1. Directed Analysis Global Analysis Functional Genomics Genetics
Networks
High throughput
data set analysis
Genetics
Biochemistry
Cell Biology

2. 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

3. Genetic Instability in Human Cancers
MIN: Microsatellite instability
(increased mutation rate)
CIN: Chromosome instability
(increased aneuploidy rate)
Yeast as an experimental model
CIN biology
CIN candidate genes, CIN cancer genes
Therapeutics

4. Budding yeast cell cycle
Chromosome cycle G1 S G2 M
START
Ze budding yeast
- The organism that I work in is the budding yeast Saccharomyces cerevisiae
- many features of budding yeast that make it a great model organism great for genetics - easy to generate mutants and study their phentoypes:
- for example you can tell what stage of the cell cycle the yeast is in by the size of the bud - Lee Hartwell got his Nobel prize in medicine this year for isolating cell division cycle (or cdc) mutants that arrest at a particular point in the cell cycle
- which brings me to a second point that although yeast is obviously different from other organisms, there is a striking conservation in its key regulatory components such as some of the Cdc proteins
Specific interest is in the regulation of chromosome segregation
FOCUS ON CENTROMERE - SPENT TOO LONG ON OTHER STUFF
- a cell must duplicate its DNA and faithfully segregate it during mitosis
- errors in chromsome transmission are a hallmark of diseases such as cancer
- I am interested in identifying proteins that regulate the fidelity of chromosome transmission
- more specifically I am interested in the centre region of the chromosome - called the centromere - which is essential for proper chromosome segregation
- the centromere and proteins that associate with it are called the kinetochore
- the kinetochore provides the site of attachment of the spindle microtubules so that sister chromatids can be pulled to opposite poles during mitosis

5. Metaphase
Anaphase

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

7. hBUB1 Spindle Checkpoint Cohesin Separase APCCdc20 Securin Bub1, Bub3,
1998
hBUB1
Improperly attached
kinetochore
Spindle
Checkpoint
Bub1, Bub3,
Mad1, Mad2, Mad3
Cohesin
Separase
Securin
APCCdc20

8. hZW10, hZwilch, hRod hBUB1 Spindle Checkpoint hMRE11 Cohesin Separase
2004
hZW10, hZwilch, hRod
1998
hBUB1
Improperly attached
kinetochore
Spindle
Checkpoint
Bub1, Bub3,
Mad1, Mad2, Mad3
hMRE11
Cohesin
Separase
Securin
APCCdc20
2004
hDING
hCDC4

9. <20% of CIN mutational spectrum in colon cancer hCDC4
2004
hZW10, hZwilch, hRod
1998
hBUB1
Improperly attached
kinetochore
Spindle
Checkpoint
Bub1, Bub3,
Mad1, Mad2, Mad3
hMRE11
Cohesin
Separase
Securin
APCCdc20
2004
hDING
<20% of CIN mutational
spectrum in colon cancer
hCDC4

10. Colony Sectoring Assay
Chromosome Transmission Fidelity (ctf) Screen
Colony Sectoring Assay
non-essential Chromosome Fragment M
SUP11 + wt
White colony
(10-5)
EMS mutagenesis
ctf mutant
Sectored colony
(10-3)
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.
138 mutants, ~50 genes

11. Summary of the 24 Cloned ctf Mutants
# alleles
Gene Name
Essential?
Function 1 30
CTF1/CHL1
Cohesion (helicase) 3 11
CTF3
Kinetochore protein 4 8
CTF4/CHL15/POB1
Cohesion (establishment) 5
CTF5/MCM21 6
CTF6/RAD61
Cohesion 7
CTF7/ECO1
Yes
CTF8
Cohesion (alternative RFC) 10
CDC6
DNA replication
PDS5 12
CTF12/SCC2 13
CTF13
Kinetochore protein (CBF3) 14
CTF14/NDC10 15
CTF15/RPB4
Subunit of RNA polymerase II 17 2
CTF17/MCM17/CHL4 18
CTF18/CHL12 19
CTF19 s3
BIM1
Microtubule binding
s127
SIC1
Cdk inhibitor
s138
SPT4
Chromatin structure
s141
NUP170
Nucleoporin
s143
MAD1
Spindle chkpt. / kinetochore
s155
MCM16
s165
SCC3
Cohesin subunit
s166
SMC1
Kinetochore proteins
Cohesion
DNA /RNA metabolism
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.

12. What are all the genes “mutable” to CIN?
Essential vs. non-essential
Dominant vs. recessive
Redundant vs. non-redundant
Karen Yuen UBC
Forrest Spencer Johns Hopkins
Cheryl Dunbar Warren

13. Global screens for candidate CIN proteins
Gene deletion set
Genetic interaction screening
Synthetic lethals
Synthetic dosage lethals
Haploinsufficiency modifiers
Direct phenotype screening
Genome instability assays
Systematic Two-hybrid
Protein complexes/ mass spectrometry

14. 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
~6,000 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 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

15. Yeast as a tool to discover drugs and their mechanism of action
Identifying “orphan drug” targets via drug induced haploinsufficiency in yeast heterozygotes

16. Identification of natural compounds as potential anti-cancer agents
Metastasis requires invasion of adjacent tissue by tumour cells
Development of cell-based screens for anti-invasive compounds
Michel Roberge, Raymond Andersen
Lianne McHardy, Cal Roskelley
Motivation: cancer: metastasis

17. DihydroMotuporamine C2 H
Motuporamine C N N N H 2 H
DihydroMotuporamine C
Xestospongia exigua
from outer reef off
Motupore Island,
Papua New Guinea
Quite simple molecules

18. Motuporamine C inhibits angiogenesis in vivo Photographs of developing CAMs incubated for 2 days with VEGF (A) or VEGF and motuporamine C at 2.5 µM (B), 5 µM (C) or 10 µM (D)

19. How to identify the mechanism of action
of motuporamines?
Motuporamines are anti-invasive and anti-angiogenic compounds with apparent in vivo activity
they are attractive drug candidates
Invasion is a complex process, incompletely understood
Structure of motuporamines gives no clue to function

20. Yeast “chemical genomics” approaches to identify drug targets
Drug-induced haploinsufficiency screen:
Collection of heterozygous diploid yeast strains in which one allele of every gene is individually deleted
Lowering the dosage of a gene from two copies to one
usually results in a strain that is sensitized to drugs
that act on the product of this gene
Proof of principle study:
Giaever et al.. Nat Genet 21, (1999)

21. Drug-Induced Haploinsufficiency
Alive
Alive
Y/Y Y Y
Drug
y∆/Y
Alive
Dead Y Y
Look for heterozygous deletion strains that are “supersensitive” to the drug…
Idnetify… TARGET
Synthetic Lethals….. Within the targetted pathway or paralell pathway
OFF-TARGTs
Can these techniques identify the target or targetted pathways
of a drug with an unknown mechanism? Can they predict the target in human cells?

22. Steps of drug-induced haploinsufficiency screen
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

23. dhMotC affects yeast growth in liquid culture
Measuring the rate of yeast growth by culture OD.
DhMotC has an affect on yeast cells…therefore it must have at least 1 target in yeast.
USE GROWTH INHIBITON as phenotype

24. Screen with or without 60 µM dhMotC
identification of strains showingincreasedsensitivity
8 strains
in duplicate
Treatment:
DMSO
dhMotC
Robotic: all genes on 5 plates containing or not cpd

25. Heterozygous deletion strains sensitive to dhMotC
ORF NAME Biological Process
YCL034W LSB5 actin filament organization
YNL314W DAL allantoin catabolism and transcription initiation from
YML099C ARG arginine metabolism
YBR078W ECM cell wall organization and biogenesis
YNL267W* PIK1 * cytokensis, post Golgi transport and signal transduction YLR286C CTS1 cytokinesis, completion of separation
YDL192W ARF1 ER to golgi transport and intra-golgi transport
YBR290W BSD2 heavy metal ion transport and protein-vacuolar targeting YLR025W SNF7 late endosome to vacuole transport
YHR147C MRPL6 protein biosynthesis
YOL040C* RPS15 * protein biosynthesis
YAL005C SSA1 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
YER140W YER140W unknown
YER188W YER188W unknown
YGR205W YGR205W unknown
YLR294C YLR294C unknown
* essential genes
21 heterozygous deletion strains sensitive to dhMotC
Which are more relevant?

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

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

31. Dihydrosphingosine rescues growth inhibition by dhMotC

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

34. Drug-induced haploinsufficiency
Determining drug mode of action in yeast
Predicting the target/ target pathway in human cells
Advantages
Systematic, unbiased and genome-wide
Adaptable to other phenotypes.
Pathway conservation = physiological phenotype
Development of chemical probes
See: Baetz et al PNAS 2004
Also: Lum et al 2004 Cell
Giaver et al 2004 PNAS