Why yeast?
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Model organism genetics and human disease With an emphasis on….. APOYG! Model Organisms The “Security Council” 1,000 Myr 500 Myr 80 Myr Human Biology Mammalian Biology Multicellular Biology Unicellular Biology Not so many genes! 6,000 14,000 19,000 21,000 Why we love yeast Model organism Eukaryotic intracellular biology Gene function conservation (e.g., human disease genes) Testbed for genomic technologies Experimental approaches Classical genetics (+biochemistry) Recombinant genetics Emerging technologies Community of “yeast people” Open exchange of ideas, reagents, results Collaboration Saccharomyces cerevisiae Budding yeast The “E.coli of eukaryotic cells” Yeast vs. Human ~50% of yeast genes have at least one similar human gene ~50% of human genes have at least one similar yeast gene Human vs. Yeast Human disease genes in model organisms Disease Human Mouse Fruit fly Worm Yeast Bacteria Ataxia ATM MEI-41 T06E4.3 TEL1 telangiectasia ATM Breast BRCA1,2 BRCA1,2 cancer Cystic CFTR CFTR MDR50 mrp-1,2 YCF1 MDL fibrosis Duchenne DMD SPEC-B F15D3.1a muscular dys DMD HNP colon MSH2 MSH2 SPEL1 msh-2 MSH2 MUTS cancer Huntingtin HD HD disease Werner syndrome Wilson disease Waardenburg syndrome (huntingtin) (huntingtin) WRN WRN MUS309 wrn-1 SGS1 RECQ WND ATP7B CA-P60A B0365.3 CCC2 f834 PAX3 PAX3 CG6716-P vab-3 Human disease genes in model organisms Disease Human Mouse Fruit fly Worm Yeast Bacteria Ataxia ATM MEI-41 T06E4.3 TEL1 telangiectasia ATM Breast BRCA1,2 BRCA1,2 cancer Cystic CFTR CFTR MDR50 mrp-1,2 YCF1 MDL fibrosis Duchenne DMD SPEC-B F15D3.1a muscular dys DMD HNP colon MSH2 MSH2 SPEL1 msh-2 MSH2 MUTS cancer Huntingtin HD HD disease Heo et al. (1999) Genes to Cells 4, 619-625. Werner syndrome Wilson disease Waardenburg syndrome (huntingtin) (huntingtin) WRN WRN MUS309 wrn-1 SGS1 RECQ WND ATP7B CA-P60A B0365.3 CCC2 f834 PAX3 PAX3 CG6716-P vab-3 APOYG and Disease: Two examples Zelwegers Syndrome Peroxisome biogenesis Colorectal Cancer Genome instability Zellweger Spectrum Zellweger syndrome Neonatal adrenoleukodystrophy Infantile Refsum Disease Zellweger Patient Cells Share a Common Phenotype with Yeast pex Mutants Control Zellweger patient Wild-type pex mutant Human PTS1) Yeast (PTS1 - GFP) Strategies for Mammalian PEX Gene Identification Functional complementation Mammalian cDNA expression libraries “Homology probing” Identify all yeast peroxins Identify all homologous human proteins Test as “candidate genes” Yeast / Human Connections Human Identification Function Yeast Discovery of Yeast and Human PEX Genes 25 Yeast PEX Genes 20 15 Human 10 5 0 1990 1992 1994 1996 1998 2000 Cancer C. Rieder If you want to understand cancer, you need answers to the many questions about the role genome instability plays. ---Bert Vogelstein, 2002 Genetic Instability in Human Cancers MIN: Microsatellite instability (increased mutation rate) CIN: Chromosome instability (increased aneuploidy rate) Metaphase Anaphase Improperly attached kinetochore Spindle Checkpoint Bub1, Bub3, Mad1, Mad2, Mad3 Cohesin Separase Securin APCCdc20 ~20% of CIN mutational spectrum in colon cancer Improperly attached kinetochore 4% 2% hBUB1 Spindle Checkpoint Bub1, Bub3, Mad1, Mad2, Mad3 hMRE11 Cohesin Separase 11% hCDC4 Securin hDING 4% APCCdc20 Yeast as a model CIN biology (gene function) CIN candidate genes- (Cancer CIN genes) Therapeutics Finding an “Achilles heel” of cancer What are all the proteins mutable to CIN? Spontaneous mutants eg, CTF mutant collection HieterLab members (20yrs) Systematic screening Karen Yuen Non-essential D mutants Ts mutants, semi-permissive Shay Ben-Aroya Chromosome Transmission Fidelity (ctf) Screen Colony Sectoring Assay non-essential Chromosome Fragment M SUP11 + White colony (10-4) wt EMS mutagenesis ctf mutant Sectored colony (10-2) 138 mutants, ~50 genes Summary of the 26 Cloned ctf Mutants ctf 1 2 3 4 5 6 7 8 9 10 11 12 18 13 14 15 17 19 s3 s127 s138 s141 s143 s155 s165 s166 # alleles 30 11 11 8 5 5 5 3 3 3 3 3 3 1 1 1 2 2 1 1 1 1 1 1 1 1 Gene Name CTF1/CHL1 TOF1/CTF2 CTF3 CTF4/CHL15/POB1 CTF5/MCM21 CTF6/RAD61 CTF7/ECO1 CTF8 SMC3/CTF9 CDC6/CTF10 PDS5/CTF11 CTF12/SCC2 CTF18/CHL12 CTF13 CTF14/NDC10 CTF15/RPB4 CTF17/MCM17/CHL4 CTF19 BIM1 SIC1 SPT4 NUP170 MAD1 MCM16 SCC3 SMC1 Essential? Yes Yes Yes Yes Yes Yes Yes Yes Yes Function Cohesion (helicase) Cohesion, replication Kinetochore protein Cohesion (establishment) Kinetochore protein Cohesion Cohesion (establishment) Cohesion (alternative RFC) Cohesion (cohesin subunit) DNA replication Cohesion (cohesin associated) Cohesion (cohesin loading) Cohesion (alternative RFC) Kinetochore protein (CBF3) Kinetochore protein (CBF3) Subunit of RNA polymerase II Kinetochore protein Kinetochore protein Microtubule binding Cdk inhibitor Chromatin structure Nucleoporin Spindle checkpoint Kinetochore protein Cohesion (cohesin subunit) Cohesion (cohesin subunit) Kinetochore proteins Cohesion DNA /RNA metabolism 13 genes 93 / 138 S. cerevisiae Genome Deletion Project •“Complete” set of yeast nonessential deletion mutants •~4,700 haploid strains •~4,700 homozygous diploid strains nonessential genes deleted with kanMX = fifty 96 well plate •~5,800 heterozygous diploid strains 96 well plate frozen glycerol stock pin 96 strains onto G418 plates condense 4 plates onto 1 The yeast gene knockout collection Yeast CIN genes ~300 non-essential genes (85% coverage) ~100 essential genes (and still counting) Human homologs? 12 yeast CIN genes have top-hit human homologs that are mutated in cancers Gene Top Human Hit E-value Cancer Type/Cancer syndromes ADE17 ATIC RAD54 RAD54L TPD3 PPP2AR 1E-133 Lungcancer RAD51 RAD51 1E-122 Susceptibility to breast cancer RDH54 RAD54B SGS1 BLM RAD1 ERCC4 1E-121 Lymphoma,n-Hodgkin; Colonadecarcima Bloomsyndrome; leukemia, lymphoma, skin squamous cell, other 1E-115 cancers Xeroderma pigmentosum, groupF; skin basal cell, skin squamous 1E-109 cell, melama MRE11 MRE11A DUN1 CHK2 BUB1 BUB1 MAD1 MAD1L CDC73 parafibromin 0 anaplastic large cell lymphoma Lymphoma,n-Hodgkin; Breastcancer, invasiveintraductal; Colon 1E-164 adecarcima 1E-108 Ataxia-telangiectasia-like disorder, colorectal cancer with CIN Li-Fraumeni syndrome; Osteosarcoma, somatic; Prostatecancer, familial; Susceptibility to breast and colorectal cancer 1E-41 Colorectal cancer with CIN 6E-55 5E-12 Lymphoma, somatic; Prostatecancer, somatic Hyperparathyroidism-jaw tumor syndrome; Hyperparathyroidism, 9E-12 familial primary; Parathyroid adema with cystic changes CIN mutational spectrum in cancer- Why? Cancer biology Tumor classification Identification of new drug targets CIN gene / Synthetic Lethal gene pairs Synthetic Lethality yfg2 yfg1 Viable Yfg2 = Drug target Yfg1 = CIN mutant Viable Normal yfg2 yfg1 Dead Tumor Yeast Genetic Interactions MRE11 (4%) BUB1 (2%) 12 yeast CIN genes have human homologs that are mutated in cancers Gene Top Human Hit E-value Cancer Type/Cancer syndromes ADE17 ATIC RAD54 RAD54L TPD3 PPP2AR 1E-133 Lungcancer RAD51 RAD51 1E-122 Susceptibility to breast cancer RDH54 RAD54B SGS1 BLM RAD1 ERCC4 1E-121 Lymphoma,n-Hodgkin; Colonadecarcima Bloomsyndrome; leukemia, lymphoma, skin squamous cell, other 1E-115 cancers Xeroderma pigmentosum, groupF; skin basal cell, skin squamous 1E-109 cell, melama MRE11 MRE11A DUN1 CHK2 BUB1 BUB1 MAD1 MAD1L CDC73 parafibromin 0 anaplastic large cell lymphoma Lymphoma,n-Hodgkin; Breastcancer, invasiveintraductal; Colon 1E-164 adecarcima 1E-108 Ataxia-telangiectasia-like disorder, colorectal cancer with CIN Li-Fraumeni syndrome; Osteosarcoma, somatic; Prostatecancer, familial; Susceptibility to breast and colorectal cancer 1E-41 Colorectal cancer with CIN 6E-55 5E-12 Lymphoma, somatic; Prostatecancer, somatic Hyperparathyroidism-jaw tumor syndrome; Hyperparathyroidism, 9E-12 familial primary; Parathyroid adema with cystic changes Yeast CIN Genes and Human Cancer CIN “candidate genes” Somatic mutations in colon cancer ~40% spectrum in 11 genes Cancer therapeutics “Achilles heel” candidate genes Validation in mammalian cells Four volumes of the Encyclopedia of Life 6000 14,000 19,000 21,000 April, 1953 April, 2003 Human genome sequence “completed” ~10,000 parts ~6,000 genes ~4,000,000 parts ~20,000 genes Power of Model Organism Research Genetics, biochemistry, genomics Basic biology Human health Human disease Therapy Preventative medicine APOYG! APOWG! APOIBE!
