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GENE PROFILES Synthetic lethality Chemical Genetic Interactions

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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

44 Thank you, And don’t worry, the cookies are caffeine, sorbitol and heavy metal free

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