Dupuy, Denis (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "High-throughput screening for alternative splicing regulation factors in vivo"

DUPUY, Denis

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

The aim of this our study is to systematically identify the trans-acting elements involved i alternative splicing regulation. Fluorescent reporters provide the opportunity to observe functional effects of a gene knock-down that may have a subtle phenotype that is undetectable with traditional observations. We are using a transgenic strain carrying a two-color reporter system in which two fluorescent reporters (GFP and RFP) are respectively fused to mutually exclusive alternatively spliced exons (Ohno, et al 2008) of the let-2 gene (exons 9 and 10). This strain has been subjected to a genome-wide RNAi screen for modifiers of the alternative splicing ratio. While previous RNAi screens have mostly consisted of visual observation of the worms by microscopy, we use the COPAS-Profiler to perform multidimensional quantitative analysis on the knocked-down worm populations in 96-well format. This enables us to detect not only variation in growth and fertility but also any modification of the relative expression of the two reporters i.e. modification of the alternative splicing balance. In this set up we are able to measure, for each individual RNAi experiment, the total number of worms, their sizes distribution, as well as collect longitudinal expression profiles in two fluorescent channels. Importantly, we are able to compare expression patterns within individual animals, thus providing an endogenous control for stochastic variations in overall expression level.

Dupuy, Denis et al. (2017) International Worm Meeting "Meta analysis of RNAseq datasets provides quantitative measurement of alternative exons usage in C. elegans."

DUPUY, Denis, Millet, Jonathan, Tourasse, Nicolas

[International Worm Meeting, 2017]

Almost twenty years after the completion of the C. elegans genome sequence, gene structure annotation is still an ongoing process with new evidence for gene variant still being regularly uncovered by more in-depth transcriptome studies. Alternative splice forms, allow a single gene to encode several protein variants, called isoforms, with altered stability, localization, specificity or activity. Here we generated a compendium of ~1,700 C. elegans RNAseq datasets to expand the dynamic range of detection of RNA isoforms and obtain robust measurement of the relative abundance of each splicing event. We detected ~700,000 exon-exon junctions and over 5,000,000 transplicing sites, but most of these sites are only supported by a very low number of reads : 98% of splicing reads come from ~60,000 high confidence-splice junctions and 88% of transpliced reads come from ~36,000 robust transplice sites. Our analysis indicates that the mechanism of transplicing generates more aspecific species than cis-splicing, but it also highlights that putative erroneous splicing only represent a minor proportion of detectable spliced RNA species. We found that rarely used splice sites within coding genes are significantly less conserved in other nematode genomes than splice sites with a higher usage frequency. We generated updated gene models including previously unreported transcription start and splice sites and including quantitative exon usage information for the entire C. elegans genome to allow users to visualize at a glance the relative expression of each isoform of their gene of interest.

Denis Dupuy et al. (2005) International Worm Meeting "The C. elegans Localizome Project: a beginning"

Jane Shingles, Robert Johnsen, Qianru Li, Ian A Hope, Jennifer Rosenberg, John Reece-Hoyes, Alain Carnec, David Lee, Rebecca Newbury, Denis Dupuy, Wiliam A Mohler, Rock Pulak, Marc Vidal, David Baillie, Nicolas Bertin, Ryan Viveiros, Don Moerman, Domena Tu

[International Worm Meeting, 2005]

The field of systems biology attempts to provide a systems level understanding of biological processes by building descriptive and mechanistic models of biological phenomena. An important aspect of the complexity of metazoan organisms lies within the differential expression of the genes that drives cell diferentiation. The long-term goal of the C. elegans localizome is to generate a gene expression and protein localization map, for most genes, across development. We present solutions for the major challenges of a C. elegans proteome-wide localizome map. We generated a genome-wide resource of C. elegans promoters needed to generate transgenic animals expressing GFP. Version 1 of the worm promoterome is a resource of ~6,500 C. elegans promoters that can be transferred rapidly into various Multisite Gateway Destination vectors to drive expression of GFP, or other markers, either directly from promoters or in fusion with a protein-encoding ORFs. We will describe a new method that uses a nematode profiler/sorter system. This system allows the high-throughput mapping of gene expression patterns along the anterior-posterior axis of each animal. As thousands of animals from a mixed population of worms are analyzed, fluorescence traces can be arranged by size into Post-embryonic Developmental Expression Chronograms (PDECs). A PDEC provides a reconstituted "time-lapse" image of gene expression throughout larval to adult development across the length of the worms body. Although the spatial resolution of the PDEC is lower than that of traditional microscopic analyses, this data format allows automated comparison and clustering of expression patterns, while also giving access to a clear temporal context of gene expression. We acquired PDECs for over 1,500 GFP-strains generated mainly by the C. elegans Gene Expression Consortium and the Hope lab. We will discuss how these data can be used in combination with interactome, transcriptome and phenome datasets, and also how they compare to manual annotations performed through microscopic observation.

Denis Dupuy et al. (2007) International Worm Meeting "Genome-scale analysis of in vivo spatiotemporal promoter activity in C. elegans."

Domena Tu, Rebecca Hunt-Newbury, Cesar Hidalgo, Ian Hope, David Lee, Ryan Viveiros, Albert-Laszlo Barabasi, Anne-Ruxandra Carvunis, John Reece-Hoyes, Denis Dupuy, David Baillie, Nicolas Bertin, Murat Tasan, Donald Moerman, William Mohler, Rock Pulak, Kavitha Venkatesan, Marc Vidal, Frederick Roth, Nenad Svrzikapa

[International Worm Meeting, 2007]

*The first 3 authors contributed equally. Correspondence should be addressed to any of the last 3 authors. Much of the complexity of metazoan organisms comes from differential expression of genes to drive cell differentiation. Characterizing the transcriptional activity of gene promoters, in time and in space, is therefore a critical step towards understanding complex biological systems. We developed a new application that enables high-throughput fluorescence expression pattern acquisition, using the nematode profiler (COPAS, Union Biometrica). Individual profiles collected from a mixed stage population are arranged by size into post-embryonic expression chronograms. A chronogram provides a reconstituted time-lapse image of gene expression during development from larva to adult across the length of the worm body. This data format allows unsupervised comparison and clustering of expression patterns, while also providing a clear temporal overview of gene expression. We present an analysis of the spatiotemporal activity of ~5% of the predicted C. elegans promoters driving the expression of green fluorescent protein (GFP) in vivo. Automated comparison and clustering of the obtained ~900 chronograms show that genes co-expressed in space and time tend to belong to common functional categories. Moreover, integration of this localizome dataset with C. elegans protein interactome datasets enables prediction of interaction territories between protein partners.

Denis Dupuy et al. (2003) International Worm Meeting "Cloning the C. elegans promoterome, a gateway to a Genome wide Localizome in the Worm (GLOW)."

Bill Mohler, Marc Vidal, Bart Deplancke, Joel Jessie, Denis Dupuy, Ian A Hope, Maria DeBarnardi, Mike Brasch, AJ Marian Walhout

[International Worm Meeting, 2003]

It has been argued that, to globally understand development, it will be crucial to generate databases describing where (in what cell) and when (at what stage of development) each protein is expressed. One possible approach is to construct transgenic C. elegans strains carrying reporter genes, such as that encoding the Green Fluorescent Protein (GFP), driven by particular promoters and examine expression patterns throughout development. Such studies have already been successfully carried out with relatively small subsets of genes, this project aims to address conceptual and technical issues that will allow us to apply this approach to a genome wide scale. We propose to clone the majority of worm promoters into a novel Gateway compatible vector. The availability of a comprehensive collection of cloned worm promoters (promoterome) will be very useful to the scientific community. We use an innovative Gateway technology with which we will transfer cloned promoters into various destination vectors. Each promoter can then be used to detect expression of reporter genes, or to drive expression of their cognate ORFs, fused to either GFP, or any other Tag-encoding sequence. These can be used to examine sub-cellular protein localization, for complex purifications or in genetic rescue experiments. To increase the throughput of generating transgenic worms to a genome scale, we will adapt the recently developed ballistic transformation protocol (Praitis, 2001) This method allows genomic integration of low copy plasmids which may allow more physiological regulation of the transgenes than extrachromosomal arrays. We will then use a semi-automated 96 well confocal microscopic platform (Atto Bioscience) for rapid imaging and analysis of multiple independent transgenic strains. All the data generated will be made available to the community on a freely accessible database. To illustrate usage of Multisite Gateway to generate promoter::ORF::GFP fusion proteins. We created a construct in which a Histone2B (B0035.8) is expressed from the myo-2 promoter, which drives expression in the muscle cells of the pharynx. We then used the microparticle bombardment technique to integrate this construct into the C. elegans genome. As expected, the pmyo-2::H2B::GFP construct expressed in the muscle cells of the pharynx, and the H2B::GFP fusion localized to the nucleus.

Quere, Cecile A.L. et al. (2013) International Worm Meeting "In vivo reporters for spatiotemporal regulation of genes by microRNA."

Quere, Cecile A.L., DUPUY, Denis

[International Worm Meeting, 2013]

Caenorhabditis elegans development is very tightly regulated, leading to the same number of cells in each individual. Part of this regulation network relies on small single strand RNAs (miRNAs), which can target homologous sequences in the 3' untranslated regions (3'UTR) of mRNAs. We want to investigate the contribution of miRNAs during neurons differentiation. In order to study the miRNA contribution to gene regulation we use double fluorescent reporters that allow us to visualize the post-transcriptional contribution to regulation throughout development. The GFP and the mCherry are expressed under the control of the gene promoter, but followed by either the 3'UTR of interest, or a control 3'UTR. We first chose as a control 3'UTR the 3'UTR of unc-54, a gene encoding a myosin class II heavy chain. It is expressed through all larval stages and in the adult worms. The prediction softwares do not predict any target site for miRNA regulation. Our observations showed that some cells were not expressing the control reporter while expressing the reporter associated to the endogenous 3'UTR. This was the case for 6 different genes. Moreover, constructions using the same 3'UTR showed a perfect colocalization of the two reporters excluding the possibility of an artifact linked to the fluorescent reporters. Therefore, we selected the 3'UTRs from act-1, ubc-1 and his-24, because of their constitutive expression and the absence of miRNA target site prediction by Pictar. To confirm the potential post-transcriptional regulation of unc-54, we will compare the expression patterns associated with these 3'UTRs with that of unc-54. We will also use the promoter of unc-119 to analyze the 3'UTRunc-54 behavior specifically in neurons. We will perform an EMS mutagenesis screen to identify the regulators involved in this post-transcriptional regulation. With permissive 3'UTRs, we will be able to extend the characterization of post-transcriptional regulation to more genes participating in neurons development. These genes will be selected based on the prediction of a target site for miRNA expressed in neurons, like lsy-6, mir-273 or mir-81.

Millet, Jonathan R. M. et al. (2013) International Worm Meeting "In vivo spatiotemporal analysis of mRNA alternative splicing during C.elegans neural development."

DUPUY, Denis, Millet, Jonathan R. M.

[International Worm Meeting, 2013]

Neuronal differentiation is a complex process involving fine-tuned gene regulatory network from neuroblast generation to terminal differentiation of well-defined neurons. Networks are defined by their components (genes) and by the relationship linking those components (regulatory mechanisms). Alternative splicing is a phenomenon that allows a single gene to produce multiple isoforms in a time- or location-dependent manner. Alternative splicing can lead to protein isoforms with altered stability, localization, specificity or activity, and affect neuronal gene regulatory network. At least 15% of C.elegans genes have two or more splicing isoforms. We compare the spatio-temporal expression of mRNA isoforms from a single gene in transgenic lines. We use a frame shift reporter fused to the gene of interest, that allows us to visualize the splicing regulation using fluorescence microscopy. This method was applied to several genes with multiple isoforms. top-1 encodes one embryo-specific and one neuron-specific mRNA splice variants of the DNA topoisomerase I. che-1 is an important transcription factor involved in ASE terminal differentiation. tax-6 encodes three different isoforms of Calcineurin and play an important role in C.elegans sensory neurons adaptation. gpa-13 encodes three mRNA isoforms of a Galpha-subunits involved in sensory neuron signal transduction. We will use EMS mutagenesis followed by COPAS-assisted screening for mutants with altered fluorescence patterns for the identification of alternative splicing regulators. Using this method, in combination with other genetic approaches, we aim to better understand splicing regulatory networks involved in neuron differenciation. We want to validate neuron-specific alternative splicing regulators and their targets and to understand the contribution of alternative splicing to Caenorhabditis elegans neuronal genetic programming. Unravelling the components of gene regulatory network involved in the development of the compact nervous system of C.elegans could lead to a better understanding of neural development.

Zniber, Ilyass et al. (2011) International Worm Meeting "An integrated approach for in vivo miRNA regulation studies in C. elegans."

DUPUY, Denis, Bothorel, Marie, ZNIBER, Ilyass, GUIGNARD, Leo

[International Worm Meeting, 2011]

MicroRNAs (miRNAs) are 20-22 nucleotides RNA molecules that function as negative regulators of gene expression in both eukaryotic and procaryotic organisms. Single stranded mature miRNA bind to target mRNAs through partly complementary sequences that are localized in their 3' untranslated region. The first discovered miRNA genes were lin-4 and let-7, which have been identified initially in Caenorhabditis elegans. PicTar, a powerfull target prediction algorithm for miRNA regulation, predicts that 10% of C. elegans genes are regulated by this mechanism. To date, very few of these predictions have been experimentaly validated. The aim of this project is to develop an approach for large scale experimental validation of over a hundred of predicted targets. I built a reporter cassette where two fluorescent proteins are expressed in a bicistronic manner under the target gene's promoter to mimic the spatio-temporal expression pattern of the studied gene. The mCherry (transcriptional output reporter) is associated to the permissive UTR of the unc-54 gene while the GFP (post-transcriptional output reporter) is regulated by the target gene's UTR. Taking advantage of C. elegans fosmid library, we can replace the ORF of the target gene by the two-color reporter cassette using recombineering (recombinaison ingeneering) for 200 miRNA regulated predicted genes by PicTar. Reporter fosmids will be injected to C. elegans and expression profile will be analysed by microscopy for fine expression and by flow cytometry using COPAS (Union Biometrica) for quantitative analysis. As a proof of principle, I present several studies performed on transgenic worms carrying the bicistronic cassette reporter specific of several let-7 targets.

Giordano-Santini, Rosina et al. (2010) Worm Breeder's Gazette "Antibiotic markers for rapid selection and easy maintenance of transgenic nematodes"

Lehner, Ben, DUPUY, Denis, Semple, Jennifer, Giordano-Santini, Rosina

[Worm Breeder's Gazette, 2010]

ZNIBER, Ilyass et al. (2009) International Worm Meeting "A high-throughput platform for quantitative analysis of RNAi phenotypes in C. elegans."

DUPUY, Denis, ZNIBER, Ilyass, Rebora, Karine, GUIGNARD, Leo

[International Worm Meeting, 2009]

Genome scale RNAi studies are generating a wealth of information on the phenotypic effects of gene depletion in C. elegans. However, for about 90% of genes no physiological effect could be observed in the initial visual screens. The use of RNAi sensitised mutant strains and fluorescent reporters allowed to uncover phenotypes for an additional 5 % of the gene tested. Provided we used the appropriate fluorescent reporter construct, it is likely that it would be possible to observe a modifying phenotype for the vast majority of genes with otherwise undetectable physiological effect on the animal. However, the number of RNAi screens to be performed to cover would be unpracticable using traditional microscopy and are extremely challenging for automated image analysis software. Here we present a high throughput platform for screening of fluorescent modulation in C. elegans. We use the COPAS-Profiler to perform multidimensional quantitative analysis on the knocked-down worm populations. This enables us to detect not only variation in growth and fertility but also any modification of the relative expression of up to three fluorescent reporters. We modified the Reflx plate handling system to increase the processing speed. We are currently able to collect quantitative information for up to 1000 worms from each well of a 96-plate in 25 minutes. In this set up we are able to measure, for each individual RNAi experiment, the total number of worms per well, their sizes distribution, as well as collect longitudinal fluorescence expression profiles. Importantly, we are able to compare expression patterns within individual animals, thus providing an endogenous control for stochastic variations in overall expression levels.