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'Edgetic' perturbation of a C. elegans BCL2 ortholog

Nature Methods volume 6, pages 843–849 (2009)Cite this article

An Erratum to this article was published on 16 November 2009

This article has been updated

Abstract

Genes and gene products do not function in isolation but within highly interconnected 'interactome' networks, modeled as graphs of nodes and edges representing macromolecules and interactions between them, respectively. We propose to investigate genotype-phenotype associations by methodical use of alleles that lack single interactions, while retaining all others, in contrast to genetic approaches designed to eliminate gene products completely. We describe an integrated strategy based on the reverse yeast two-hybrid system to isolate and characterize such edge-specific, or 'edgetic', alleles. We established a proof of concept with CED-9, a Caenorhabditis elegans BCL2 ortholog. Using ced-9 edgetic alleles, we uncovered a new potential functional link between apoptosis and a centrosomal protein. This approach is amenable to higher throughput and is particularly applicable to interactome network analysis in organisms for which transgenesis is straightforward.

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Figure 1: Schematic representations of genotype-phenotype associations.
Figure 2: Isolation of ced-9 alleles insensitive to EGL-1.
Figure 3: Outline of the edgetic strategy.
Figure 4: Edgetic and non-edgetic residues in CED-9 and CED-9/CED-4 structures.
Figure 5: Positioning edgetic residues in CED-9 structures.
Figure 6: Node removal and edgetic perturbation in vivo.

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Change history

  • 16 November 2009

    In the version of this article initially published, the schematic in Figure 5a was misaligned. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

This paper is dedicated to the memory of Stan Korsmeyer. We thank the members of the Vidal Lab and of the Dana-Farber Cancer Institute CCSB and particularly A.-R. Carvunis for helpful discussions. This work was supported by US National Institutes of Health (NIH) grants R01 HG001715 from the National Human Genomics Research Institute (NHGRI) and National Institute of General Medical Sciences (NIGMS) and grants R33 CA105405, R33 CA132073 and R21/R33 CA081658 from the US National Cancer Institute (NCI) (M.V.), U01 CA105423 from the NCI (principal investigator, S. Orkin; project leader, M.V.) and by Institute Sponsored Research funds from the Dana-Farber Cancer Institute Strategic Initiative awarded to CCSB. M.D. and G.L. were supported by a 'Research Fellow' fellowship from the Fonds de la Recherche Scientifique (FRS-FNRS, French Community of Belgium). B.C. was supported by the Belgian Program on Interuniversity Attraction Poles initiated by the Federal Office for Scientific, Technical and Cultural Affairs (IAP P6/19 PROFUSA). S.M. was supported by an NIH National Research Service Award training grant fellowship (T32CA09361). P.-O.V. was supported by a European Molecular Biology Organization long-term fellowship 61-2002. Support was provided by the Leukemia Research Foundation to M.B. M.V. and R.B. thank the 'Fonds de la Recherche Scientifique (FRS-FNRS, French Community of Belgium)'. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NCI, NHGRI, NIGMS or the NIH.

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Author notes

  1. Stuart Milstein, Pierre-Olivier Vidalain, Nenad Svrzikapa & Mike Boxem

    Present address: Present addresses: Alnylam Pharmaceuticals, Cambridge, Massachusetts, USA (S.M. and N.S.), Laboratoire de Génomique Virale et Vaccination, Centre National de la Recherche Scientifique, Unité de Recherche Associée 3015, Institut Pasteur, Paris, France (P.-O.V.) and Utrecht University, Utrecht, The Netherlands (M.B.).,

  2. Matija Dreze, Benoit Charloteaux, Stuart Milstein and Pierre-Olivier Vidalain: These authors contributed equally to this work.

Authors and Affiliations

  1. Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, and Department of Genetics, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA

    Matija Dreze, Benoit Charloteaux, Stuart Milstein, Pierre-Olivier Vidalain, Muhammed A Yildirim, Quan Zhong, Nenad Svrzikapa, Viviana Romero, Géraldine Laloux, Mike Boxem, Michael E Cusick, David E Hill & Marc Vidal

  2. Unité de Recherche en Biologie Moléculaire, Facultés Universitaires Notre-Dame de la Paix, Namur, Wallonia, Belgium

    Matija Dreze, Géraldine Laloux & Jean Vandenhaute

  3. Centre de Biophysique Moléculaire Numérique, Faculté Universitaire des Sciences Agronomiques de Gembloux, Gembloux, Wallonia, Belgium

    Benoit Charloteaux & Robert Brasseur

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Contributions

M.D., B.C., S.M., P.-O.V., M.A.Y., Q.Z., R.B., J.V., M.B. and M.V. conceived the experiments and analyses. S.M. and P.-O.V. generated the ced-9 mutant library and performed Y2H screens and R-Y2H selections. M.D. and P.-O.V. cloned the alleles. P.-O.V. and M.D. performed the co-APs. M.D. and G.L. developed and implemented the modified Y2H assay. B.C. performed the structural analyses. B.C. and M.A.Y. performed the statistical analyses. N.S. generated the transgenic worms under the supervision of S.M. and M.B. S.M. performed survival and apoptosis challenge experiments. M.D. and V.R. performed the mutant alleles stability experiment. M.D., B.C., S.M., P.-O.V., M.E.C. and M.V. wrote the manuscript. All authors discussed the results. D.E.H. and M.V. conceived and co-directed the project.

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Correspondence to Marc Vidal.

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Dreze, M., Charloteaux, B., Milstein, S. et al. 'Edgetic' perturbation of a C. elegans BCL2 ortholog. Nat Methods 6, 843–849 (2009). https://doi.org/10.1038/nmeth.1394

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