1. GENE PROFILES Synthetic lethality Chemical Genetic Interactions
Pleiotropy
Zohar Itzhaki
Advanced Seminar in Computational Biology
November 2005

2. What’s a gene profile ?
Vector: Discrete information regarding a gene; characterization of
a gene according to defined parameters
Can be clustered
Main Goals:
A better understanding of different mechanisms
Annotation of unknown genes
Gene A
Gene B

3. Some reminders and definitions
Synthetic lethality (genetic interactions)
death of double mutants
Chemical genetic interaction
Hypersensitivity of a mutant to a sub-lethal compound
Pleiotropy
one single mutant gene causes multiple mutant phenotypes

4. The Genetic Profiles in the studies
Dudely AM. Janse DM. Tanay A. Shamir R. Church GM. A global View of Pleiotropy and Phenotypically Derived Gene Function in Yeast
Molecular Systems Biology 2005
Pleiotropy
Tong AH … Boone C. Global Mapping of The Yeast Genetic Interaction Network Science 2004
Genetic interactions
Synthetic lethality
Parsons AB … Boone C. Integration of Chemical-genetic and genetic Interaction data links bioactive compounds to cellular target pathways
Nat Biotechnol 2004
Chemical genetic
interactions

5. General procedures - A yeast array
Every spot is a yeast colony (different strains)
Treatment
Computational detection, usually for growth changes

6. genetic interactions (GGI) synthetic lethality
Is it really an interaction ? (PPI)
Parallel pathways
Same pathways
Trait
Two ways of interpretation:
Parallel pathways (ie: gene duplication)
Same pathway / complex (ie: membrane receptor + signal transducer)
Essential gene GGI is checked by temperature sensitive (partial) mutations
Davierwala … Boone C. The synthetic genetic interaction spectrum of essential gene Nature genetics 2005
בתמונה: שני גנים הקובעים את צבע הפרח, מוטציה בכל אחד מהם לא תשנה בהרבה את צבעו, בשניהם – יהפוך ללבקן
יכולים להיות מצבים הרבה יותר מסובכים כגון loops

7. GGI – Article & Technology
Tong AH … Boone C. Global Mapping of The Yeast Genetic Interaction Network Science 2004
SGA (synthetic genetic array) analysis
an array that contains ~4700 haploids, every strain is mutated
in a different gene
A query: a single mutant haploid strain is crossed
A computational analysis for detecting the growth changes
The selected query genes are related to cytoskeleton & DNA repair
לגנים נחוצים מוטציות מותנות, לגנים לא נחוצים – מוטציות החסרה
Tong & Boone 2004

8. Synthetic lethality – GGI assay
132 SGA screens (cytoskeleton & DNA repair mutated genes)
X 3
Pass 3
Candidate synthetic lethal pairs
Pass 1 *
Further evaluation
~4000 interactions
of ~1000 genes
Results
34 interactions/gene
8 int/gene in PPI
מי שעבר אחד והוא מיוחד: ללא אנוטציה או עם אנוטציות דומות של 2 הגנים
Spore analysis – הערכה נוספת
False negative: 17-41%
Evaluation: ~100,000 GGI
Tong & Boone 2004

9. GGI and Function (GO annotations)
Significant correlations between the GGI and their GO annotations:
GGI to GO:
12% of the GGI has the same GO annot’ (pVal=10-296)
27% of the GGI has a same or similar GO annot’ (pVal=10-322)
Bridging GGI:
1755 out of ~285,500 (n2/2) possible pairs of GO attributes were “bridged” by a GGI (pVal<0.05)
גישור: כלומר שלאחד הגנים יש אנוטציה אחת ברורה ואילו לשני אחת אחרת
CONCLUSION: The GGI map represents a global map
of functional relationships between genes
Tong & Boone 2004

10. GGI and Function – Bridging GGI
Tong & Boone 2004

11. More validations of the concept
GGI were significantly more abundant between:
Genes sharing the same phenotype mutant (pVal=10-316)
Genes encoding proteins with the same localization (pVal=10-70)
Genes encoding proteins within the same complex (pVal=10-68)
מבחן פישר
Tong & Boone 2004

12. Gene profiles and profile clustering
Gene profiles: Foreach “array” gene create a 132d
binary vector, regarding GGI with the query genes
Create a 132*1000 matrix out of these vectors
Bicluster the matrix + GO annotations for every cluster
Tong & Boone 2004

13. The GGI Bi-cluster
Tong & Boone 2004

14. What can we learn from the Bi-Cluster ?
A. Connecting pathways
DNA damage
Checkpoint
Microtubule
Dynamic
Spindle
Checkpoint
DNA replication
Checkpoint
Recombination
Sister chromatid cohesion during chromosome replication
גני השאילתא שקשורים להצמדת הכרומטידות במטרה שייקשרו היטב לסיבי הכישור ולפני פרידתן
Tong & Boone 2004

15. What can we learn from the Bi-Cluster ?
B. Predict biological functions
Profile similarity of csm3 (un-annotated) to tof1 and mrc1
tof1 and mrc1 products response to stress and interact to the DNA replication machinery
“Wet” phenotype checks (related to cell cycle):
∆tof1 ∆rad9 = ∆csm3 ∆rad9
∆mrc1 ∆rad9 = ∆csm3 ∆rad9
PPI assays (Y2H): Csm3 interacts Tof1
Tong & Boone 2004

16. GGI as predictors for PPI
Analyze the GGI network and a 15,000 PPI network:
# of common GGI neighbors
between a pair of genes
PPI between the pair
of the product proteins
Within a complex
Parallel pathways a b
ctf18
ctf8
ctf18 & ctf8 don’t interact as genes but their proteins products do
הבדלים בין PPI ל- GGI
רשת PPI מניסויים large scale
even though there is a potential for similarity (feed forward loops)
Tong & Boone 2004

17. The GGI network features
Similarity to the PPI, WWW and other famous networks:
The degrees of the nodes follow the power law distribution (many with little, few with lots)
HUBS - important for fitness: gim3-5 – chaperones for actin
Small world (small shortest path)
למשל רשת שדות התעופה בארה"ב
מס' גנים עם דרגה מסוימת... הגרף הקטן לוגריתמי
Tong & Boone 2004

18. GGI networks: summary & conclusion
represent the functional relationships between the genes
help understand and connect pathways
help annotate new genes
predicting PPI
potential for understanding human multi gene syndromes (ie: Alzheimer)
Tong & Boone 2004

19. Chemical Genetic Interactions (CGI)
Parsons AB … Boone C. Integration of Chemical-genetic and genetic Interaction data links bioactive compounds to cellular target pathways
Nat Biotechnol 2004
CGI - A hypersensitivity of a mutant to a sub-lethal
compound.
Integrate GGI and CGI data using profiles in order
to understandi the targets of different compounds
במקרה הזה ניתן לומר שהתוצר של גן B הוא ככה"נ המטרה של חומר X
Parsons & Boone 2004

20. Chemical Genetic Interactions (CGI)
CGI (chemical genetic) array analysis
an array contains ~4700 diploids, every strain has a mutation
in a different gene
A query: an inhibitory chemical compound
A computational analysis for detecting the growth changes
In this assay 12 compounds were checked, among them:
Microtubule depolymerization agent
A protein glycosylation inhibitor
aa biosynthesis inhibitor
Signaling inhibitors
A topoisomerase inhibitor
etc…
לגנים נחוצים מוטציות מותנות, לגנים לא נחוצים – מוטציות החסרה
Parsons & Boone 2004

21. The CGI assay 12 screens against different inhibitors
Measure colonies’ sizes: same or 3 degrees of reduction
Sensitive strains were grown separately
with different compound concentrations
FP detection
Comparison between the rapamycin results and
another rapamycin (not large scale) former study
FN detection
185 61 24
בבדיקה לניסויים שעברו על רפמיצין: 246 זנים רגישים התגלו פה, 85 בעבר, 61 משותפים (כלומר פוספסו 24)
Profile creation and bi-clustering (12 drugs X 647 relevant genes)
Parsons & Boone 2004

22. The CGI bi-cluster
Parsons & Boone 2004

23. Multi drug resistant gene (MDR)
65 genes are associated with sensitivity to multiple compounds (>4/10
critical compounds):
Known MDR genes as: drug pumps, ABC proteins
Membrane lipid composition genes (erg family)
New multidrug sensitivity strains (vph2)
ABC= ATP bindign cassete proteins - טרנספורטרים טרנס ממברנליים
בתמונה: גנים לסינתזה של ארגוסטרול: שהכרחיים למבנה תקין של הממברנה
It seems that MDR genes are also conserved in mammals
Parsons & Boone 2004

24. The MDR network & enrichments
Lipid metabolism
Vacuoles
H+ pumps
Vesicular
transport
וקואלה – וזיקולה.
Parsons & Boone 2004

25. Comparison of GGI and CGI results
Perform a GGI assay (SGA array) for several genes, known as drug targets
pVal=10-56
ERG11 הוא מטרה לתרכובת פלוקונזול, הוא גם קשור בביוסינתזה של הממברנה, כמו חבריו מקודם (3,4,6)
Parsons & Boone 2004

26. Comparison of GGI and CGI results
High significance but not a total overlap:
Statistics (drug) vs. complete (mutation)
presence of the complex (drug + protein) may prevent alternative
pathways
Genes that are inhibited by the CGI and not by the GGI may be
involved in cellular import or export
Red = MDR a better overlap without them
לדוגמא בציור: האדומים הם MDR והסרתם מגבירה את החפיפה
Parsons & Boone 2004

27. Comparison of GGI and CGI results
Perform 57 GGI assays (SGA array):
cytoskeleton, DNA related, secretion and more
Filter out the MDR genes (of the CGI DB)
Create gene profiles and a combined matrix
Bicluster of the matrix (803 X 69)
הגנים שנבדקו ב-SGA הם גנים לציטוסקלטון, הפרשה, תיקון וסינטזת DNA וכו'
Parsons & Boone 2004

28. The GGI-CGI Bi-cluster
The ERG11 and the fluconazole
Were clustered together
Other examples of clustering of genes and the drugs against them
Parsons & Boone 2004

29. What can we learn from the Bi-Cluster ?
Good correlation between CGI and GGI
link compounds to their cellular pathways
link compounds to their gene targets
Reveal uncharacterized gene functions (ie: similar patterns of GGI and CGI reflects a functional similarity).
Parsons & Boone 2004

30. CGI - summary & conclusion
CGI profile summaries GGI profiles & is easier to perform
Uncharacterized gene functions
MDR gene detector
Parsons & Boone 2004

31. Pleiotropy
Drosophila: sex related genes; fertility & development
Cats: being white and deaf
Pleiotropy: a single mutant gene - multiple mutant phenotypes
Humans: single mutated gene - disease with several symptoms (heart & limbs defects)
Good or bad ??
Good usage of small genomes – good for low organisms
Disadvantage for higher organisms: problematic adaptation, gene evolution, contradiction to the modular nature of the evolution (ie: fused domains)
דוגמא לגן ומחלות: TBX5 מוטנט: מחלות בלב ובגפיים (holt oram syndrom).

32. Pleiotropy – the article
Dudely AM. Janse DM. Tanay A. Shamir R. Church GM. A global View of Pleiotropy and Phenotypically Derived Gene Function in Yeast
Molecular Systems Biology 2005
The challenge: loss of single function or loss of multiple functions ?
(difficult to answer experimentally)
The technique: check the mutant strains under different conditions
Dudely & Church 2005

33. Pleiotropy – large scale experiments
use the former arrays (~4700 diploids, every strain has a mutation in a
different gene):
Every array is put in a different condition (total of 21)
A computational analysis - detect the growth changes (full,slow,no)
Every experiment was performed twice and the results were compared to former studies.
The 21 conditions, among them:
Nutrient limiting conditions
Stress conditions (high ethanol, low pH, high salt etc…)
Heavy metals
Inhibitors of cellular functions (cytoskeleton…)
השוואה של כל מערך למערך בקרה בתנאים רגילים (YPD).
767 גנים הראו לפחות בתנאי אחד שינוי פנוטיפי בגידול
סוכריות - סורביטול
767 genes
were detected
Dudely & Church 2005

34. Gene profiles and clustering
767 strains
with growth changes
551 strains influenced
By 1-2 conditions
Cluster (sort) into
65 groups
216 strains influenced
By 3-14 conditions
Bi-cluster
overlapping groups
הצבעים לא רלוונטים, מתייחסים לאנוטציות GO
למעשה הגרף הימני היא הגנים הפלאותרופיים
כל שורה במטריצה מייצגת קבוצת גנים, כל טור מייצג אפיון
Dudely & Church 2005

35. GO enrichments- low pleiotropy clusters
“Proper and logical” clusters:
‘galactose only’ cluster was enriched for “galactose” (e-18)
‘UV only’ cluster was enriched for “response to DNA damage” (e-17)
Some new insights:
‘caffeine’ cluster was enriched for “cell cycle regulation”
Other significant clusters of unknown genes (and known condition)
הנקודה האחרונה מסייעת להכרה של אנוטציות חדשות
Dudely & Church 2005

36. GO enrichments- high pleiotropy clusters
Consistency with Parson’s research (CGI) regarding drugs:
Vacuole, golgi, intracellular transport
All of the set of genes:
Vacoular organization and biogenesis;
Protein transport and degradation
Maintaining pH (H+ transport)
Other significant enrichments:
Conditions related to RNA pol (inhibitors mainly) – transcription
MDR genes
גם פה התקבלו תוצאות הגיוניות כפי שציפינו
Understand gene functions for known and unknown genes
Dudely & Church 2005

37. Phenotypic profiles, GGI and complexes
Use of Gavin complexes
complex 113 – transcriptional elongation
complex 137 – histone deacetylase
Phenotypic profiles (boxes/black arrows): same complex proteins have:
same phenotype (dep1 pho23) – similar function
different phenotype (cti6 dep1) – distinct groups for distinct conditions
GGI - Synthetic lethal (blue arrows)
backup inside a complex (leo1 rtf1)
similar functions of components of the complexes (cdc73 sap30)
קומפלקסים חלבוניים שמראים בד"כ על PPI - אפשר להתעלם מהקופסאות
הפעם מדברים על פרופילים פנוטיפים: למס' גנים יש את אותו הפנוטיפ
Dudely & Church 2005

38. Complexes vs. phenotypic profiles
The similarity between the genes within a complex (Average the phenotype profiles)
עשו מדד להתאמת פרופילי הגנים בקומפלקסים:
הנוסחא: X*Y אותם מחלקים בערך המוחלט של X ובערך המוחלט של Y (מעין קוסינוס)
Conclusion: Phenotype profiles represent functional relationships
Dudely & Church 2005

39. Detecting multi functions genes
Chromatin
modification
snf1 is assigned to
Both clusters
Vesicle
transport
לכל ביקלסטר נמצא העשרת GO
ל-snf1 יש שיוך לשני ביקלסטרים ולכן יש לו 2 פונקציות שונות, כ"א מתבטאת בזמנים שונים
Indeed snf1 has several functions (targeting substrates, response
to stress, regulation of filamentations etc…)
Dudely & Church 2005

40. Multi-function genes Indeed multi functions
סידרו את הגנים על פי מספר הקלסטרים בהם הם משתתפים
Indeed multi functions
Overlapping clusters – biological redundancy
Dudely & Church 2005

41. Conclusions
New annotations and perspectives: relationship between phenotype, pathways and genes
Another perspective regarding MDR genes
Distinguish between genes with one vs many functionalities
A large number of pleiotropic genes (pVal<10-9). Were thought to be a disadvantage, maybe an advantage ?
שאלת מספרם של הגנים הפלאותרופיים – שאלה לדיון
Dudely & Church 2005

42. However…
“Small scale”: only 132 genes (GGI), 12 compounds (CGI) and 21 conditions.
Selected genes had similar “narrow” annotations (cytoskeleton, DNA repair)
Only growth rates were measured, what about other phenotypes ?
Binary systems: influenced or not. (Even when quantitatively measured)
Severe thresholds and problematic attitude, in some cases (CGI), only
genes that “passed” an initial stage, were tested using different
compound concentration.
ניסויים שבהם נבדקו גם ריכוזי חומר (CGI), ופרופילי גידול שונים (פלאותרופיה)
Tong & Boone 2004
Parsons & Boone 2004
Dudely & Church 2005

43. Summary and conclusions
Relatively simple methods
large scale
unsupervised
Integration of the DBs + use other data: Get a more complex view on genes, functions and pathways.
A less central field, with high potential
Phenotypic
profiles
GGI
CGI
A better understanding
CGI ואינטרקציה פנוטיפית הם אותו ניסוי
הבנה יותר טובה : מסלולים, מנגנונים ואנוטציות
Tong & Boone 2004
Parsons & Boone 2004
Dudely & Church 2005

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