protein/metabolism
10.
J Clin Invest ; 122(1): 178-91, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22182838

ABSTRACT

Epoxyeicosatrienoic acids (EETs) are small molecules produced by cytochrome P450 epoxygenases. They are lipid mediators that act as autocrine or paracrine factors to regulate inflammation and vascular tone. As a result, drugs that raise EET levels are in clinical trials for the treatment of hypertension and many other diseases. However, despite their pleiotropic effects on cells, little is known about the role of these epoxyeicosanoids in cancer. Here, using genetic and pharmacological manipulation of endogenous EET levels, we demonstrate that EETs are critical for primary tumor growth and metastasis in a variety of mouse models of cancer. Remarkably, we found that EETs stimulated extensive multiorgan metastasis and escape from tumor dormancy in several tumor models. This systemic metastasis was not caused by excessive primary tumor growth but depended on endothelium-derived EETs at the site of metastasis. Administration of synthetic EETs recapitulated these results, while EET antagonists suppressed tumor growth and metastasis, demonstrating in vivo that pharmacological modulation of EETs can affect cancer growth. Furthermore, inhibitors of soluble epoxide hydrolase (sEH), the enzyme that metabolizes EETs, elevated endogenous EET levels and promoted primary tumor growth and metastasis. Thus, our data indicate a central role for EETs in tumorigenesis, offering a mechanistic link between lipid signaling and cancer and emphasizing the critical importance of considering possible effects of EET-modulating drugs on cancer.


Subject(s)
Eicosanoids/metabolism , Neoplasm Metastasis/physiopathology , Neoplasms, Experimental/metabolism , Animals , Aryl Hydrocarbon Hydroxylases/genetics , Aryl Hydrocarbon Hydroxylases/metabolism , Cytochrome P-450 CYP2C8 , Cytochrome P-450 CYP2J2 , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Endothelium, Vascular/metabolism , Epoxy Compounds/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Neoplasm Metastasis/pathology , Neoplasms, Experimental/blood supply , Neoplasms, Experimental/pathology , Neovascularization, Pathologic , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Vascular Endothelial Growth Factor A/metabolism
11.
PLoS One ; 4(5): e5478, 2009.
Article in English | MEDLINE | ID: mdl-19424492

ABSTRACT

BACKGROUND: Bovine tuberculosis is a highly prevalent infectious disease of cattle worldwide; however, infection in the United States is limited to 0.01% of dairy herds. Thus detection of bovine TB is confounded by high background infection with M. avium subsp. paratuberculosis. The present study addresses variations in the circulating peptidome based on the pathogenesis of two biologically similar mycobacterial diseases of cattle. METHODOLOGY/PRINCIPAL FINDINGS: We hypothesized that serum proteomes of animals in response to either M. bovis or M. paratuberculosis infection will display several commonalities and differences. Sera prospectively collected from animals experimentally infected with either M. bovis or M. paratuberculosis were analyzed using high-resolution proteomics approaches. iTRAQ, a liquid chromatography and tandem mass spectrometry approach, was used to simultaneously identify and quantify peptides from multiple infections and contemporaneous uninfected control groups. Four comparisons were performed: 1) M. bovis infection versus uninfected controls, 2) M. bovis versus M. paratuberculosis infection, 3) early, and 4) advanced M. paratuberculosis infection versus uninfected controls. One hundred and ten differentially elevated proteins (P < or = 0.05) were identified. Vitamin D binding protein precursor (DBP), alpha-1 acid glycoprotein, alpha-1B glycoprotein, fetuin, and serine proteinase inhibitor were identified in both infections. Transthyretin, retinol binding proteins, and cathelicidin were identified exclusively in M. paratuberculosis infection, while the serum levels of alpha-1-microglobulin/bikunin precursor (AMBP) protein, alpha-1 acid glycoprotein, fetuin, and alpha-1B glycoprotein were elevated exclusively in M. bovis infected animals. CONCLUSIONS/SIGNIFICANCE: The discovery of these biomarkers has significant impact on the elucidation of pathogenesis of two mycobacterial diseases at the cellular and the molecular level and can be applied in the development of mycobacterium-specific diagnostic tools for the monitoring progression of disease, response to therapy, and/or vaccine based interventions.


Subject(s)
Blood Proteins/analysis , Tuberculosis, Bovine/diagnosis , Tuberculosis, Bovine/microbiology , Animals , Biomarkers/analysis , Cattle , Chromatography, Liquid , Isotope Labeling , Mycobacterium avium subsp. paratuberculosis/physiology , Mycobacterium bovis/physiology , Reproducibility of Results , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tuberculosis, Bovine/blood , Vitamin D-Binding Protein/analysis
SELECTION OF CITATIONS
SEARCH DETAIL
...