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Synthetic Lethality

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  1. Synthetic Lethality X A X Viable! Y B X Viable! Z C Product Dead! • Inactivating two interacting pathways • causes lethality (or sickness)

  2. A A aD aD X X B B bD bD Wild-type Viable Viable Lethal Synthetic Lethality • Synthetic Lethality Identifies Functional Relationships • Large-Scale Synthetic Lethality Analysis Should Generate a • Global Map of Functional Relationships between Genes • and Pathways • Gene Conservation = Genetic Network Conservation

  3. A A A A X X X X B B B B Y Y Y Y C C C C Z Z Z Z Essential Product Essential Product Essential Product Essential Product Similar Patterns of Genetic Interactions Identify Pathways or Complexes X X X X X X Dead Dead Dead Genetic Interaction Network

  4. or A B or regulates Scenarios That May Give Rise to Synthetic Interaction • Interpretation depends on context • Each synthetic interaction must be interpreted on a case-by-case basis (Guarente (1993) TIG, 9:362) A B A B etc. etc.

  5. MATa MATa X bni1 xxx Mating D a/a wild-type Sporulation MATa Haploid Selection (MFA1pr-HIS3) Double Mutant Selection

  6. Synthetic Gene Array (SGA) Statistics • 132 query gene mutations were crossed into ~4700 yeast deletion mutants. • Query genes derived from 3 basic functional groups: (1) actin/polarity/secretion, (2) microtubule/mitosis, and (3) DNA synthesis/repair. • Number of interactions per query varied from 1 to 146 with an average of 36. • (Genes, Genetic Interactions): ~1000 nodes and ~4000 edges. • 17 to 41% false negative rate • False positive rate? • Data quality is good

  7. Making Sense of Genetic Interaction Network • Correlation with GO annotations • Hierarchical clustering groups according to their SGA profile • Useful for inferring function of unknown genes • Correlation with protein-protein interactions? • Only 30/4039 encode physically-interacting proteins • Statistical properties of genetic interaction network graph

  8. Network of GO Attributes

  9. Cell polarity • Actin patches • Endocytosis • Cell wall synthesis • Cell integrity (PKC) Clustering Array Query

  10. Cell Polarity BEM1 BEM2 BEM4 BUD6 SLA1 CLA4 ELM1 GIN4 NAP1 SWE1 Cell Wall Maintenance BCK1 SLT2 BNI4 CHS3 SKT5/CHS4 CHS5 CHS7 FAB1 SMI1 Cell Polarity 20% Cytokinesis 6% Unknown 22% Cell Wall Maintenance 18% Mitosis 16% Cell Structure 6% Vesicular Transport SNC2 VPS28 YPT6 Mitosis ARP1 ASE1 DYN1 DYN2 JNM1 PAC1 NIP100 NUM1 Unknown BBC1/YJL020c NBP2 TUS1 YBL051c YBL062w YDR149c YHR111w YKR047w YLR190w YMR299c YNL119w Cytokinesis BNR1 CYK3 SHS1 Others PCL1 ELP2 ELP3 Cell Structure ATS1 PAC11 YKE2/GIM1 bni1D: Genome-Wide Synthetic Lethality Screen

  11. Cell Polarity BEM1 BEM2 BEM4 BUD6 SLA1 CLA4 ELM1 GIN4 NAP1 SWE1 Cell Wall Maintenance BCK1 SLT2 SMI1 CHS3 SKT5/CHS4 CHS5 CHS7 BNI4 SMI1 Cell Polarity 20% Cytokinesis 6% Unknown 22% Cell Wall Maintenance 18% Mitosis 16% Cell Structure 6% Vesicular Transport SNC2 VPS28 YPT6 Mitosis ASE1 ARP1 DYN1 DYN2 JNM1 PAC1 PAC11 NIP100 NUM1 Unknown BBC1/YJL020c NBP2 TUS1 YBL051c YBL062w YDR149c YHR111w YKR047w YLR190w YMR299c YNL119w Cytokinesis BNR1 CYK3 SHS1 Others PCL1 ELP2 ELP3 Cell Structure ATS1 PAC11 YKE2/GIM1 bni1D: Genome-Wide Synthetic Lethality Screen

  12. DNA Repair 46% Unknown 4% Cell Polarity 4% Chromatin Structure 13% DNA Synthesis 13% Meiosis 4% sgs1D : Genome-Wide Synthetic Lethality Screen (24 Interactions) DNA Repair ASF1 HPR5 POL32 RAD27 RAD50 SAE2 SLX1 MMS4/SLX2 MUS81/SLX3 SLX4 WSS1 Meiosis CSM3 Others PUB1 RPL24A SWE1 SIS2 SOD1 Unknown YBR094w Chromatin Structure ESC2 ESC4 TOP1 DNA Synthesis RNR1 RRM3 YNL218w

  13. Cell Polarity Cell Wall Maintenance Cell Structure Mitosis Chromosome Structure DNA Synthesis DNA Repair Unknown Others 8 SGA Screens: 291 Interactions 204 Genes

  14. Extension of SGA: E-MAP • E-MAP = epistatic miniarray profiles • Quantitative measurement of phenotype (e.g. growth rate) • Measure both aggravating and alleviating genetic interactions • Hypomorphic alleles (not null mutations) • Focus on subset of genes • Maya Schuldiner/Jonathan Weissman

  15. Organizing Complexes into Pathways Using Genetic Interactions Complex A Complex X X Complex B Complex Y X X = Negative Positive= Complex C Complex Z P

  16. “RNA World” E-MAP (600 genes)

  17. Positive Genetic Interactions Negative Genetic Interactions

  18. Positive Genetic Interactions Negative Genetic Interactions

  19. Proteasome Mutants Suppress Deletions in THP1/SAC3 WT ∆sem1 ∆thp1 ∆thp1 ∆sem1 rpn11-DAmP ∆thp1rpn11-DAmP rpt6 ts ∆thp1rpt6 ts

  20. Proteasome Mutants Suppress mRNA Export Defects of thp1∆ ∆thp1∆sem1 WT ∆thp1 polyA RNA polyA RNA Nuclei Merge

  21. Proteasome is Required for Efficient polyA mRNA Export WT ∆sem1

  22. Organizing Complexes into Pathways Using Genetic Interactions Complex A Complex X X Complex B Complex Y = synthetic lethality epistatic/ suppressive= X X Complex C Complex Z P What about essential genes??????

  23. Essential vs. Non-essential Genes in Budding Yeast Non-Essential Genes (~4800) Essential Genes (~1050)

  24. CREATING MUTANT ALLELES OF ESSENTIAL GENES 1. TET-Promoter Shut-Off Mutants 2. DAmP Alleles 3. Conditional point mutants

  25. 1. TET-Promoter SHUT-Off Strains -Hughes and colleagues created a library of promoter-shutoff strains comprising nearly two-thirds of all essential genes in yeast (602 genes)

  26. 1. TET-Promoter SHUT-Off Strains -the library was subjected to morphological analysis, size profiling, drug sensitivity screening and microarray expression profiling

  27. rRNA Processing Cdc53 1. TET-Promoter SHUT-Off Strains Cell Morphology Cell Size

  28. 1. TET-Promoter SHUT-Off Strains Gene Expression Analysis

  29. Protein Secretion Ylr440c Mitochondrial Regulation Yol026c Ribosome Biogenesis Ymr290c, Ykl014c, Yjr041c 1. TET-Promoter SHUT-Off Strains

  30. 1. Genetic Analsyis using the TET-Promoter SHUT-Off Strains -30 different mutants X TET-promoter collection -found many interactions between dissimilar genes -claimed that there are five times as many “negative” genetic interactions for essential genes when compared to non-essential genes -however, the cause of this may be due to the fact that the TET strains were very sick (and they were not quantitatively assessing the growth of the double mutant by considering the growth of the two single mutants)

  31. 2. DAmP Alleles (Schuldiner et al., Cell, 2005)

  32. 2. DAmP Alleles

  33. 3. Point Mutants of Essential Genes

  34. Genetic Profiling of Point Mutants Reveals Insight on Structure-Function PCNA (Pol30) -PCNA is important in many aspects of DNA metabolism, including DNA replication and DNA repair -PCNA interacts with CAF-1, a three-subunit protein, to couple DNA replication or DNA repair to nucleosome deposition -Two mutants of PCNA (pol30-8 and pol30-79) generated by Stillman and colleagues

  35. Genetic Profiling of Point Mutants Reveals Insight on Structure-Function PCNA (Pol30) -PCNA is important in many aspects of DNA metabolism, including DNA replication and DNA repair -PCNA interacts with CAF-1, a three-subunit protein, to couple DNA replication or DNA repair to nucleosome deposition -Two mutants of PCNA (pol30-8 and pol30-79) generated by Stillman and colleagues

  36. What is “Chemical Genetics?” Chemical genetics is the use of exogenous ligands to alter the function of a single gene product within the context of a complex cellular environment. • Find ligands that affect a biological process (forward) • Optimize ligands to study protein function (reverse)

  37. Forward Chemical Genetics • Goal is target identification • Screening large sets of small molecules • Those that cause a specific phenotype of interest are used to isolate and identify the protein target

  38. Plate with cells Identify protein Target (deconvolution) Add one compound per well Select compound that produces phenotype of interest Forward Chemical Genetics Target Identification

  39. Reverse Chemical Genetics • Goal is target function and validation • Screen for compounds that bind to a given protein • Optimize for selectivity

  40. Find ligand for protein of interest Optimize for selectivty Assay for phenotype Add ligand to cells Reverse Chemical Genetics Target Validation

  41. FORWARD Chemical Genetic Studies in Yeast 1. Screening the deletion set for drug sensitivities 2. Comparing mutant profiles to drug profiles 3. Haploinsufficieny analysis

  42. Organizing Complexes into Pathways Using Genetic Interactions Complex A Complex X X Complex B Complex Y = synthetic lethality X Complex C Complex Z P X= Drug

  43. Deletion Mutants Sensitive to a Particular Drug Should be Synthetically Lethal with the Drug Target Synthetic Lethal Interactions Synthetic Chemical Interactions Alive Alive Drug Alive Alive Drug Dead Dead

  44. 1. Screening the deletion set for drug sensitivities

  45. 1. Screening the deletion set for drug sensitivities

  46. FORWARD Chemical Genetic Studies in Yeast 1. Screening the deletion set for drug sensitivities 2. Comparing mutant profiles to drug profiles 3. Haploinsufficieny analysis

  47. 2. Comparing mutant profiles to drug profiles

  48. 1. Clustering of the Drug Profiles: Camptothecin and Hydroxyurea have a similar mode of action: they both inhibit DNA replication Parsons et al., 2004, Nature Biotechnology

  49. CAMPTOTHECIN: causes single-stranded DNA nicks and inhibits DNA replication Also known as : Hycamtin (GlaxoSmithKline) and Camptosar (Pfizer) -used as an anti-cancer agent RFA1 RTT105 POL30-79 POL30-879 POL32 RAD27 RFC5 POL30 ELG1 RFA2 PRI1 RFC4 CDC9 TSA1 CAMPTOTHECIN (15 g/ml) CAMPTOTHECIN (30 g/ml) DNA Replication Factors 2. Comparison of drug profiles to mutant profiles: