1. Yeast as a model organism Model eukaryote –Experimental genetics –Gene function – Orthologs, family members –Pathway function - “Biological synteny” Testbed for genomic technologies –Genome sequenced (4/96) relatively less complex –Ability to assess biological relevance of the data

4. Genomics technology development Yeast as a testbed Gene expression patterns –DNA microarrays, SAGE Genomic DNA scans –Mapping complex traits (SNPs) Phenotype screening –Genome-wide knockouts Genetic interaction networks –Synthetic lethals Protein interaction networks –Two-hybrid, mass spectrometry

5. Affymetrix whole genome yeast array Each gene is probed by multiple oligonucleotide probes (>19). A control probe is synthesized adjacent to each actual probe ~120,000 different oligonucleotide sequences for the entire genome. Entire yeast genome is on 5 arrays (~ 65,000 25 mers on each). 2 kb Gene 1 Gene 2 25mers Lisa Wodicka, Dave Lockhart, Affymetrix

6. Assigning function by analyzing gene expression Isolate mRNAIsolate mRNA Label mRNALabel mRNA Hybridize to arrayHybridize to array Detect hybridizationDetect hybridization Measure the abundance of every mRNAMeasure the abundance of every mRNA –Test different growth conditions –Test different genetic backgrounds

7. Measuring gene expression using the Affymetrix array Whole genome mRNA expression pattern for yeast grown in rich media Lisa Wodicka, Dave Lockhart Affymetrix

8. Transcriptional analysis of the yeast cell cycle M G1 S G2 Events of cell growth, DNA replication, cell division and chromosome segregation are tightly controlled. Events of cell growth, DNA replication, cell division and chromosome segregation are tightly controlled. Cultures can be synchronized with respect to the cell cycle. Cultures can be synchronized with respect to the cell cycle. Cyclic regulation of transcription expected. Cyclic regulation of transcription expected.

9. Cell cycle transcriptional regulation of two genes CLN1YML027W late G1 phase S phase G1 phase S phase

10. Genes induced in S and M phase Normalized Transcript Level G1 S G2 M G1 S G2 M 2.52.01.51.00.50 0 20 40 60 80 100 120 140 160 Time (minutes) Time (minutes)

11. Cell-cycle transcription in yeast 425 open reading frames were identified that displayed cell-cycle dependent fluctuations in transcript levels. 40% were of unknown function. 30% are located next to other cell-cycle transcribed genes (possible enhancer effect). Correlation with known and unknown promoter elements.

12. Mapping complex traits Construct a SNP genetic map Perform cross Analyze rare segregants Identify regions inherited solely from one parent

13. YJM789 Isolated from the lung of an AIDS patient. Able to grow at 42 °C, form pseudohyphae and undergo colony- morphology switching. Hypersensitive to cycloheximide. Polymorphic –one difference every 150 bases relative to sequenced strain Laboratory strain YJM789 parent

14. Allelic variation between two strains can be detected using arrays. Laboratory strain (non-pathogenic) YJM789 (virulent wild isolate) 2 kb Gene 1 25mers Mismatch control probe (position 13 of 25) 2 kb Gene 1 25mers * * ** missing signals = markers Polymorphisms Yeast Array Since probe locations are known, a genetic map can be constructed: interesting loci (virulence) can be mapped and positionally cloned for study.

15. Allelic variation in YJM789 3808 markers detected by automated analysis of scanned images. –Largest gap = 56 kb –Average frequency = 3000 bases (1.0 cM) More markers identified in one hybridization than in the past 40 years of yeast genetics.

16. Verification of markers by tetrad analysis Expect 90 cross-overs per genome. Expect 90 cross-overs per genome. Expect clear recombination breakpoints Expect clear recombination breakpoints Expect most markers to segregate 2:2. Expect most markers to segregate 2:2.

17. Segregation of markers in one tetrad (one chromosome) 96 crossovers (90 expected). 96% of markers segregate 2:2. Clear breakpoints observed. Markers segregate as expected

18. spore 1 spore 2 spore 4 spore 3 Laboratory strain (S96) genotype: MATa, lys5, LYS2, ho, CYH Wild Isolate (YJM789) genotype MAT  LYS5, lys2, ho::hisG, cyh Diploid Haploid 1 16 1 1 1... (mat  lys2, LYS5, ho, cyh)

19. Inheritance of markers in 10 lys2 segregants

20. Results of mapping five phenotypic loci in 10 segregants. Five regions identified that were inherited solely from one parent. Four encompassed known locations of MAT, LYS5, LYS2, and HO. Minimum intervals ranged from 12 to 90 kb.

21. Cycloheximide sensitivity = pdr5 Cycloheximide sensitivity maps to remaining 56 kb interval on Chromosome XV adjacent to pdr5. PDR5 is deleted in YJM789. Wildtype strain, deleted for pdr5 is unable to complement YJM789.

22. Mapping Complex Traits: Feasibility Summary Identified 3808 genetic markers. Demonstrated that traits can be mapped using these markers. Next step: Map virulence loci.

23. Virulence in YJM789 Virulence is a multigenic trait with 5 loci contributing. –Only 5 of 200 segregants from crosses between YJM789 and laboratory strain are virulent. Genes cannot be cloned by complementation. Hybridization with arrays is an appropriate way to map all contributing loci simultaneously.

24. Assigning Function through Mutational Analysis Inactivate gene product (delete gene). Grow mutant strain under different selective or stress conditions. Identify mutants with growth defects. Function of gene product may be revealed. –UV sensitivity = DNA repair protein –Adenine auxotrophy = Adenine biosynthesis

25. Construction of yeast deletion strains KanR plasmid Deletion Cassette Chromosomal Gene Amplify selectable marker gene using primers with yeast gene homology at 5’ ends Replace yeast gene by homologous recom- bination yeast sequence

26. International Deletion Consortium Members Mike Snyder, Jasper Rine, Mark Johnston, Jef Boeke, Howard Bussey, Rosetta, Acacia, Peter Philippsen, Hans Hegemann, Francoise Foury, Guido Volckaert, Bruno Andre, Giogio Valle, Jose Revuelta, Steve Kelly, Bart Scherens 24,000 strains in 3 years

28. Serial analysis of deletion strains Apply Selection Identify deletion strains with growth defects 1 2 3 6,000

29. Molecular tags as strain-identifiers Unique 20-mers Unique 20-mers Good hybridization properties Good hybridization properties Similar melting temperatures Similar melting temperatures More than 5 base differences between each More than 5 base differences between each 1.1 x 10 12 possible 20mers12,000 best Shoemaker et al., 1996. Nature Genetics, 14: 450 -456 Can be introduced during strain construction Can be introduced during strain construction Two different tags (UPTAG and DOWNTAG) per strain Two different tags (UPTAG and DOWNTAG) per strain

30. Detecting molecular tags in yeast pools PCR-amplify tags from pooled genomic DNA using fluorescently-labeled primers Hybridize labeled tags to oligonucleotide array containing tag complements Each tag has unique location

31. Tags can be used to perform negative selections on pools Growth in minimal media identifies all known auxotrophic strains Winzeler et., 1999 Science 285:901-906

34. Genomic profiling of drug sensitivities via “induced haploinsufficiency” Decreased gene dosage from two copies to one copy in heterozygous strains results in increased sensitivity, or drug- induced haploinsufficiency

37. Strains that are heterozygous for drug target are haploinsufficient in the presence of drug: Giaever et al., 1999. Nature Genetics, 21: 278-283

38. Tunicamycin sensitivity Analysis of pools of heterozygous (and homozygous) strains reveals primary and secondary drug targets G. Giaever, unpublished results

39. Saccharomyces cerevisiae Genome Deletion Project Collaboration of eight North American and eight European labs to generate a complete set of yeast nonessential deletion mutants ~4,700 nonessential genes deleted with kanMX = fifty 96 well plates ~6,000 heterozygous diploids also available 96 well plate frozen glycerol stock condense 4 plates onto 1 pin 96 strains onto G418 plates

40. Examples- global screens Synthetic lethals Synthetic dosage lethals Heterozygous diploids Haploinsufficiency modifiers Increased drug sensitivity- (target ID) Direct phenotype screening

41. Yeast as a tool to discover drugs and their mechanism of action

42. Metastasis responsible for 90% of cancer deaths Metastasis requires invasion of adjacent tissue and blood vessels by tumour cells Lianne McHardy, Cal Roskelley, Shoki Dedhar, Ali Karsan,David Williams, Raymond Andersen, Michel Roberge Identification of natural compounds that inhibit invasion

43. N N H NH 2 Motuporamine C Xestospongia exigua from outer reef off Motupore Island, Papua New Guinea N N H NH 2 DihydroMotuporamine C

44. - Invasion is a complex process, incompletely understood - Structure of motuporamines gives no clue to function -Motuporamines are not good candidates for biochemical approaches How to identify the mechanism of action of motuporamines?

45. Drug-Induced Haploinsufficiency Y/Y y∆/Y YYYY YY Alive Dead Drug

46. Proof of principle study: Giaever et al. Genomic profiling of drug sensitivities via induced haploinsufficiency. Nat Genet 21, 278-83. (1999) Can these techniques really identify the target or targetted pathways of a drug with an unknown mechanism? Can they predict the target in human cells? Drug-induced haploinsufficiency

47. 1- selection of a drug-induced phenotype 2- systematic high-throughput drug-induced phenotypic screen of yeast heterozygous deletion diploid set 3- quantitative ranking of drug sensitivity - PRIORITIZATION 4- confirmation of drug mode of action in yeast 5- assessment of cognate mode of action in the mammalian system Steps of drug-induced haploinsufficiency screen

48. dhMotC affects yeast growth liquid culture

49. 8 strains in duplicate Screen with or without 60 µM dhMotC and identification of strains showing increased sensitivity Treatment:DMSOdhMotC

50. Heterozygous deletion strains sensitive to dhMotC ORFNAME Biological Process YCL034WLSB5 actin filament organization YNL314WDAL82 allantoin catabolism and transcription initiation from YML099CARG81 arginine metabolism YBR078WECM33 cell wall organization and biogenesis YNL267W*PIK1 * cytokensis, post Golgi transport and signal transduction YLR286C CTS1 cytokinesis, completion of separation YDL192WARF1 ER to golgi transport and intra-golgi transport YBR290WBSD2 heavy metal ion transport and protein-vacuolar targeting YLR025WSNF7 late endosome to vacuole transport YHR147CMRPL6 protein biosynthesis YOL040C*RPS15 * protein biosynthesis YAL005CSSA1 protein folding and protein-nucleus import, translocation YIL047C SYG1 signal transduction YBR265W*TSC10 * sphingolipid biosynthesis YMR296C*LCB1 * sphingolipid biosynthesis YJR007W*SUI2 * translation initiation YML092C*PRE8 * ubiquitin-dependent protein catabolism YER140WYER140Wunknown YER188WYER188Wunknown YGR205WYGR205Wunknown YLR294CYLR294Cunknown * essential genes Which are more relevant?

51. Ranking of strain sensitivity in liquid culture using low dhMotC concentration (20 µM)

52. Supersensitive strains (Integrated Growth Curve Difference >2)

56. Dihydrosphingosine rescues growth inhibition by dhMotC

57. Can these techniques really identify the mode of action of a drug? YES Can they predict the target/target pathway in human cells? YESAdvantages -systematic, unbiased and genome-wide -adaptable to other phenotypes. -pathway conservation = physiological phenotype -development of chemical probes

58. Examples- global screens Synthetic lethals Synthetic dosage lethals Heterozygous diploids Haploinsufficiency modifiers Increased drug sensitivity- (target ID) Direct phenotype screening

59. Method for genomic synthetic lethal (SL) screen Tong et al., 2001 Science,Vol. 294, 2364-2368--- (Boone Lab) YF mutation, plasmid,reporter,…… each deletion strain in quadruplicate Final double mutant selection MAT a deletion set no growth potential SL interaction

62. DONE

68. CSG2 deletion rescues growth inhibition by dhMotC dhMotC reduces cellular ceramide levels

69. Ceramide partially rescues dhMotC toxicity in mammalian cells