Canadian Bioinformatics Workshops

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Canadian Bioinformatics Workshops

• www.bioinformatics.ca

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(No Transcript)
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Large-scale Mapping of Genetic and
Chemical-Genetic Interactions in Yeast
Part I Synthetic Lethal Genetic Interaction in
Yeast Part II Mapping Chemical-genetic
Interactions (Comparing Chemical-genetic
Interactions and Synthetic Lethal Genetic
Interactions) Part III Conservation of Genetic
Interactions Yeast to Humans
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University of Toronto Yeast Genomics Labs
Jack Greenblatt Nevan Krogan
Brenda Andrews
Tim Hughes
Andrew Emili
Corey Nislow
Guri Giaever
Gary Bader
Igor Stagljar
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Part I Large-scale Mapping of Genetic
Interactions in Yeast

• 6000 Yeast Genes
• 1000 Essential Genes
• 5000 Nonessential Genes
• Genetic Array 5000 Viable Yeast Deletion
  Mutants
• 1000 conditional alleles of essential genes
• Automated Genetics
• Examine 36 Million Double Mutants

Collaboration with Brenda Andrews
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Genetic Interactions
A Genetic Interaction occurs if an allele of one
gene combines with the allele of another gene to
generate an unexpected double mutant phenotype.
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Synthetic Lethality
Functional Relationships
Pathway B
Pathway A
Complex A
Complex B
B1
A1
A2
B2
A3
B3
SL interaction
Essential biological function
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Genetic Networks

• analysis of double mutations in inbred
  experimental organisms suggest principles that
  may apply to natural variation in outbred
  populations
• 
  
• 
  

Hartman, Garvik, and Hartwell, Science, 9
February (2001), Pg. 1001
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MATa
MATa
X
xxx?
bni1?
Mating
D
a/a
wild-type
Sporulation
MATa Haploid Selection (can1DMFA1pr-HIS3)
Canavanine, His- Medium
Double Mutant Selection
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SGA a General Method
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Large-scale Mapping of Genetic Interactions in C.
elegans
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Plastic Pads (wash-free) for Rapid Array
Construction and Replication
96 to 1536 Replicate at 1536 Density
Singer Instruments
1536 Replicating Pad
1536 Yeast Array
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Synthetic Lethal Scoring Program
Query
Wildtype control
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SGA Synthetic Lethal Screen with bni1D Query
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BNI1 Encodes a Formin that Controls Actin
Assembly BNR1 also Encodes a Formin bni1D is
alive bni1D bnr1D is synthetic lethal
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bni1D Genome-Wide Synthetic Lethality Screen
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bni1D Genome-Wide Synthetic Lethality Screen
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Genetic Network
8 Queries x 5000 viable D mutants
bni1D (actin/polarity) arp2 arc40 bbc1D nbp2D (si
gnaling) sgs1D (DNA) rad27D bim1D (mitosis)
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8 SGA Screens 291 Interactions 204 Genes
Tong et al., Science 2942364-2368 (2001)
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Genetic Interaction Network 132 Screens 4000
Interactions 1000 Genes 100,000
Interactions/genome
Amy Tong, Fritz Roth et al., Science 303808-813
(2004)
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• General Principles Yeast Genetic Interaction Map
• 4-fold more complex than the protein-protein
  interaction map.
• Interactions occur amongst functionally related
  genes.
• Genetic Network is a Small World Network.
• SGA Screens often Identify complexes/pathways.
• Genes encoding components of the same
  pathway/complex show
• similar patterns of genetic interactions.
• Genetic Interaction Patterns can Predict Precise
  
• Molecular Function.

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Similar Patterns of Genetic Interactions Identify
Pathways or Complexes (Viable Deletion Mutants)

• A, B, and C Share the Same Genetic
• Interaction Profile
• A, B, C, Do Not Interact with Each Other

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Array Genes
Two Dimensional Hierachical Clustering Identifies
Similar Patterns of Genetic Interactions Cluste
rs of Query Genes Y axis Clusters of Array
Genes X axis
MMS4 MUS81 RTT107 YBR094W HST3 TOP1 CDC8 RAD50 RAD
52 CDC45 CDC7 DBF4 CSM3 MRC1 TOF1 ELG1 POL32 RAD27
ORC2 ORC5 RAD9 RAD53 ESC2 SGS1 RAD24 ARP1 DYN1 PA
C1 NBP2 NUM1 BIK1 TUB3 CHL1 MAD2 CHL4 YDR332W CTF1
8 CTF8 CTF4 DCC1 BIM1 KAR3 GIM3 YKE2 GIM4 PAC10 TU
B2 CLB4 KAR9 KIP3 ARP6 GOD1 HTZ1 SET2 ARC40 ARP2 B
NI1 CLA4 KRE1 SLT2 SHS1 SMY1 BNI4 PHO85 BBC1 CDC42
-118 YDR437W CNB1 CNB1/1 ERG11 RIC1 YPT6 HST1 YJR0
70C APP1 RAS2
CDC7 DBF4 ORC2 ORC5
DNA Replication
Query Genes
GIM3 YKE2 GIM4 PAC10
Actin/Tubulin Chaperone
Arc40 Arp2
Actin Assembly
RIC1 YPT6
Secretion
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Kinetochore
Dynein/Dynactin
ARP1 JNM1 NUM1 DYN1 PAC1 NIP100 PAC11 DYN2 YMR299c
CHL4 CTF3 MCM22 CTF19 MCM21 IML3 MCM16
MAD2 CHL1
CTF4
GIM3 GIM4 YKE2 PAC10
CLB4 KAR9 KIP3
BNI1
YMR299c DYN3 Tony Bretscher John Cooper
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Large-scale Mapping of Genetic Networks (From 4
to 100 of the Total Network)

• Deletion Mutant Collection in the SGA Query
  Strain
• Conditional Alleles of Essential Genes
• Second Generation Robotics
• Scoring Genetic Interactions Quantitatively
  from Colony

Images
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Display Wall Princeton
Chad Myers
Olga Troyanskaya
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Genetic Interactions
What is the expected combined effect of two
single mutations? Fritz Roth PNAS 2008
A Genetic Interaction occurs if an allele of one
gene combines with the allele of another gene to
generate an unexpected double mutant phenotype.
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Two Basic Types of Genetic Interactions
doublings per hour
wt
1
a
0.5
b
0.5
ab
Neutral Expected Result ?
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Two Basic Types of Genetic Interactions
doublings per hour
wt
1
a
0.5
b
0.5
ab
0.25
Neutral Expected Result Multiplicative
Model
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Two Basic Types of Genetic Interactions
doublings per hour
wt
1
a
0.5
b
0.5
ab
0.25
Negative Synthetic Lethal
Neutral Expected Result
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Two Basic Types of Genetic Interactions
doublings per hour
wt
1
a
0.5
b
0.5
ab
0.5
Neutral Expected Result
Positive e.g. two genes whose products are
in the same nonessential complex/pathway
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Two Basic Types of Genetic Interactions
doublings per hour
wt
1
a
0.5
b
0.5
ab
0.5
Neutral Expected Result
Positive Within Pathway
Negative Synthetic Lethal
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SGA scoring
Query

• Goals
• measure single and double mutant
  fitness
• quantify the genetic interaction
• Problems
• Experimental variation
• Growth and positional effect
• Data normalization procedures complicate
  single mutant fitness measurements

e xy x ? y
xy expected e 0 no interaction xy gt
expected e gt 0 positive interaction xy lt
expected e lt 0 negative interaction
Wildtype
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Modeling Quantitative Genetic Interactions From
Double Mutant Colony Growth

• Model double mutant colony size is
  multiplicative in biological and experimental
  factors

Single mutant fitnesses
Genetic Interaction
Log-normal error
Experimental factors
Time
Challenge Most of the variation in colony sizes
is due to experimental factors, not biological
effects, but these effects are largely systematic.
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SGA Genetic Interaction Matrix Secretion
Vacuole protein sorting
Protein glycosylation
Vesicle-mediated transport
Autophagy
GIM complex
TRAPP, GARP, COG, vacuole ATPase AP-1,
AP-2 complexes
ESCRT, vacuole assembly complexes
Mitochondrial protein targeting
aggravating
alleviating
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SGA Genetic Interaction Matrix Secretion
Retromer complex
negative
(synthetic lethal)
Clathrin-associated Complex AP-1 AP-2
positive
Vacuolar ATPase
Retromer complex
Vacuolar ATPase
ESCRT
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Two Color SGA Genetic Interaction Network
Vacuolar H ATPase
Retromer complex
ER assembly
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Surveying the Global Genetic Interaction Map of
Yeast Cells
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Organizing the genome into 10 functional
neighborhoods
Polarity, cell wall
Functional neighborhoods
Polarity/cell wall
Cell cycle
Metabolism
Mitochondria
DNA rep/repair
Transcription
RNA processing
Transport
5000 genes
Ribosome/protein biosynth.
Secretion
5000 genes
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Screening Progress
Mar. 2008
Aug. 2008
Random screen assignments
15-25 neighbourhood coverage
30 neighbourhood coverage

• Current throughput 200 genome-wide
  screens/month
• By Aug. 2008 1500 genome-wide screens completed
  (25 of genome)

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Global Mapping of Genetic Interactions in Yeast

• Systematically Test descending GO levels

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Large-scale Mapping of Genetic and
Chemical-Genetic Interactions in Yeast
Part I Synthetic Lethal Genetic Interaction in
Yeast Part II Mapping Chemical-genetic
Interactions (Comparing Chemical-genetic
Interactions and Synthetic Lethal Genetic
Interactions) Part III Conservation of Genetic
Interactions Yeast to Humans
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Synthetic Genetic Interactions
Chemical Genetic Interactions
A
Drug Target
Gene X
Phenotype
Drug Target
Gene X
Phenotype
Alive
Drug
Alive
Drug
Dead
B
Compendium of Genetic Interactions
Deletion Array
Chemical-Genetic Profile
1D
2D
3D
4D
5D
6D
7D
8D
9D
Query Genes
Deletion Array
Gene A
1D
2D
3D
4D
5D
6D
7D
8D
9D
Gene B
Gene C
Gene D
Chemical-Genetic Interaction
Synthetic Genetic Interaction
Parsons et al., Nature Biotech. (2004)
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Compounds Cluster with Their Pathways or Targets
Calcineurin Function
CsA FK506 CNB1 ARC40 ARP2 BNI1 SMY1 CLA4
FKS1 GAS1 CHS3 CHS7 CHS6 PHO85 ROT2 ERG11
Fluconazole BBC1 DYN1 PAC1 NUM1 NBP2 CIN4
BIK1 TUB3 MAD2 CIN1 BIM1 KAR3 CTF4 CTF8
CIN8 TUB2 GIM3 KAR9 KIP3 Benomyl GYP1
RIC1 HTZ1 SET2 Camptothecin Hydroxyurea
POL32 RAD27 SGS1 ORC2 ORC5 RAD53 RAD24
MMS4 MUS81 RTT107 RAD50 RAD52 LAG2 YUR1
CNE1 KRE2 HOG1 RAS2 KRE9 ALG6 SKN1 ERV29
APP1
CSA, FK506, CNB1
Ergosterol Biosynthesis
Fluconazole, ERG11
Yeast Labs
Microtubule Function
Benomyl, TUB2
Camptothecin, Hydroxyurea, POL32, RAD27
DNA Synthesis Repair
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700 SGA Screens Caffeine Clusters with TOR2
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Barcodes Enable Chemical-Genetic Profiling with
Small Amounts of Precious Compounds
- Drug Control
Drug Hypersensitivity Screen

• Grow Deletion Mutants in 10mls Drug (80 wt
  fitness)
• Isolate Cells
• PCR Barcodes, Drug Differentially Labeled
  Cy3/Cy5
• Competitive Hyb Barcode Chip

Cy3/Cy5
KANMX4
Cy3/Cy5

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Compounds
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Linking Antifungal Compounds to Targets 1.
Generate Chemical-Genetic Profile 2. Compare to
Synthetic Lethal Matrix 3. Compare to
Chemical-Genetic Compendium
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Linking Antifungal Compounds to Targets 1.
Generate Chemical-Genetic Profile 2. Compare to
Synthetic Lethal Matrix 3. Compare to
Chemical-Genetic Compendium 4. Identify
Drug-Resistant Mutation
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Resistant Mutants Identify Drug Targets
Yeast TOR1/TOR2 - Rapamycin FKS1 -
Caspofungin ACT1 - Latrunculin Human
Tubulin - Taxol
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Construction of a Barcode ORF Library Cloning
Drug Resistant Mutants
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Constructing Each Barcoded ORF (2)
Homologous Recombination in S. cerevisiae
kanMX
kanMX
pr-ORF-3 UTR
I-SceI
I-SceI
URA3
oriT
CmR
CEN
ori R6Kg
CmR
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Complementation Cloning of Drug-Resistant Mutant
by Barcode Microarray Analysis

CYH2
A
X
X
cyh2
X
C
D
A
C
E
X
X

Cycloheximide resistant mutant
CYH2
D
F
E
F
X
X
Transformed 96 Barcoded Vectors
YEPDG418 Cycloheximide
YEPDG418
CYH2
A
A
X
X
X
C
D
C
D
X
X
X
X
E
F
E
F
X
X
X
X
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Natural Product Extracts
Natural Products from Marine Organisms are a Rich
Source of Novel Bioactive Compounds
Ray Andersen (UBC)
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Two Extracts with Highly Similar Chemical-Genetic
Profiles
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Profiles of Two Seemingly Unrelated Natural
Product Extracts are Virtually Identical
r 0.81
Sea cucumber, Dominican Rep.
Extract 00-192
Stichloroside
Extract 00-132
Marine sponge, Indonesia
Theopalauamide
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Linking Antifungal Compounds to Targets 1.
Generate Chemical-Genetic Profile 2. Compare to
Synthetic Lethal Matrix 3. Compare to
Chemical-Genetic Compendium 4. Identify
Drug-Resistant Mutation
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Complementation Cloning of Stichloroside/Theopal
auamide Resistant Mutant by Barcode Microarray
Analysis
MVD1
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Sterol-Recognizing Molecules
Proteins
gt 20 family members of pore forming toxins
Theopalauamide
Minoru Yoshida RIKEN
Saponines
Cell 1997, 89, 685.
a-tomatine
q-toxin from Clostridium perfringens
Stichloroside
Polyene antifungals
Amphotericin B
Nystatin A1
Filipin III
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Large-scale Mapping of Genetic and
Chemical-Genetic Interactions in Yeast
Part I Synthetic Lethal Genetic Interaction in
Yeast Part II Mapping Chemical-genetic
Interactions (Comparing Chemical-genetic
Interactions and Synthetic Lethal Genetic
Interactions) Part III Conservation of Genetic
Interactions Yeast to Humans
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Conservation of Synthetic Lethal Genetic
Interactions?
Yeast
Humans
Phenotype
Genotype
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Synthetic Lethality and Cancer Target ID
(exploit synthetic lethal interactions to
selectively kill tumor cells)
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Conservation of Synthetic Lethal Genetic
Interactions?
Yeast
Humans
Phenotype
Genotype
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Yeast
Worms?
6 / 837 1
9 / 21 42
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Assessing Genetic Network Conservation over 1
Billion Years of Evolution Estimate the extent
of genetic network conservation using the
distantly related, single-celled fission yeast,
Schizosaccharomyces pombe.
S. cerevisiae Budding Yeast
S. pombe Fission Yeast
16 chromosomes, 5800 Genes Cell Cycle Ground
State G1
3 chromosomes, 5200 Genes Cell Cycle Ground
State G2
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Michael Costanzo S. cerevisiae SGA Scott
Dixon, Dan Durocher S.pombe SGA Huiming Ding,
Chad Myers, Olga Troyanskaya Bioinformatics
Brenda Andrews