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Michael Boutros Heidelberg

Mapping synthetic genetic interactions in Drosophila and human cells. Michael Boutros Heidelberg. Linking Genes and Phenotypes. Perturbations. Phenotypes. Genetic networks. Genetic interactions by RNAi. no interactors. -90 270. positive interactor. Features.

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Michael Boutros Heidelberg

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  1. Mapping synthetic genetic interactions in Drosophila and human cells Michael Boutros Heidelberg

  2. Linking Genes and Phenotypes Perturbations Phenotypes Genetic networks

  3. Genetic interactions by RNAi no interactors -90 270 positive interactor

  4. Features • Size, eccentricity, angle • Shape (rugged, smooth…) • Actin intensity • Tubulin spatial distribution Intensity = 1778 Cell size = 421 Eccentricity = 1.08 Actin.intensity = 124 Tubulin.intensity = 94 DNA.intensity = 74 Nucleus.size = 46 Actin.hz11 = 17.4 Actin.hz12 = 11.3 Tubulin.hz11 = 8.4 Tubulin.hz12 = 7.5 Nucleus.hz11 = 3.4 ... Image analysis pipeline Image Segmentation Cells Feature extraction Feature maps Classification Cell phenotypes Phenoprints Perturbation (gene) phenotypes Bioconductor/R EBImage/imageHTS with Wolfgang Huber (EMBL/EBI)

  5. Single-cell phenotyping ...

  6. Subset of Drosophila kinases, phosphatases, adaptors • Each targeted by two independent dsRNA designs • Validation of knock-down by qPCR • >70.000 experiments, >2x109 cells • Phenotypes of 4.600 distinct gene pairs Thomas Horn, Thomas SandmannBernd Fischer, Wolfgang Huber Genetic interactions in Drosophila cells Drosophila S2 cells

  7. Screen plot of one phenotypic read-out (number of cells) Quality control: quantitative and reproducible phenotypes within screen technical replicates (R2 = 0.968) between biological screen replicates (R2 = 0.948) between sequence-independent dsRNA (R2 = 0.902)

  8. Genetic interactions of Ras pathway components

  9. Observing different features revealsnon-redundant sets of interactions FDR < 0.05 Significant enrichment of annotated Drosophila in vivo genetic interactions (Flybase) and orthologous human protein-protein interactions

  10. JNK JNK Clustering genes by similarity of their interactions Ras Count Ras Size

  11. Machine-learnable pathway compositions I know your friends and I'll tell you (or somebody else) who you are

  12. Identification of modules linking signaling to chromatin 140+ dsRNAs … 1600 384-well plates ~400.000 experiments ~20,000 individual cells per experiment >300 features per cell ~ 3000 dsRNAs (template) …

  13. Quality control of RNAi designs detection of possible off-target effects:2 independent dsRNA designs per genecor. of multi-phenotype interaction profile between designs1293 genes passed QC

  14. Genetic interactions in multiple phenotypes

  15. Selection of phenotypes and ‘query’ genes - PCA of 328 phenotypic features- selection of ‘query’ genes with medium viability effect and distributed well over phenotypic space

  16. Genetic interactions by phenotype 1% FDR pos. and neg. interactions per feature fraction of gene pairs that interact cumulative over features fraction of gene pairs that interact 21 non-redundant features

  17. Genetic interaction map clustering of genetic interaction profilesred: ribosome biogenesisgreen: kinetochoreblue: centrosome

  18. Co-complexity score matrix Chaperonin-containing T-complex anaphase-promoting complex γ-tubulin ring complex DNA−directed RNA polymerase II core complex 26S proteasome

  19. Interaction profiles of the mediator complex

  20. Multi-phenotype interactions resolve biological modules Interaction networks based on all phenotypes Interaction networks based on cell number

  21. Cka / Striatins - a family of putative adaptor proteins Genetic interactions with many Ras pathway components

  22. sty Cka is a novel modulator of Ras/ERK signaling qRT-PCR Western blot Co-immunoprecipitation Student’s t-test, *p<0.05, ** p<0.01, n = 4 Drosophila S2 cells, starved O/N in serum-free medium Drosophila S2 cells We also showed that Striatins are similarly required for Ras/MAPK signaling in human cells

  23. Gain-of-function Ras signaling leads to a rough eye phenotype A P wt sev>RasV12 sev>RasV12 / Cka1 sev>RasV12 / Cka4 Cka loss-of-function alleles suppress the RasV12 phenotype

  24. The STRIPAK complex regulates RAS/MAPK signaling STRN/Cka STRIPAK complex GCKIII PP2A Raf/Phl RAS MEK

  25. Genetic interactions in human cells ~300 genes >200 features colon cancer cells

  26. Genetic interaction of PHF16 and SETD8

  27. Summary • Scalable approach for synthetic genetic interaction mapping by RNAi • Multi-phenotype genetic interaction maps provide a fine-grained resource to dissect functional modules • Epistasis analysis by differential genetic interactions

  28. Acknowledgements Collaborators Wolfgang Huber Bernd Fischer, Greg Pau Thomas Horn Thomas Sandmann Max Billmann Florian Fuchs Max Billmann Christian Laufer Marco Breining Funding: HFSP, DFG, EU, EMBO YIP, SFB

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