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Remarkably divergent regions punctuate the genome assembly of the Caenorhabditis elegans Hawaiian strain CB4856
Thompson, O.A. ; Snoek, L.B. ; Nijveen, H. ; Sterken, M.G. ; Volkers, R.J.M. ; Brenchley, R. ; Hof, A. van 't; Bevers, R.P.J. ; Cossins, A.R. ; Yanai, I. ; Hajnal, A. ; Schmid, T. ; Perkins, J.D. ; Spencer, D. ; Kruglyak, L. ; Andersen, E.C. ; Moerman, D.G. ; Hillier, L.W. ; Kammenga, J.E. ; Waterston, R.H. - \ 2015
Genetics 200 (2015)3. - ISSN 0016-6731 - p. 975 - 989.
natural variation data - c. elegans - arabidopsis-thaliana - gene - polymorphism - populations - diversity - nematodes - dna - evolutionary
The Hawaiian strain (CB4856) of Caenorhabditis elegans is one of the most divergent from the canonical laboratory strain N2 and has been widely used in developmental, population and evolutionary studies. To enhance the utility of the strain, we have generated a draft sequence of the CB4856 genome, exploiting a variety of resources and strategies. The CB4856 genome when compared against the N2 reference has 327,050 single nucleotide variants (SNVs) and 79,529 insertion-deletion events (indels) that result in a total of 3.3 megabasepairs (Mb) of N2 sequence missing from CB4856 and 1.4 Mb of sequence present in CB4856 not present in N2. As previously reported, the density of SNVs varies along the chromosomes, with the arms of chromosomes showing greater average variation than the centers. In addition, we find 61 regions totaling 2.8 Mb, distributed across all six chromosomes, that have a greatly elevated SNV density, ranging from 2% to 16% SNVs. A survey of other wild isolates show that the two alternative haplotypes for each region are widely distributed, suggesting they have been maintained by balancing selection over long evolutionary times. These divergent regions contain an abundance of genes from large rapidly evolving families encoding F-box, MATH, BATH, seven-transmembrane G-coupled receptors, and nuclear hormone receptors suggesting that they provide selective advantages in natural environments. The draft sequence makes available a comprehensive catalog of sequence differences between the CB4856 and N2 strains that will facilitate the molecular dissection of their phenotypic differences. Our work also emphasizes the importance of going beyond simple alignment of reads to a reference genome when assessing differences between genomes.
Systemic Regulation of RAS/MAPK Signaling by the Serotonin Metabolite 5-HIAA
Schmid, T. ; Snoek, L.B. ; Fröhli, E. ; Bent, M.L. van der; Kammenga, J.E. ; Hajnal, A. - \ 2015
Plos Genetics 11 (2015)5. - ISSN 1553-7404 - 16 p.
caenorhabditis-elegans - c-elegans - natural variation - vulvar induction - complex disease - receptor - protein - gene - kinase - activation
Human cancer is caused by the interplay of mutations in oncogenes and tumor suppressor genes and inherited variations in cancer susceptibility genes. While many of the tumor initiating mutations are well characterized, the effect of genetic background variation on disease onset and progression is less understood. We have used C. elegans genetics to identify genetic modifiers of the oncogenic RAS/MAPK signaling pathway. Quantitative trait locus analysis of two highly diverged C. elegans isolates combined with allele swapping experiments identified the polymorphic monoamine oxidase A (MAOA) gene amx-2 as a negative regulator of RAS/MAPK signaling. We further show that the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA), which is a product of MAOA catalysis, systemically inhibits RAS/MAPK signaling in different organs of C. elegans. Thus, MAOA activity sets a global threshold for MAPK activation by controlling 5-HIAA levels. To our knowledge, 5-HIAA is the first endogenous small molecule that acts as a systemic inhibitor of RAS/MAPK signaling.
|Bar-1 loss-of-function causes a developmental delay in intertwined with a DAF-16 stress response
Sterken, M.G. ; Bent, M.L. van der; Volkers, R.J.M. ; Riksen, J.A.G. ; Schmid, T. ; Hajnal, A. ; Kammenga, J.E. ; Snoek, L.B. - \ 2014
In: Proceedings of the Berlin C. elegans Meeting 2014. - - p. 241 - 241.
The Wnt/â-catenin pathway is a key development pathway in metazoans. C. elegans has been extensively used as a model species to study the Wnt pathway. Four â-catenins in C. elegans are important gene expression regulators in Wnt signalling. One of these, bar-1, is part of the canonical Wnt pathway. Together with Wnt effector POP-1, BAR-1 forms a transcription activation complex which regulates the transcription of downstream genes. The effects of bar-1 loss-of-function mutations on many phenotypes, like vulval development and neuronal migration, have been studied well. However, the effects on global gene expression remain largely unknown. The transcriptional profiles of the strain EW15, carrying bar-1(ga80)lof, and Bristol N2 were measured in the L4 stage of age-synchronized populations at 48 hours. Gene expression differences were combines whit ChiP-seq data from the modENCODE project to pinpoint involved transcription factors. Genes differentially expressed between EW15 and N2 showed strong signs that development of bar-1(ga80)lof was slowed down. We verified this observation by measuring the timing of egg-hatching and egg-laying. The development delay increased over time, ranging from 0 hours at the moment of egg-hatching to 6 hours at the moment of the egg laying. After correcting for this development delay, gene expression analysis revealed that the expression of many genes involved in the Wnt pathway were activated in the bar-1 mutant. Furthermore, the genes up-regulated in the bar-1 mutant showed enrichment for transcription factor activity. These up-regulated genes show strong indications of DAG-16 involvement by the combination of modENCODE ChiP-seq data as well as known DAF-16 regulated genes. Together our findings show that bar-1 loss-of-function leads to delayed development and an activated Wnt pathway, and indicate that the DAF-16 stress-response is causative of this development delay.
Loss-of-function of b-catenin bar-1 slows development and activates the Wnt pathway in Caenorhabditis elegans
Bent, M.L. van der; Sterken, M.G. ; Volkers, J.M. ; Riksen, J.A.G. ; Schmid, T. ; Hajnal, A. ; Kammenga, J.E. ; Snoek, L.B. - \ 2014
Scientific Reports 4 (2014). - ISSN 2045-2322 - 7 p.
natural variation data - c-elegans - signaling components - neuroblast migration - negative regulator - vulval development - gene-expression - cell-migration - proteins - endoderm
C. elegans is extensively used to study the Wnt-pathway and most of the core-signalling components are known. Four beta-catenins are important gene expression regulators in Wnt-signalling. One of these, bar-1, is part of the canonical Wnt-pathway. Together with Wnt effector pop-1, bar-1 forms a transcription activation complex which regulates the transcription of downstream genes. The effects of bar-1 loss-of-function mutations on many phenotypes have been studied well. However, the effects on global gene expression are unknown. Here we report the effects of a loss-of-function mutation bar-1(ga80). By analysing the transcriptome and developmental phenotyping we show that bar-1(ga80) impairs developmental timing. This developmental difference confounds the comparison of the gene expression profile between the mutant and the reference strain. When corrected for this difference it was possible to identify genes that were directly affected by the bar-1 mutation. We show that the Wnt-pathway itself is activated, as well as transcription factors elt-3, pqm-1, mdl-1 and pha-4 and their associated genes. The outcomes imply that this response compensates for the loss of functional bar-1. Altogether we show that bar-1 loss-of function leads to delayed development possibly caused by an induction of a stress response, reflected by daf-16 activated genes.
|Uncovering hidden Wnt/beta-catenin enhancer in Caenorhabditis elegans
Rodriguez, M. ; Snoek, L.B. ; Schmid, T. ; Riksen, J.A.G. ; Samadi, N. ; Bent, L. van der; Grolleman, J. ; Hajnal, A. ; Kammenga, J.E. - \ 2013
In: Proceedings of 1st Annual Meeting, COST FA 1208, 09-11 October 2013, Birnam, Scotland. - - p. 55 - 55.
Background: Wnt/ß-catenin pathway is well conserved along metazoans and plays an essential role in important cellular functions such as specialization, migration, adhesion and development. Mutant analyses in C. elegans strain Bristol N2 have been widely studied. However mutations in a single genetic background do not reveal genome-wide allelic effects in natural populations and they are of limited value in the approach of complex human disease pathways, for which C. elegans is an important model species. Observations: Mutations in natural variants may revealed hidden polymorphic regulators, thus we investigated the phenotypic effects of bar-1(ga80) in a population of different C. elegans genotypes. Each genotype carries the bar-1 mutation in a genetic mosaic background resulting from the recombination of two of the most divergent C. elegans strains genotypes: Bristol N2 and Hawaii CB4856. We quantified genome-wide gene expression and measured the vulval development index (determined by the number of vulval precursor cells that undergo vulval development cell fate) and gonad migration across all genotypes. Quantitative genetics analysis identified loci on chromosome I and II which are associated to vulval development and gonad migration phenotypes. The two loci spanned regions of 300 Kbp on chromosome I harbouring 100 genes and 55 Kbp containing 15 genes on chromosome II. Experiments will be presented to reveal the candidate gene that plays the causal modifier role. Continue Conclusions: By applying forward genetics in different genotypes we have revealed hidden genetic modifiers affecting Wnt signalling pathway. We show that natural genetic variation provides a powerful means to study the cryptic variation harbouring new players in Wnt/ ß-catenin signalling.
|Natural variation in Monoamine Oxidase A modulates RAS/MAPK pathway activity during C. elegans vulval development
Schmid, T. ; Snoek, L.B. ; Rodriguez, M. ; Bent, L. van der; Despot Slade, E. ; Kammenga, J.E. ; Hajnal, A. - \ 2013
In: Proceedings of the 5th EMBO meeting, 21-24 September 2013, Amsterdam, the Netherlands. - - p. 49 - 49.
Background: The genetic background has a strong influence on the pathogenesis of many complex, polygenic human diseases. Therefore, detailed genetic studies are needed to decipher the underlying genetic risk factors in human diseases. We use the Nematode C. elegans as a model to study the interplay between cancer-related signaling pathways and genetic background. We focus on the RAS, Notch and WNT pathway genes, whose human orthologues have been shown to be involved in several complex diseases. Obervations: We are addressing the question of how the genetic background influences the output of these signaling networks. For this purpose, we crossed mutants in the RAS pathway originally isolated in the N2 Bristol background into the CB4856 strain isolated in Hawaii and established mutation included recombinant inbred lines (MIRILs). We then measured pathway activity of RAS of each RIL by scoring the number of differentiated vulval cells. A RIL sets containing a mutation in the RAS homologue let-60(gf) in a mixed Hawaii/Bristol background show strong variation in the penetrance and expressivity of the phenotype when compared to the mutant let-60(n1046) in the “pure” Bristol background. This indicates the presence of polymorphic modifiers affecting RAS signaling. Subsequently, we have identified by QTL mapping several genomic regions that contain those modifiers. Using RNAi knockdown and transgenesis, we could identify amx-2, homologous to human MAOA, as being important for suppression of RAS pathway activity. We are now further evaluating the link between the metabolism of biogenic amines such as Serotonin and Dopamine and their role in cancer signaling. Conclusions: By using a quantitative genetics approach, combined with classical genetics we identified amx-2/MAOA as a polymorphic modifier of RAS signaling during C. elegans vulval development.
|Uncovering genotype variation of Wnt signaling in C. elegans
Rodriguez, M. ; Snoek, L.B. ; Schmid, T. ; Samadi, N. ; Bent, L. van der; Hajnal, A. ; Kammenga, J.E. - \ 2013
In: Proceedings of the 19th International C.elegans Meeting, 26-30 June 2013, Los Angeles, California, USA. - - p. 330 - 331.
WormQTL—public archive and analysis web portal for natural variation data in Caenorhabditis spp
Snoek, L.B. ; Velde, K.J. van der; Arends, D. ; Li, Y. ; Beyer, A. ; Elvin, M. ; Fisher, J. ; Hajnal, A. ; Hengartner, M. ; Poulin, G. ; Rodriguez Sanchez, M. ; Schmid, T. ; Schrimpf, S. ; Xue, F. ; Jansen, R.C. ; Kammenga, J.E. ; Swertz, M.A. - \ 2013
Nucleic acids research 41 (2013)D1. - ISSN 0305-1048 - p. D738 - D743.
life-history traits - c. elegans - systems biology - qtl - polymorphism - genotype - environment - plasticity - platform - genome
Here, we present WormQTL (http://www.wormqtl.org), an easily accessible database enabling search, comparative analysis and meta-analysis of all data on variation in Caenorhabditis spp. Over the past decade, Caenorhabditis elegans has become instrumental for molecular quantitative genetics and the systems biology of natural variation. These efforts have resulted in a valuable amount of phenotypic, high-throughput molecular and genotypic data across different developmental worm stages and environments in hundreds of C. elegans strains. WormQTL provides a workbench of analysis tools for genotype–phenotype linkage and association mapping based on but not limited to R/qtl (http://www.rqtl.org). All data can be uploaded and downloaded using simple delimited text or Excel formats and are accessible via a public web user interface for biologists and R statistic and web service interfaces for bioinformaticians, based on open source MOLGENIS and xQTL workbench software. WormQTL welcomes data submissions from other worm researchers.
|Identification of polymorphic modifiers of ras and wnt signaling in C. elegans
Schmid, T. ; Largey, F. ; Rodriguez Sanchez, M. ; Snoek, L.B. ; Kammenga, J.E. ; Hajnal, A. - \ 2012
In: 4th International conference on Quantitative Genetics: Understanding Variation in Complex Traits, Edinburgh, Scotland, 17-22 June, 2012. - Edinburgh, Scotland : ICQG2012 - p. 224 - 224.
|Combining natural genetic variation and vulval development mutants in Caenorhabditis elegans to understand complex human disease pathways
Rodriguez Sanchez, M. ; Snoek, L.B. ; Schmid, T. ; Hajnal, A. ; Kammenga, J.E. - \ 2012
In: Model Organisms to Human Biology: Cancer Genetics, Washington, USA, 17-20 June, 2012. - Washington, USA : The Genetics Society of America - p. 89 - 90.
|“All” the sequence polymorphisms between Hawaii (CB4856) and Bristol (N2)
Snoek, L.B. ; Brenchley, R. ; Hof, A. van 't; Bevers, R.P.J. ; Bono, M. de; Hajnal, A. ; Schmid, T. ; Weber, K. ; Francesconi, M. ; Lehner, B. ; Cossins, A. ; Kammenga, J.E. - \ 2012
In: Abstracts of papers presented at the Evolution of Caenorhabditis and Other Nematodes, Cold Spring Harbor Laboratory, New York, USA, 3-6 April 2012. - - p. 47 - 47.
“All” the sequence polymorphisms between Hawaii (CB4856) and Bristol (N2) Basten L Snoek¹, Rachel Brenchley², Arjen van 't Hof², Roel P Bevers¹, Mario de Bono³, Alex Hajnal4, Tobias Schmid4, Kate Weber³, Mirko Francesconi5, Ben Lehner5, Andy Cossins², Jan E Kammenga¹ 1Wageningen University, Nematology, Wageningen, 7608PB, Netherlands, ²University of Liverpool, Institute of Integrative Biology, Liverpool, L69 7ZB, United Kingdom, ³Medical Research Council, Molecular Biology, Cambridge, CB2 0QH, United Kingdom, 4 University of Zurich, Molecular Life Sciences, Zurich, CH-8057 , Switzerland, 5EMBL-Centre for Genomic Regulation, Systems Biology, Barcelona, 08003, Spain The decreasing costs and increasing throughput of next generation sequencing over the last 5 years has put sequencing of different genotypes of one species within reach , also for C. elegans. The availability of the full genome sequences of individual genotypes will identify “all” the genetic polymorphisms between them. These can be used in several ways: I) to clone new mutations, II) to calculate genetic distances, III) to find genomic sites of positive selection and IV) to find the genes (and polymorphisms) causal for natural phenotypic variation. Since most induced mutations are made in an N2 background a different stain is used to find the exact site of this mutation, often this is CB4856. Most studies on the sources of natural variation have been done on a population derived from a cross between CB4856 and N2. Even though CB4856 is so frequently used it yet has to be sequenced. Here, we present the full genome sequence of CB4856 in a format that enables easy access to the worm community. Three independent strains of CB4856 were sequenced in three different labs (Kammenga/Cossins, De Bono and Hajnal). The Kammenga/Cossins approach used the sequences of 102 recombinant inbred lines, including CB4856 and N2, which together contain the full N2 and CB4856 genome. This led to a mean coverage over 500x. The de Bono and Hajnal labs both sequenced the CB4856 with 10x coverage. These were independently aligned to the N2 reference sequence after which polymorphisms were extracted. We found almost 93,000 SNPs between N2 and CB4856. Of those almost 11,000 led to an amino acid change in almost 6,300 genes. The polymorphisms were not distributed equally over the genome. Differences in SNP frequency between, as well as within, chromosomes were found. Also loci with a high frequency of amino acid polymorphisms could be identified. Several groups of genes were enriched for SNPs and amino acid changes; these include the chemo receptors, c-type lectins and f-box proteins. Funded by: ERASysBio+ project GRAPPLE - Iterative modelling of gene regulatory interactions underlying stress, disease and ageing in C. elegans EU 7th Framework Programme under the Research Project PANACEA, contract nr 222936.
|WormQTL – Public archive and analysis web portal for C. elegans natural variation data
Snoek, L.B. ; Li, Y. ; Velde, J.K. van der; Arends, D. ; Beyer, A. ; Elvin, M. ; Fisher, J. ; Hajnal, A. ; Hengartner, M. ; Poulin, G. ; Rodriguez Sanchez, M. ; Schmid, T. ; Schrimpf, S. ; Xue, F. ; Zheng, X. ; Jansen, R.C. ; Kammenga, J.E. ; Swertz, M.A. - \ 2012
In: Abstracts of papers presented at the Evolution of Caenorhabditis and Other Nematodes, Cold Spring Harbor Laboratory, New York, USA, 3-6 April 2012. - - p. 48 - 48.
WormQTL – Public archive and analysis web portal for C. elegans natural variation data Basten L Snoek¹, Yang Li², Joeri K van der Velde², Danny Arends², Antje Beyer³, Mark Elvin 4, Jasmin Fisher³, Alex Hajnal5, Michael Hengartner5, Gino B Poulin4, Miriam Rodriguez¹,Tobias Schmid5, Sabine Schrimpf5, Feng Xue4, Xue Zheng5 , Ritsert C Jansen², Jan E Kammenga¹, Morris A Swertz2,6 1Wageningen University, Nematology, Wageningen, 7608PB, Netherlands, ²University of Groningen, Bioinformatics Centre, Groningen, 9747AG, Netherlands, ³Microsoft Research, Programming Principles and Tools, Cambridge, CB3 0FB, United Kingdom, 4 University of Manchester, Life Sciences, Manchester, M13 9PT, United Kingdom, 5University of Zurich, Molecular Life Sciences, Zurich, CH-8057, Switzerland, 6University Medical Centre, Genomics Coordination, Groningen, 9700RB, Netherlands Over the past decade increased efforts have been made to explore the model species C. elegans as a platform for molecular quantitative genetics and the systems biology of natural variation. These efforts have resulted in a huge amount of phenotypic and genotypic data across developmental worm stages and environments. To facilitate the accessibility, comparative analysis and meta-analysis of all these data we present WormQTL, a public web portal for the management of all these new data and for integrated development of suitable analysis tools. The web server provides a rich set of data and analysis tools available for direct use, based on R/qtl software. But users can also upload and share new scripts as ‘plugin’ for colleagues in the community. Furthermore, new data can be uploaded and downloaded using XGAP, which is an extensible text format for genotypes and phenotypes (Swertz et al. 2010a). All data and tools can be accessed via web user interfaces and programming interfaces. Large consortia as well as individual researchers can make use of a private area that is under embargo for publication. All software is free for download as MOLGENIS ‘app’ (Swertz et al. 2010b). WormQTL is freely accessible without registration and is hosted on a large computational cluster enabling high throughput analyses. We present WormQTL as an online scalable system for QTL exploration to service the worm community. WormQTL provides many publicly available datasets and welcomes submissions from other worm researchers. Funding: The Centre for BioSystems Genomics (CBSG) and the Netherlands Consortium of Systems Biology (NCSB), both of which are part of the Netherlands Genomics Initiative / Netherlands Organisation for Scientific Research and the EU 7th Framework Programme under the Research Project PANACEA, contract nr 222936.
|Combining Genetic Variation with Targeted Knock-downs to Construct Gene Networks of Complex Human Diseases in C. elegans
Kammenga, J.E. ; Fisher, J. ; Li, Y. ; Swertz, M.A. ; Elvin, M. ; Poulin, G. ; Snoek, L.B. ; Rodriguez Sanchez, M. ; Beyer, A. ; Schrimpf, S. ; Velde, J. van de; Escobar, J. ; Schmid, T. ; Zheng, C. ; Hajnal, A. ; Hengartner, M. ; Jansen, R. - \ 2011
In: Abstract Book of the 12th International Conference on Systems Biology, Heidelberg/Mannheim, Germany, 28 August - 1 September 2011. - Heidelberg : IBM System Storage Solutions - p. 116 - 116.
The nematode worm C. elegans has intensively been studied for complex human disease pathways. Within the EU FP7 funded PANACEA consortium we perform a quantitative pathway analysis of natural variation in complex disease signalling in C. elegans. Here, we present a robust approach for selecting candidate genes associated with the Notch, Wnt and RAS pathway using a combination of recombinant inbred lines (RILs), derived from a cross of the wildtypes N2 and CB4856, and RNAi. The consortium focuses on the transcriptome, proteome and cellular development. We tested 180 different RNAi’s targeted against genes within these three pathways in a set of 50 RILs. We highlight a case of this cryptic genetic variation in genome wide gene expression levels underlying a gld-1 knock-down (a gene involved in germline development) This revealed new candidate regulators of gld-1 affected gene expression. Furthermore we will show how mutations of the RAS pathway in different genetic backgrounds lead to the discovery of hidden modifiers affecting vulva-development, an important readout of cancer signalling pathways. All this (transcriptomics, proteomics and phenotypic) data will feed into a predictive model of vulva development which can be used to investigate the relative contributions of various recombinants.