Model organism genetics and human disease With an emphasis on….. APOYG!

The “Security Council” Model Organisms The “Security Council” 500 Myr 80 Myr 1,000 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 Saccharomyces cerevisiae Budding yeast The “E.coli of eukaryotic cells”

Yeast vs. Human Human vs. Yeast ~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

Human disease genes in model organisms Heo et al. (1999) Genes to Cells 4, 619-625.

APOYG and Disease: Two examples Zelwegers Syndrome Peroxisome biogenesis Colorectal Cancer Genome instability

Zellweger Spectrum Zellweger syndrome Neonatal adreno- leukodystrophy Infantile Refsum Disease

Zellweger Patient Cells Share a Common Phenotype with Yeast pex Mutants Control Zellweger patient Human  PTS1) Wild-type pex mutant 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 Identification Function Yeast

Discovery of Yeast and Human PEX Genes 5 10 15 20 25 Yeast PEX Genes Human 1990 1996 1992 1994 1998 2000

Cancer If you want to understand cancer, you need answers 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

Spindle Checkpoint Cohesin Separase APCCdc20 Securin Bub1, Bub3, Improperly attached kinetochore Spindle Checkpoint Bub1, Bub3, Mad1, Mad2, Mad3 Cohesin Separase Securin APCCdc20

spectrum in colon cancer 2% hBUB1 ~20% of CIN mutational spectrum in colon cancer 2% hBUB1 Improperly attached kinetochore Spindle Checkpoint 4% Bub1, Bub3, Mad1, Mad2, Mad3 hMRE11 Cohesin Separase Securin APCCdc20 11% hDING hCDC4 4%

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 Systematic screening Non-essential D mutants Ts mutants, semi-permissive HieterLab members (20yrs) Karen Yuen Shay Ben-Aroya

Colony Sectoring Assay Chromosome Transmission Fidelity (ctf) Screen Colony Sectoring Assay non-essential Chromosome Fragment M SUP11 + wt White colony (10-4) EMS mutagenesis ctf mutant Sectored colony (10-2) 138 mutants, ~50 genes Phetype screening based on marker stability in yeast has provided a powerful approach for identifiying mutants in genes that act to preserve geme integrity. The Hieter lab has utilized a colony sectoring assay to monitor chromosome transmission fidelity. Basically, a non-essential chromosoem fragment was introduced to yeast cells. Cells containing the chromosome fragment are white, and cells that don’t have it are red. Wildtype cells maintain the chromosome fragment stably, developing into white colonies. Back in 1989, Forrest Spencer, who was a post-doc in the Hieter lab at the time, pursued a screen by random mutagenesis, to look for mutants that lose the chromosome fragment at a high rate, resulting in sectoring colonies. This chromosoem transmission fidelity screen has identified 136 mutants, representing about 50 genes. The ctf screen was done using a n-essential artificial chromosome fragment and a visual colony sectoring assay to monitor chromosome loss. The starting haploid strain has an ochre mutation in the ADE2 gene, resulting in an accumulation of a red pigment. The n-essential chromosome fragment contains a cnetromere, a marker for selection & a tRNA gene, SUP11, which suppresses the ochre mutation, preventing the red pigment accumulation. The chromosome fragment also contains 2 telomeres at the ends & ~100kb of gemic sequence. And several chromosome fragments were constructed with different gemic sequence. So, when the chromosome fragment is introduced to this (ade2-101) strain, cells are white. In wild-type cells, the artificial chromosome fragment is quite stable, and lose at a frequency of 10 to the minus 4, which is only 10 fold higher than the loss rate of natural chromosomes. Therefore, wild-type cells develop into colonies that are mostly white. But if cells lose the chromosome fragments during mitosis, the mitotic progeny become red. This strain is subject to EMS mutagenesis. Mutants that increased the loss rate of the chromosome fragment will develop into white colonies with red sectors. And here is an example of how one of the ctf mutant strain looks. In total, 136 ctf mutants were isolated (from screening ~600,000 colonies, I.e. 10 geme coverage) & they were put into 16 complementation groups, and there were 36 single mutants. In total, the ctf collection represents ~50 genes. Over the past 10 years, several secondary screens (CEN transcriptional readthrough assay, dicentric chromosomal stability test, synthetic dosage fidelity) were put through the ctf collection in order to prioritize the cloning and characterization of these CTF genes.

Summary of the 26 Cloned ctf Mutants # alleles Gene Name Essential? Function 1 30 CTF1/CHL1 Cohesion (helicase) 2 11 TOF1/CTF2 Cohesion, replication 3 CTF3 Kinetochore protein 4 8 CTF4/CHL15/POB1 Cohesion (establishment) 5 CTF5/MCM21 6 CTF6/RAD61 Cohesion 7 CTF7/ECO1 Yes 9 CTF8 SMC3/CTF9 Cohesion (alternative RFC) Cohesion (cohesin subunit) 10 CDC6/CTF10 DNA replication PDS5/CTF11 Cohesion (cohesin associated) 12 CTF12/SCC2 Cohesion (cohesin loading) 18 CTF18/CHL12 13 CTF13 Kinetochore protein (CBF3) 14 CTF14/NDC10 15 CTF15/RPB4 Subunit of RNA polymerase II 17 CTF17/MCM17/CHL4 19 CTF19 s3 BIM1 Microtubule binding s127 SIC1 Cdk inhibitor s138 SPT4 Chromatin structure s141 NUP170 Nucleoporin s143 MAD1 Spindle checkpoint s155 MCM16 s165 SCC3 s166 SMC1 Kinetochore proteins Cohesion DNA /RNA metabolism Yes 13 genes 93 / 138 Over the past 10 years or so, 23 ctf mutantswere cloned, and they mainly function in 3 categories: the kinetochore, sister-chromatid cohesion and DNA/RNA metabolism. However, it is kwn, from the number of alleles found, that random mutagenesis rarely achieve saturation. And the mutability varies among genes due to differences in size, base composition, and the frequency of mutable sites that can lead to viable cells with detectable phetype. Also, 1 phetypic screen would capture a subset but t all of the genes important for chromosome stability. So multiple phetypic screens would be more comprehensive in isolating the whole set of genes involved in chromosome maintenance. 20 of the ctf mutants were cloned (in 10 years/since 1989), and they mainly function in 3 categories: the kinetochore function, sister-chromatid cohesion and DNA/RNA metabolism. (15/23 ctf genes are n-essential.) When I decided to stay in this lab, I started by cloning 3 of the 35 uncloned ctfs that are in the complementation groups or single mutants with interesting phetypes: CTF6, CTF11 and s3, hoping to find some vel genes for study. However, they turn out to be kwn genes. In fact, CTF6 is the gene Ben has been studying, and he identified it by Two-hybrid Interaction with Sgt1, a kinetochore regulator. And you will hear his story shortly. Synthetic lethal with CTF17 & SS CTF19 & CTF3 Ctf6 diploids have G2 accumulation & bemyl resistant S134 has a nsense mutation at ~1200b, truncating ~half of the protein PDS5 (Precocious Dissociation of Sisters) identified by Viccent Guacci and Doug Koshland. Essential Required for sister chromatid cohesion & chromosome condensation BIM1 (Binding to Microtubules) Identified by a two-hybrid interaction with Tub1 (encoding alpha-tubulin) SL with Bub1,2,3, Mad1,2,3, Ctf8, Ctf19, Mcm21, Mcm22, Chl4 Null mutant is bemyl sensitive Associate Kar9p with microtubules Mediate microtubule capture & position the mitotic spindle Synthetic lethal (SL) with spindle-assembly checkpoint genes & kinetochore genes Involved in attachment of microtubules to the kinetochore ~33%) identical to the human homolog EB1, which binds the tumor suppressor APC protein Therefore, I did t pursued them further. The ctf mutant collection indicates that the screen was working - the chromosome fragment mimics the behavior of natural chromosomes, and the visual sectoring assay is sensitive. However, the ctf screen was t saturated since there are 36 single mutants and some gene mutants that are kwn to cause sectoring were t identified in the screen. Mutability varies among genes due to differences in gene size, base composition and the frequency of mutable sites leading to viable cells with detectable phetype. For example, Ctf1 has 30 alleles, whereas s3 has only 1, so the variability in mutability is quite high. Therefore, I’d like to identify ctf mutants in a comprehensive manner and do it in a gemic scale, and I can intersect this primary screen result with other global screen result.

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 All of the budding yeast genome has been sequence - all of the nonessential ORFs have been deleted - a collaborative effort between 16 labs in Europe and North America - now available to yeast researches in 96 well plates - can get all the deletion strains in 50 96 well plates - these arrive from the company as frozen glycerol stocks which need to be thawed then “patched” onto a plate - have a pinning tool which dips into each well then dips onto a plate that selects for the knockouts (G418 selects for kan resistance) - maybe I should know something about these antibiotics???? - although 96 strains per plate may seem like a lot, often want to do an experiment in duplicate…. Therefore can condense four plates onto one 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 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 Top Human Hit E-value Cancer Type/Cancer syndromes ADE17 ATIC anaplastic large cell lymphoma RAD54 RAD54L 1E-164 Lymphoma,n-Hodgkin; Breastcancer, invasiveintraductal; Colon adecarcima TPD3 PPP2AR 1E-133 Lungcancer RAD51 1E-122 Susceptibility to breast cancer RDH54 RAD54B 1E-121 Lymphoma,n-Hodgkin; Colonadecarcima SGS1 BLM 1E-115 Bloomsyndrome; leukemia, lymphoma, skin squamous cell, other cancers RAD1 ERCC4 1E-109 Xeroderma pigmentosum, groupF; skin basal cell, skin squamous cell, melama MRE11 MRE11A 1E-108 Ataxia-telangiectasia-like disorder, colorectal cancer with CIN DUN1 CHK2 6E-55 Li-Fraumeni syndrome; Osteosarcoma, somatic; Prostatecancer, familial; Susceptibility to breast and colorectal cancer BUB1 1E-41 Colorectal cancer with CIN MAD1 MAD1L 5E-12 Lymphoma, somatic; Prostatecancer, somatic CDC73 parafibromin 9E-12 Hyperparathyroidism-jaw tumor syndrome; Hyperparathyroidism, familial primary; Parathyroid adema with cystic changes Bsides gaining insight aboout the functions of the genes identified, it’s important to te that about 40% have significant homology with human proteins. And if I looked at the top human hits, 12 of them are mutated in cancer or associated with cancer susceptibility syndromes. Some n-top hits, but with significant e-values, are also mutated in cancers. We believe that other genes identified would also be good candidates for mutation testing. In fact, Christoph Lengauer’s group at Johns Hophins has recently tested 100 candidate genes for mutation in colorectal cancer patients. Those genes have homologs in yeast or flies that are shown to cause CIN when mutated. And they found somatic mutations in 5, which were shown in my first few slides.

CIN mutational spectrum in cancer- Why? Cancer biology Tumor classification Identification of new drug targets CIN gene / Synthetic Lethal gene pairs

Synthetic Lethality Normal Tumor Viable Viable Yfg2 = Drug target Yfg1 = CIN mutant yfg2 yfg1 Viable Viable yfg2 yfg1 Dead

Yeast Genetic Interactions MRE11 (4%) BUB1 (2%)

12 yeast CIN genes have human homologs that are mutated in cancers Top Human Hit E-value Cancer Type/Cancer syndromes ADE17 ATIC anaplastic large cell lymphoma RAD54 RAD54L 1E-164 Lymphoma,n-Hodgkin; Breastcancer, invasiveintraductal; Colon adecarcima TPD3 PPP2AR 1E-133 Lungcancer RAD51 1E-122 Susceptibility to breast cancer RDH54 RAD54B 1E-121 Lymphoma,n-Hodgkin; Colonadecarcima SGS1 BLM 1E-115 Bloomsyndrome; leukemia, lymphoma, skin squamous cell, other cancers RAD1 ERCC4 1E-109 Xeroderma pigmentosum, groupF; skin basal cell, skin squamous cell, melama MRE11 MRE11A 1E-108 Ataxia-telangiectasia-like disorder, colorectal cancer with CIN DUN1 CHK2 6E-55 Li-Fraumeni syndrome; Osteosarcoma, somatic; Prostatecancer, familial; Susceptibility to breast and colorectal cancer BUB1 1E-41 Colorectal cancer with CIN MAD1 MAD1L 5E-12 Lymphoma, somatic; Prostatecancer, somatic CDC73 parafibromin 9E-12 Hyperparathyroidism-jaw tumor syndrome; Hyperparathyroidism, familial primary; Parathyroid adema with cystic changes Bsides gaining insight aboout the functions of the genes identified, it’s important to te that about 40% have significant homology with human proteins. And if I looked at the top human hits, 12 of them are mutated in cancer or associated with cancer susceptibility syndromes. Some n-top hits, but with significant e-values, are also mutated in cancers. We believe that other genes identified would also be good candidates for mutation testing. In fact, Christoph Lengauer’s group at Johns Hophins has recently tested 100 candidate genes for mutation in colorectal cancer patients. Those genes have homologs in yeast or flies that are shown to cause CIN when mutated. And they found somatic mutations in 5, which were shown in my first few slides.

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

the Encyclopedia of Life Four volumes of the Encyclopedia of Life 14,000 6000 19,000 21,000

April, 1953 April, 2003 Human genome sequence “completed”

~10,000 parts ~4,000,000 parts ~6,000 genes ~20,000 genes

Genetics, biochemistry, genomics Power of Model Organism Research Genetics, biochemistry, genomics Basic biology Human health Human disease Therapy Preventative medicine APOYG! APOWG! APOIBE!