Phenotype and the Interaction of Genetic Perturbations

1. Phenotype and the Interaction ofGenetic Perturbations Informatics for System Genetics

2. Phenotype and the Interaction ofGenetic Perturbations • Introduction • Generalized derivation of genetic-interaction networks • Generation of a yeast invasiveness network • Local and global interaction patterns

3. Phenotype and the Interaction ofGenetic Perturbations Introduction

4. Network element activities and phenotype • microarray/proteomics: expression and physical interactions of each constituent • phenotype: a system variable • biomedicine

5. Directed Perturbations • Many systems have deletion projects/consortia/databases • yeast, worm, mouse, fly • Molecular biology methods can target large numbers of genes • antisense oligos, including morpholinos • RNA interference • inducible promoters

6. RNAi • Fraser et al. (Nature 408, p. 35, 2000) targeted 90% of genes on C. elegans chromosome I using RNA interference experiments, and classified resulting phenotypes.

7. Synthetic Genetic Array analysis • Systematic construction of double deletion mutants • A mutant is crossed to an array of ~5000 deletion mutants. • Observing synthetic lethal genetic interactions, generated a network of 291 interactions between 204 genes. • Tong et al. (Science 294, p. 2364, 2001)

8. SGA

9. Genetic-Interaction Databases

10. Phenotype Ontologies

11. Open Microscopy Environment (Sorger Lab)

12. What’s Needed • Parallel advances in in concepts and computational methods • Generalized derivation of genetic-interaction networks • Quantitative (at least ordered) phenotype data • Analysis of local and global interaction patterns

13. Phenotype and the Interaction ofGenetic Perturbations Generalized derivation of genetic-interaction networks

14. Genetic Interaction • - Interaction of two genetic perturbations in the determination of a phenotype • - Observed in the phenotypes of four genotypes: • a reference genotype, the “wild type” • a perturbed genotype, A • a perturbed genotype, B, with a perturbation of a different gene • a doubly perturbed genotype, AB. • - Perturbations may be of any form (null, loss-of-function, gain-of-function, dominant-negative, etc.). • - Two perturbations can interact in different ways for different phenotypes or under different environmental conditions.

15. Example

16. Hereford-Hartwell 1974

17. Hereford-Hartwell double mutant epistasis analysis

18. Hartwell: Synthetic Defects and Phenotype Buffering

19. Example

20. 75 Phenotype Inequalities in 9 (A)Symmetric Interaction Modes

21. Phenotype and the Interaction ofGenetic Perturbations Generation of a yeast invasiveness network

22. Dimorphic Fungal Pathogens S. cerevisiae Magnaporthe grisea

23. Filamentous-Form System Properties • altered cell-cycle progression • cell elongation • unipolar distal budding • adhesion • host (substrate) invasion • altered metabolism

24. Key Pathways

25. Large-scale Genetic Perturbation Transformation of Knockout Strains with Multicopy Plasmids Rsr1 Bem1 Cdc42 Ras2 • We transformed 118 homozygous diploid knockout strains plus a wildtype control strain with plasmids for constitutive overexpression of genes involved in regulation of filamentous growth. Phd1 Ste11 Kss1 Ste12 Tec1 Flo8 Gln3 Msn1

26. Phenotype Analysis Wash Assay for agar invasion: • Strains: • Diploid S1278b mutants transformed with multicopy plasmids • Phenotype: • Agar invasion • Conditions: • High glucose, low nitrogen prewash colony postwash colony

27. MATa MATa X xxx yyy Mating a/a Sporulation Haploid Selection P-MFA1::HIS3 Homozygous Double Mutant xxx yyy Strain Construction Mata xxx::NatMX x Mata yyy::KanMX Mata xxx::HygMX x Mata yyy::KanMX Mata xxx::NatMX yyy::KanMX Mata xxx::HygMX yyy::KanMX Mate and select for HygR NatR to get diploid xxxD yyyD

28. Phenotype Analysis • Strains are pinned onto solid media in a 384-spot format. • Each strain is represented by 4 independent constructions. • 4 replicates of each plate are pinned. • Each plate contains 48 spots of a wildtype vector control strain.

29. Analysis of agar invasion phenotypes of diploid mutant strains on low-nitrogen media Incubate 4 days at 30o C Wash plate Pin strains onto low-nitrogen media Scan plate Scan washed plate Prewash image Postwash image

30. Quantitation of Invasiveness

31. flo1D flo11D dia4D isw1D dfg16D Non-Invasive Invasive Hyper rim9D hmi1D bud6D tpk2D bud8D Agar invasion phenotypes of diploid mutant strains on low-nitrogen media Prewash image Postwash image Agar invasion can be visualized in the composite image.

32. Phenotype Data Analysis • Calculate ratios of postwash signal/prewash signal • Raw data file from dapple processed to ID spots and subtract background. • Output contains X = prewash signal and Y = postwash signal for each spot. • Calculate the ratio Y/X for each spot. • Normalize data to allow comparison of strains on different plates • Each plate contains 48 wildtype controls. • Calculate the median Y/X ratio for the wildtype vector controls on each plate = Mn for plate n. • The correction factor for plate n is [median (all Mvalues)/Mn]. • Phenotype Error = MAX(MAD, 10%MAD)

33. Invasiveness Quantitative Phenotypes

34. Example: Image Data

35. Example: Data Analysis

36. Phenotype Error

37. Data Subset

38. Entire Network

39. Interaction-Mode Distribution

40. Error Parameter Insensitivity

41. Phenotype and the Interaction ofGenetic Perturbations Local and global interaction patterns

42. Local Interaction,with Biological Processes - Is there “monochromatic” interaction with modules?

44. Local Interaction, with Biological Processes As noted for epistasis and synthesis…the results suggest there are characteristic network mechanisms to be found underlying the various modes of genetic interaction.

45. Global Interaction Patterns • genetic-interaction complexity • map similarities among perturbations in interaction patterns

46. Global Interaction Patterns

47. Mutual Information and and

48. Global Interaction Patterns

50. A Mutual-Information Network