1 / 31

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.

marinel
Télécharger la présentation

Michael Boutros Heidelberg

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related