© 2011 Nature America, Inc. All rights reserved. Brenda Andrews Received Received 13 September 2010; accepted 25 March 2011; published online ([email protected]). B.A. or ([email protected]) C.B. to addressed be should Correspondence work. York, USA. New Bronx, Medicine, of College Einstein Albert Immunology, and Microbiology of Department Canada. Ontario, Toronto, USA. Maryland, Bethesda, Health, of Institutes National Institute, Cancer National Canada. Ontario, Toronto, Toronto, of University Research, Biomolecular and Cellular for Centre Donnelly Canada. 1 progression and initiation disease in implicated are and mutations amplification gain-of-function gene which in cancer, as such diseases of nisms effect a gain-of-function it called to tends in an cause gene activity increase gene provides a dosage Increasing means of as probing gene function of a cell. could generate insights into the functional wiring diagram of dosage suppression studies with other interaction networks frequencies. This work suggests that integrating the results mechanisms, which provided insight into their relative total interactions into four general types of suppression library, MoBY-ORF 2.0. We classified 87% of the 1,640 with a high-copy molecular-barcoded open reading frame populations of mutant cells temperature-sensitive transformed identified dosage suppressors for 29 genes from pooled generality of these network properties, we experimentally types of genetic or physical Tointeractions. confirm the related genes and that the majority do not overlap with other dosage suppression interactions occur between functionally interactions from the literature. Analyses revealed that many to 437 essential genes in yeast. For 424 genes, we curated analyze a network of interactions linking dosage suppressors might illuminate global cellular functions is unclear. Here we functional connections among genes, the extent to which it another gene. Although dosage suppression is known to map of overproduction one gene rescues a mutant phenotype of Dosage suppression is a genetic interaction in which Anna Kobylianski Bahr Sondra MagtanongLeslie enhance functional wiring diagrams of the cell Dosage suppression genetic interaction networks nature biotechnology nature 15 May 2011; Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Ontario, Toronto, Toronto, of University Genetics, Molecular of Department 4 1, Department of Pharmaceutical Sciences, University of Toronto, Toronto, of University Sciences, Pharmaceutical of Department 2 2 . Gene overexpression is relevant to the molecular mecha molecular the to relevant is overexpression Gene . Banting and Best Department of Medical Genetics, Terrence Terrence Genetics, Medical of Department Best and Banting 3 doi:10.1038/nbt.185 . In wild-type yeast cells, systematic analysis of gene gene of analysis systematic cells, yeast wild-type In . 1 , 2 , , Andrew M Smith 1 , 1 2 5 2 3 , Present address: Howard Hughes Medical Institute, Institute, Medical Hughes Howard address: Present , , Zhijian Li Genetic Branch Center for Cancer Research, Research, Cancer for Center Branch Genetic & Boone Charles 2 , 5 ,

6 advance online publication online advance , Cheuk , HeiCheuk Ho 6 These authors contributed equally to this this to equally contributed authors These 5 1 , 2 , , Michael Costanzo 1 , 2 , Lawrence , E Lawrence Heisler 1 , 2 1 , 2 , 6 , Sarah , L Sarah Barker

1

, 2 , , Munira A Basrai - 2 , , John S Choy of known function is an effective way to probe gene activity activity gene probe to way effective an is function known of mutations gene containing cells overexpression in in sensitized genes phenotype when overexpressed ectopically that ­overexpression has only revealed a of subset genes cause an overt as these have shown that dosage suppression analysis can delineate delineate can analysis suppression dosage that shown have these as signaling by Rsr1p to activated be that seems Cdc42p of allele (ref. temperature-sensitive a GTPases, suppresses Ras-related respectively, or Rho-related the Rsr1p, encoding or genes Cdc42p the either of overexpression example, semipermissive For a at temperature. normally grow to cells mutant the allows sor suppres dosage the of overexpression cases, many In temperature. restrictive at higher, a observed is phenotype mutant defect exaggerated an growth temperature-sensitive a has that gene tial essen an of allele an using often studies, diverse many in examined been has interaction genetic of type This suppression. dosage called cell. the of diagram wiring functional the of understanding into insight the genes roles and further of improve specific our global provide should quent of of integration interactions new types genetic cell remains incomplete, and thus systematic identification and subse the of knowledge our networks, genome-scale these of mapping the network individual either than informative more is work a show that pairs interact also physically interaction gene genetic of fraction small a only as network, action interaction network largely genetic complement those found the in the protein-proteinin inter edges The graphs. network biological in ‘edges’ are called or products gene genes between interactions Direct network for yeast has been assembled from multiple data sources interaction physical large-scale a network, genetic the to In addition onexpected the basis of the combined effect of the single mutations than or fit more less either is mutant double the when an interaction scoring interactions, genetic positive and negative both maps work for has been described budding yeast interaction network based on loss-of-function double mutant analysis ring both within and pathways. cellular between An extensive genetic unexplored. largely been have interactions of types other and it between relationships the and suppression age dos systematic utility, functional their Despite interactions. genetic relevant that demonstrating gene functionally dosage studies identify 1 , 2 Rescue of a mutant phenotype through overexpression of a gene is overexpression of through a mutant Rescue phenotype occur connections functional map networks interaction Genetic , 6 , , Wei Jiao 18), which encodes the guanine nucleotide exchange factor for for factor exchange nucleotide guanine the encodes which 18), 3 1 , Elena , KuzminElena 3 , 2 , , Guri Giaever , , Anastasia Baryshnikova 1 1 1 , , 2 2 1 . . This quantitative genetic net , , Kerry , Andrusiak Kerry 4 , Corey , NislowCorey 1 1,2,4– 1 . . Thus, an integrated net 6 . However, examining 1 ysis i s ly a n a , 2 1 , 9

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© 2011 Nature America, Inc. All rights reserved. capable of covering novel network space. network novel covering of capable Thus, dosage suppression seems to often a identify type of interaction terms Ontology Gene that gold-standard shared for pairs gene were enriched interactions suppression dosage unique ( edge network pression interactions (68%) did not overlap with any previously sup most dosage interactions, and lap genetic mappednegative physical with for positive genetic interactions ( ( (protein- physical the found that the dosage suppression network was enriched significantly networks: interaction ­protein) other three with inte genetic suppression dosage of overlap the Weexplored edges network other with overlaps suppression Dosage map phenotypic connections between functionally diverse genes. vesicle-mediated transport. Thus, dosage suppression interactions can tional categories, including those that act at various steps in intracellularsuppressed by overexpression of genes involved in several containing differentmutations in cell polarityfunc and morphogenesis pathways are related( processes yet distinct between interactions suppression dosage of number tial ­dosage suppression interactions. Notably, a substanwe observed also genes involved in the same biological process were highly enriched for networks biological other of connectivity the inter actions within and across different genetic cellular processes. Consistent with suppression dosage of occurrence the examined We processes cellular across distribution suppressor Dosage interrelatedness. of on functional basis genes the cluster to independently used can be functioning in these different processes. This suggests that dosage dosage like other suppression forms interactions, of that interactions genetic suggests This processes. different these in functioning genes between that occur of number interactions suppression dosage a substantial with along network, the in clusters gene corresponding are by morphogenesis indicated the and relatively proximity close of their growth cell and exocytosis transport, vesicle-mediated among shared functionality ( net genetic synthetic work the to Similar processes. biological specific containing each clusters, nine tified analysis clustering Markov clusters. distinct formed actions layout 1 Fig. a although small set of suppressors, genes has a large number of interactions ( dosage few a only have genes query Most ( interactions 1,293 and genes 768 Supplementary Table 1 Saccharomyces essential genes, which we call ‘query’ genes, that were annotated in the for 424 interactions genetic suppression of dosage a set We collected network interaction genetic suppression dosage A global RESULTS yeast the in interactions suppression dosage of mapping high-throughput facilitate that reagents develop and networks interaction suppression dosage of properties analyze we here issue, this Toaddress examined. been not has interactions these mapping of systematically value but potential the components, the A  P

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architecture of signaling pathways and identify novel pathway pathway novel identify and pathways signaling of architecture 1 and and 1 2 , the relative distance between these clusters seems to reflect reflect to seems clusters these between distance relative the , 0 , such that genes sharing common dosage suppression inter suppression dosage common sharing genes that , such −16 13–1 Supplementary Table 1 Supplementary ) for both physical and negative genetic interactions but not 6 , negative genetic and positive genetic networks genetic positive and genetic negative , Genome Database (SGD, Fig. 2 Fig. Fig. 2 Fig. Fig. b and and ). These interactions form a network containing a ). For example, the growth defects of strainsofFor ). example,defects growth the 1 ). For example, the functional relationships Supplementary Table1 Supplementary P = 0.52) ( Fig. Fig. ). Saccharomyces cerevisiae Saccharomyces ≥ 1 h 30 genes that correspond to to correspond that genes 30 and and t t p Table : / / Supplementary Data Supplementary w 11,13–1 w 2 1 w 2 ). ). Despite this over (55%; (55%; . y e 6 ). Notably, these these Notably, ). Supplementary Supplementary , we found that found we , a s t g P e n << 10 o r m 2 actions actions 1 . 1 e iden 1 . 10,1 o . We . −16 r g 1 ). ). ). ). / ­ ------­ ; ,

Because the literature-curated network could be based on selected selected on based be could network literature-curated the Because 2.0 MoBY-ORF with analysis suppression dosage Systematic clusters. distinct form interactions suppression dosage common share that (nodes) genes that such layout, force-directed a using distributed were nodes The terms. indicated the by summarized processes biological Ontology Gene for enriched genes of sets are nodes Colored edges. as represented are interactions and nodes as represented are Genes interactions. genetic suppression dosage of diagram Network 1 Figure published published dosage suppression interactions ( previously no or few relatively had genes query the of majority The ( segregation and ( degradation protein to, limited not but including, processes cellular of variety a in participate that temperature. semipermissive at out the carried are dilutions spot and strain, temperature-sensitive cognate the into transformed individually are plasmids suppressor dosage candidate interactions, ( background above significantly intensity hybridization raw have barcodes that their fact the by identified are suppressors micro dosage barcode Candidate a array. to pool preselection control a of those with ized are by cells of amplified pool PCR hybrid and selected competitively the from derived barcodes Then, temperature. semipermissive a at mutation, the temperature-sensitive pressors or a wild-type copy of the essential gene (which complements MoBY-ORF 2.0 library, and transformants carrying either dosage sup genes ( essential of alleles temperature-sensitive conditional of sors readout. barcode-microarray a using a dosage suppressor gene can be monitored in a pool of transformants reagent set tailored for gene dosage analysis because the abundance of yeast ular ORF barcoded (MoBY-ORF provides 2.0) library plasmid a molecular barcode tags ( downstream genomic sequences, along with two unique 20-nucleotide a yeast open single frame (ORF) reading with its native upstream and of composed a DNA insert contains plasmid each which in plasmid) 2 yeast on the (based library plasmid a high-copy we developed sis, identified be through an must additional round of gene experiments. For more suppressing efficient analy the that meaning large, often random carrying fragments plasmids genomic of libraries high-copy using ductively pro out carried been have yeast in studies suppression dosage Classic gene genes. diverse functionally of subset a on analysis pression sup dosage a systematic out carried we biased, therefore and results Supplementary Fig. 3 Fig. Supplementary & pr P We used this protocol to screen for suppressors of 41 query genes genes query 41 of suppressors for screen to protocol Wethis used suppres dosage identifying for protocol a developed also We ost-translation modif ot SEC26 ein degradation V

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© 2011 Nature America, Inc. All rights reserved. Positive Positive genetic Negative genetic Physical Type ofinteraction Table 1 ORF sequence, and downstream upstream native carries plasmid 2.0 immediately next to it ( clones carried the complementingwild-type ORF in addition to genes functional are genes PCR-amplified the of all not but (~93%) most and strains, three of these those including for of ORFs, ~20% yeast plasmids type comple observe not 2 the because mentation would we which in cases few a expected We strains. query three but all for recovered was ORF complementing ( genes 41 query the for interactions literature-curated reported of previously (60%) 6/10 ences in conditions screening and we genetic backgrounds, identified differ However, despite colonies. background general some include This procedure involved the washing plates completely,colonies. and therefore may suppressor candidate the harvested we how to part, in attributed, be may rate confirmation low relatively The screens. which represent 23% of the dosage suppressors identified in the initial validate 214 different suppressing plasmids ( We carried out confirmation transformations and spot dilution assays to interactions. physical and genetic negative suppression, dosage showing pairs gene yellow, interactions; physical and suppression dosage showing pairs gene blue, dark interactions; genetic negative and suppression dosage showing pairs gene red, only; interactions suppression dosage showing pairs gene blue, Light considered. were interactions physical and genetic both for tested be to known pairs gene Only (PPI). interactions protein-protein and genetic negative show also that pairs gene suppression dosage of ( (0.0005). chance by expected interactions of frequency scale, color in line Red interactions. within-process function. gene suppressor Dosage pairs. random of frequency the than greater yellow, sets gene functional defined broadly 19 for measured was interactions suppression dosage showing pairs gene of frequency The network. suppression dosage the for processes 2 Figure nature biotechnology nature of query-dosagesuppressor genepairs( a P valuesbasedonahypergeometric test. a Query ORF AA biosynth.&transport/nitrogenutilization

Chrom. seg./kinetoch./spindle/microtub. Overlap Overlap of dosage suppression interactions with other types of interactions Properties of the yeast dosage suppression network. ( network. suppression dosage yeast the of Properties DNA replication&repair/HR/cohesion 2 4 . . Of the 168 dosage extragenic suppressors, three plasmid Protein degradation/proteasome Golgi/endosome/vacuole sorting Nuclear−cytoplasmic transport Cell cycleprogression/meiosis Lipid/sterol/fatty acidbiosynth Cell polarity/morphogenesis Signaling/stress response Metabolism/mitochondria Chromatin/transcriptio Supplementary Table 2 Supplementary Glycosylation/cell wall Ribosome/translation Supplementary TableSupplementary 2 No. testedpairs Drug/ion transport µ RNA processing ER−Golgi traffic MoBY-ORF library does not wild- MoBY-ORFcontain does library 1 1,503

1 advance online publication online advance ; blue, less than the frequency of random pairs; black, statistically indistinguishable from a random set of gene pairs; pairs; gene of set a random from indistinguishable statistically black, pairs; random of frequency the than less ; blue, 254 254 Supplementary Table 1 Autophagy b Query-dosage suppressor genepairsannotatedintheSGDas‘dosage rescue’inwhichthebaitisanessentialgene ( b c c n .

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), ), Metabolism/mitochondria - - value 0.52 Cell cycle progression/meiosis −16 −16 and interactions; interactions; however, most edges were unique ( genetic positive with not but interactions genetic negative and tions interac protein-protein with significantly overlapped interactions suppression dosage our Moreover, genes. query respective their to and tated with the term same Gene Ontology 137 these dosage suppression interactions linked pairs of genes anno (~40%, portion significant a network, pression suppressors dosage ( known previously no had that genes query 13 for including genes, 29 suppression interactions query dosage ported unre previously 137 found we total, In gene. query per suppressors ( 24 gene—to wild-type the only of recovery gene. suppressor same the recovered and alleles multiple we is genes, screened query For several small. region relatively intergenic the if plasmid single a on occur can ORFs two ER−Golgi traffic Supplementary Tables 1 Supplementary a Golgi/endosome/vacuole sorting Similar to what we observed for the literature-curated dosage sup dosage for literature-curated the to what we Similar observed is, 0—that from widely varied suppressors dosage of number The 2 Supplementary TableSupplementary 2 Ribosome/translation ), ), suggesting that the dosage suppressors are relatedfunctionally Nuclear−cytoplasmic transport x axis; query ORF gene function, function, gene ORF query axis; 1 Fraction 1 . No. testedpairs 0 0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 0.009 0.01 150 57 57 c c Dosage suppression(thisstudy) b and ), ), but on average, we recovered ~5 dosage b No. overlappingpairs ) Scaled square Venn diagram of fraction fraction of Venn diagram square ) Scaled 2 ). 254 dosagesuppressioninteractions 33 PPI 162 nonoverlappinginteraction 27 y 8 0 axis. The diagonal represents represents diagonal The axis. 2 2 ( negative and PPI Supplementary Table 1 genetic Supplementary Tables Supplementary 1 29 Supplementary Fig. 1 Fig. Supplementary Table P 30 negative < 2.6 × 10 × 2.6 < ysis i s ly a n a 1 genetic 1.03 1.03 x 10 ). ). On the basis <<10 P value s N/A −16 ). a c −5 −10 Subset ) of of )  - - - -

© 2011 Nature America, Inc. All rights reserved. literature (dotted arrows). Thirteen percent of percent Thirteen all arrows). gene (dotted pairs could not literature be by the primary examining a relationship supporting functional evidence (i), related we were not found additional genes themselves functionally but the interacted not their physically gene because be products classified in For or gene RNA synthesis. pairs that protein processing could otherwise the query mutant A can gene product. also suppressor dosage participate or stability,protein such as that a might protein, act to heat-shock stabilize A is chaperone a that a encodes suppressor dosage for gene required RNA or an suppressor dosage suppressor.dosage unknown synthesis protein as a an categorized RNA chaperone, processing/ were mechanistically Gene pairs that interaction. protein did not fit into either of these categories with the same term Gene were and/or Ontology also by linked a protein- gene First, pairs to were analyzed categories. see if they were annotated gene into pairs one were classified of suppression Dosage four mechanistic 4 Figure interaction genetic negative a show each (ref. both of transcription upregulate directly to dicted HCM1 both for fied ( genes query two these on impinging genes 28 and interactions 31 of for previously mapped (MAPs) and motors proteins microtubule-associated kinetochore and heterochromatin ( complex chore kineto (MIND) Nnf1p-Nsl1p-Dsn1p the Mtw1p including essential the of components two screened we analysis, systematic our in ple, exam For interactions. suppression dosage by revealed be can ways and complexes pathways particular into insight mechanistic new provide can interactions suppression dosage specific of examination detailed A Dosage suppression links PKA signaling and kinetochore function space. network relevant tionally dosage suppression of interactions cover majority relatively unexplored the and func that confirms analysis systematic our notably, and, network suppression global the of properties general the represents of these observations, we conclude that the literature-curated network n as described genotype of each colonies diploid grown freshly with rates loss chromosome to measure out carried were tests Fluctuation function. kinetochore proper for be may required signaling PKA ( biogenesis. ribosome red, signaling; PKA blue, function; kinetochore Green, gene. mutated to the suppressors link Arrows complex. of suppressors ( 3 Figure A  and fell classified into a (ii). unknown labeled category a Supplementary Table 2 Supplementary = 3 for

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BCY1/BCY1 Decision tree used to categorize dosage suppression interactions. interactions. tree used Decision suppression dosage to categorize A genetic requirement for PKA signaling in kinetochore function. function. in kinetochore signaling PKA for requirement A genetic 1 8 NSL1 . In particular, the regulatory targets of signaling path signaling of targets regulatory the particular, In . NSL1 NSL1 nsl1(ts) hcm1 nsl1(ts) Fig. 3 Fig. HCM FCF1 or and and and and 25,2 1 DSN1 DSN1 a 6 ), which participates in bridging centromeric centromeric bridging in participates which ), SPC105 bcy1 . Although dosage . suppressors dosage have Although not been NSL1 DSN1 ). Among the dosage suppressors we suppressors identi ). Among dosage the , two components of the MIND kinetochore kinetochore MIND of the , components two ∆ ∆ or or GPB2 /BCY1 and and were the S-phase transcription factor factor transcription S-phase the were GPB1 GIS2 DSN1 , , dsn1(ts) hcm1 dsn1(ts) n Attentuation = 2 for signaling of PKA , we identified a subnetwork subnetwork a identified we , 1 FCF1 1 3 ; therefore, our results are are results our therefore, ; 2 . Values are mean . Values mean are ctf19 . . b HCM1 Chromosome loss rate ∆ ∆ (per cell division) /ctf19 double mutants mutants double 5.0 1.0 1.5 NSL1 b Genotype ) Attenuation of ) Attenuation has been pre been has × × ×

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in a diploid query strain that enables a quantitative analysis of chro of analysis quantitative a enables that strain query diploid a in ity, we deleted To possibil this phenotype. test loss to a chromosome lead may also phenotype loss a chromosome with associated often are genes of kinetochore alleles DASH complex–encoding genes suppresses the temperature-sensitive mutant phenotypes of function. chore kineto proper to contributes signaling of PKA attenuation which in a model support our Thus, results complex. and MIND the signaling PKA of downregulation between relationship functional a identify and complex microtubule-associated has been observed kinetochore- DASH) (or Dam1p-Duo1p the and pathway PKA the to similar regulator, a negative as act to thought but is characterized well not is identified as dosage suppressors of pathway, the in steps at various signaling complex linker kinetochore essential an as acts also complex this and Kre28p, with a complex forms Spc105p We phenotypes. identified also the both with consistent genes linked genes by linked dosage suppression as interactions being functionally Tables 1 ( unknown as classified (13%) interactions the remaining underlying of with the mechanism the four categories, one into interactions suppression dosage of the (87%) of 1,640 1,422 classifying work, this in experimentally observed and literature the from curated interactions of network integrated the analyze to tree in yeast into four categories general mechanistic ( interactions genetic suppression dosage classifying for tree decision a developed we genome-wide, observed is mechanisms the of each pression genetic interactions and to explore the with frequency which described been has sion suppres genetic of second-site categorization mechanistic A general interactions suppression dosage of categories Mechanistic it. potentiates signaling decreased and activity kinetochore antagonizes signaling increased which in functions, kinetochore DASH and MIND over control rheostat-like provide to act may signaling PKA that suspect diploid control ( compared with the type wild-type bcy1 frequencies loss mosome query gene? Same GOfringetermas component Complex Because overexpression of of regulators overexpression negative the PKA pathway Because (PKA) A kinase protein downregulate that genes Tworedundant The decision tree first classified 1,285/1,640 (~80%) of all pairs of pairs all of (~80%) 1,285/1,640 classified first tree decision The Yes PPI ∆ /BCY1 ? advance online publication publication online advance Yes and PDE2 relationshi Functional diploids showed a pronounced chromosome loss pheno 2 No ). BCY1 3 , , in PKA signaling 0 p , we hypothesized that hyperactive PKA signaling signaling PKA hyperactive that hypothesized we , Dosage suppresso , a negative regulator of cAMP-protein kinase of cAMP-protein regulator , a negative 9,3 in silico in 3 3 2 . To extend this analysis to dosage sup dosage to analysis this Toextend . . Indeed, we found that heterozygous heterozygous that found we Indeed, . r SPC105 or 2 Chaperone predictions and the double mutant mutant double the and predictions 9 PPI 2 and because temperature DSN1 Yes (i 8 or . . Although a genetic link between protein synthesi RNA processing/ ? N ) Yes as a No ( 2 6 Fig. 3 GPB1 nature biotechnology nature . Yes DSN1 o Fig. Fig. s Role instability a and and No ). ). A third gene, Fig. Fig. dosage suppressor.dosage 4 Fig. Fig. 3 ; ; or proteinsynthesis? Role inRNAprocessing No Unknown (ii) Supplementary Supplementary GPB2 4 ). ). We used the 2 9 b , , our results ? ). ). Thus, we , were also also were , DSN1 - sensitive sensitive GIS2 and 3 1 ­ - - - - - , ,

© 2011 Nature America, Inc. All rights reserved. Table 2 ( essential were suppressor dosage the and mutant query the both which in screens our in interactions suppression age dos 28 We identified complex. protein same the to belonging ucts mutant query gene which (in suppression dosage reciprocal genes, essential other containing complexes form to tend genes essential because and hypothesis this of basis the On pro protein. a mutant query the stabilize can containing complex tein suppressor dosage the suppressor that dosage indicate its also and may product gene query mutant a between Cdc28p activate and to bind cyclins Cdc28p kinase dependent of allele gene temperature-sensitive the the essential encoding cyclin- of suppressors dosage as discovered initially were which ples of the include G1 genes in cyclins category this interaction ( protein-protein physical a gene by linked whose also term are Ontology products Gene same the with between annotated interactions genes suppression dosage as defined component,’ ( process or pathway biological general same the in genes between interactions pression and of set terms a gold-standard within term Ontology Gene same the with annotated were they because related viable. be to cell the for protein mutant functional enough to leading degraded, of instead translated be then can which product, mRNA the of quality the improve to possible be might it however, transcription, of aspect some increasing By degraded. be will probably but translated be may that product allele mutant of transcription temperature, semipermissive the At transcription. of aspect some in involved normally A is protein suppressor dosage the example, this In translation. or transcription during acts that a gene be can suppressor A dosage synthesis. protein ( function. essential its out carry to it enabling protein, mutant the stabilize and refold can A), (protein a chaperone as such suppressor, a dosage of overexpression but unfolded, b is protein mutant the temperature, semipermissive the At product. gene mutant the with interact normally not does protein suppressor dosage the example, this In product. gene mutant the of amount the affect can ( pathway. the activate to allele mutant the of upstream act gene by encoded suppressor, A dosage effector. a downstream to information transmit allele mutant the of function the temperature, semipermissive the at example, this In process. biological same the in allele query mutant respective its of downstream or upstream either function can suppressor A dosage relationship. ( function. essential its execute can complex physical the that so protein mutant folded properly of amount increases of a Overexpression suppressor,dosage A, protein partner(s). physical normal its with interact cannot therefore it and unstable, product gene the renders mutation the because probably state, unfolded an in occurs b predominantly protein mutant the temperature, semipermissive the At function. normal its for required is and product gene mutant the with interacts that a protein encodes that a gene be can suppressor ( yeast. in 5 Figure nature biotechnology nature ( sion Fig. 5 Fig. The first mechanistic category of dosage suppression is ‘complex ‘complex is suppression dosage of category mechanistic first The 2 ). ). We two considered that mechanisms may explain dosage sup b Supplementary Table 3 Supplementary gene product is impaired and cannot cannot and impaired is product gene a ). Of the 28 gene pairs, 9 showed reciprocal dosage suppres dosage reciprocal showed 9 pairs, gene 28 the Of ).

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5 A ′ temperature Chaperones, such as heat-shock proteins (HSPs) or RNA stability stability RNA or (HSPs) proteins heat-shock as processes. such biological Chaperones, different in participating as annotated between genes interactions suppression dosage 15 of subset a to applied product gene mutant the for substitute functionally can suppressor dosage the of activity ( function among duplicated genes and gene pairs that share the same molecular brane SSO2 t-SNAREs, membrane plasma redundant functionally two of of either to by overexpression suppressed are network membrane, plasma the trans-Golgi the from vesicles secretory transports that SEC3 ( interactions physical without ‘functionally related’ genes that show dosage suppression interactions complex. protein the of stability increased by mediated is suppression phenotypic which in a mechanism supporting evidence provides interactions physical and suppression dosage reciprocal between overlap strong the Thus, the ( in interaction share a also physical literature reported interactions suppression dosage reciprocal of 75% ( by chance occurred this that improbable highly is it products; gene their among action inter physical a shared also pairs gene nine all Notably, partner. its by overexpressing be suppressed gene could in mutations either with Permissive A Viable Viable Viable Viable A The third mechanistic category is ‘chaperone suppressor,’ which suppressor,’which ‘chaperone is category mechanistic third The 760 remaining the included category mechanistic second The A b b b , , involved in to vesicles the of fusion the mem plasma secretory , which encodes an essential member of the exocyst complex complex exocyst the of member essential an encodes which , 3 b b 6 . Dosage suppression interactions have also been identified identified been also have interactions suppression Dosage . 3 ′ Supplementary Table 1 Table Supplementary A Semipermissive 5 A b ′ temperature Supplementary Table 3 Supplementary b 8 A Arrested Arreste Arrested Arrested . Degradation A b b d b 3 A ′ Fig. 5 Fig. b ); in both cases, the biochemical biochemical the cases, both in ); b leads to a poor-quality mRNA mRNA a poor-quality to leads b ). For example, mutations in in mutations example, For ). Supplementary Table 4 Supplementary Semipermissive temperature A ; ; P A + dosagesuppressor 5 << 10 A ′ b b A 5 A A ysis i s ly a n a A A ′ A Viable Viable Viable Viable A A D b −16 b egra A A A b A ). Similarly, ). Similarly, A 3 A d ′ SSO1 at A 3 i o A ′ n b and ). ).  - - © 2011 Nature America, Inc. All rights reserved. the wild-type copy of an interacting gene, whereas in the context context the in whereas gene, interacting an of copy wild-type the overexpressing by improved is mutant query the of fitness the sion, show often interactions complex genetic essential negative an within interact genes because products whose interactions protein-protein with overlap the with two the defects given fitness mutant expected, single than stronger significantly statistically is that defect fitness a double-mutant as defined is interaction genetic negative A interactions. genetic negative with overlap interactions genetic suppression dosage of fraction significant a that found also suppressor protein mutant query the dosage containing its complex a and stabilize may product gene query mutant a between 1 Tables ( interactions protein-protein with lap cell. the of diagram wiring functional a enhance should sets data global other with integration whose edge, network of type distinct a defines suppression dosage Thus, actions. inter genetic negative and protein-protein with overlap 32% only as capture actions that relationships are by not other methods, observed ( genes related functionally between occur (~80%) interactions suppression dosage of majority sification of the dosage suppression interactions that revealed the vast ( genes among relationships tional func of network coherent a generated data literature-curated from derived interactions genetic suppression dosage of mapping Global DISCUSSION suppression. dosage of mechanisms 10% we of reclassified into the interactions unknown one of the other so, doing By category. mechanistic a into interaction particular a of that reclassification might information support other functional find between literature in an the we to primary gene effort their products, revisited interactions physical known without and with both pairs gene including category, unknown the in placed initially interaction ( common in terms Gene Ontology gold-standard any share not do genes whose but interaction suppression gene pairs whose protein products have a known physical egory. Notably, this category,(14) within dosage several we identified category. ribosome pression interactions belonged in the chaperone and RNA processing/ Table 1 ( mutant phenotypes the associated can genes rescue genes subunit ribosomal eral genome which lack the lone copy of the type II myosin heavy chain in the yeast subunit genes. For example, transcriptional profiling of profiling transcriptional For example, genes. subunit temperature- tion that represses of transcription RNA the processing and/or ribosomal of product ( gene the query ­sensitive of translation or transcription stood, increased dosage of the suppressing genes may lead to increased are not under well cases in these suppression of dosage mechanisms ( query of genes variety a for suppressors dosage as identified were which factors, RNAand processing subunits ribosomal encoding genes ing ( relationship of sets diverse genes pressed that do not share any obvious functional a of levels ( product gene query the stabilizing by suppressors dosage as act can factors, A 

The observation that some dosage suppression interactions over interactions suppression dosage some that observation The ‘unknown’ cat the to interactions 236 remaining the We assigned The The fourth mechanistic category applied to 104 interactions involv ysis i s ly a n ). ). We found that a relatively small (7%) fraction of dosage sup Supplementary Tables 1 1 Tables Supplementary 3 , 7 2 2 , has shown downregulation of many genes, including sev including of many genes, downregulation shown , has and Supplementary Tables 1 Supplementary 5 ) supports the hypothesis that a direct interaction interaction direct a that hypothesis the supports ) Fig. 5 Fig. Fig. 5 Fig. c ). Indeed, increased dosage of HSPs sup dosage increased ). Indeed, d Fig. Fig. 3 ), thereby compensating for a a for compensating thereby ), 10,12,3 8 1 , and overexpression of some of these these of some of overexpression and , 2 Supplementary Table 1 Table Supplementary . In the context of dosage suppres dosage of context the In . 5 ). Most dosage suppression inter suppression dosage Most ). and Fig. Fig. 9 . This observation is consistent consistent is observation This . 2 and Table Table 1 ). Indeed, mechanistic clas mechanistic Indeed, ). ). Although the molecular molecular the Although ). 2 ). 1 and and Supplementary Supplementary Supplementary Supplementary myo1 ). For every every For ). ts ∆ muta 3 cells, cells, 4 . We. ­ ------­

features of dosage suppression networks. However, the mechanistic mechanistic the However, networks. suppression dosage of features age suppression network are not and, biased in represent fact, general that the network properties associated with the literature-curated dos interactions. genetic pheno (loss-of-function) types dominant-negative to lead can dosage gene increased instances rare in although phenotypes, of-function gene function of another loss of with associated defect fitness the suppresses one of gene function of loss the which in the those are on interactions genes positive of subset query Another genes. the essential are of network suppression all dosage because dosage our interactions with overlap suppression to interactions positive these genes would We expect not complex. between physical occurs nonessential a form interactions products whose genetic positive of subset A mutants single of on of the corresponding the basis the fitness expected than that is severe mutant less double defect shows a fitness ( edges work net suppression dosage and interactions genetic positive between gene. interacting the of allele loss-of-function a by enhanced is mutant the of defect fitness the interaction, genetic negative a of Methods and any associated references are available in the online online the in version of the paper at http://www.nature.com/nbt/index.html.available are references associated any and Methods M interactions. physical and genetic of types all comprising landscape cellular complete a of type of ‘edge’functional that could be integrated into the construction distinct and prevalent highly a adds map suppression dosage global a that We conclude systems. model metazoan and cells mammalian in possible be should interactions genetic of mapping analogous an in yeast, interaction of genetic of type this utility the we demonstrate Although connections. and information functional new of potential interactions per query gene, the dosage suppression network offers the a of background context lethal the synthetic within gene each of alleles of creation temperature-sensitive the through network suppression dosage nonessential the of on majority genes the to extend could effort mapping This genes. of essential spectrum entire for the mapped be could network in yeast genes results. relevant functionally consider authors the what include only that studies focused from omitted be may genes these because simply suppressors as genes synthesis protein and ing process RNA more identify may screen unbiased our that suspect reported been have genes ribosomal for tions func some extraribosomal and roles, because specialized have paralogs ribosomal pathway-specific be may suppression the however, process assembly protein the complexes, of protein case the or, in folding protein synthesis, protein of steps different in sis synthe protein and RNA in processing involved of genes expression in decrease transient and general a induces temperature restrictive a to mutants temperature-sensitive of exposure with associated stress heat the because suppressors synthesis protein and processing RNA (25% literature the 5%; versus in than analysis systematic our in suppressors dosage of fraction larger a represented genes synthesis protein processing and RNA that revealed suppressors dosage of classification ethods The The results of our dosage systematic suppression suggested screens overlap significant statistically a observe not did we contrast, In Conditional Conditional alleles are being developed for the majority of essential 4 1 . Moreover, some some Moreover, . 2 , and thus we do not expect a strong overlap with positive positive with overlap strong a expect not do we thus and , advance online publication publication online advance Supplementary Tables 1 Supplementary Table Table 46,4 12,4 7 , and thus a dosage suppression genetic inte genetic , and suppression thus a dosage 0 1 . Gene overexpression typically leads to gain- to leads typically overexpression Gene . ). Positive genetic interactions occur when a when occur interactions genetic Positive ). ts mutations may lead specifically to defects defects to specifically lead may mutations 1 1 . With about five dosage suppression suppression dosage five about With . and 2 nature biotechnology nature ). We expected to identify identify to We). expected 43–4 5 . Furthermore, we we Furthermore, . 4 2 . In some cases, cases, . In some r 10,12,3 action action 9 ------.

© 2011 Nature America, Inc. All rights reserved. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.html. http://www.nature.com/ at online available is information permissions and Reprints Published online at http://www.nature.com/nbt/index.html. The authors declare no competing interests.financial construction of the MoBY-ORF 2.0 library and wrote the manuscript. edited the manuscript; B.A. wrote the manuscript; C.B. conceived and planned the edited the manuscript; G.G. and C.N. provided microarray data analysis and in temperature-sensitive strain generation; M.C. wrote the manuscript; M.A.B. edited the manuscript; A.K. outcarried analysis; experimental Z.L. was involved the chromosome loss assays; E.K. and K.A. outcarried analysis experimental and A.M.S. and L.E.H. were involved in microarray data analysis; J.S.C. outcarried temperature-sensitive strain generation and outcarried analysis; experimental out computational analysis and wrote the manuscript; S.B. was involved in in MoBY-ORF construction and wrote the manuscript; W.J. and A.B. carried outcarried analysis experimental and wrote the manuscript; S.L.B. was involved and wrote the manuscript; C.H.H. was involved in MoBY-ORF construction, L.M. was involved in MoBY-ORF outconstruction, carried analysis experimental Canada (RGPIN 204899-06). (MOP-57830) and the Natural andSciences Engineering Research Council of the Ontario Genomics Institute C.B. was (2004-OGI-3-01). supported by the CIHR CIHR (MOP-84305). C.B. and B.A. were supported by Genome Canada through agreements 020380 and MOP-81340, respectively). C.N. was supported by the G.G. was supported by the Canadian and Cancer Society the CIHR (research Program of the US National Cancer Institute, US National Institutes of Health. open fellowship. J.S.C. and M.A.B. were supported by the Intramural Research (CIHR) doctoral research award. A.M.S. was supported by a University of Toronto analysis support. L.M. was supported by a Canadian Institutes of Health Research microarray technical support, and C. Myers and J. Bellay for advice and data We thank S. Dixon for commentscritical on the manuscript, M. Gebbia for Note: Supplementary information is available on the E-TABM-1147. code accession under ArrayExpress codes. Accession nature biotechnology nature C AU A ckn O

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© 2011 Nature America, Inc. All rights reserved. Plasmid pool preparation. Plasmid pool Biosystems. Open from available be will library only. downtag The have 4% and a unique only uptag have 5% a unique codes, ORFs; ing 4,499 ( obtained MoBY-ORF the database from be can barcodes, and sequence ORF complete with along digests, using BamHI and HindIII was carried out. ORF fragment sizes for both double digest a secondary was ambiguous, digest XhoI-EcoRI validity. If the primary clone were and to compared the results determine independently, gel analysis ( sizes fragment insert and vector confirm to gel agarose 0.8% a on resolved and (Fermentas) EcoRI and XhoI using digested colony, doubly bacterial single a from prepared was 50 200 glucose, (wt/vol) 0.2% containing medium 50 (wt/vol) 0.2% (wt/vol), 2 reaction, lacI (genotype: P5530 °C. 37 at h ~16 for grown were Cultures chloramphenicol. 5 containing 100 had well each which in plate 96-well shallow a into MAGIC. using strains bacterial v1.0 construction clone library 2.0 MoBY-ORF chloride) or in YE medium (0.5% (wt/vol) yeast extract, 1% (wt/vol) NaCl).(wt/vol) yeast extract (Difco), 1.6% (wt/vol) (Fisher),tryptone (Difco),2% (wt/vol) 0.5% (wt/vol)glucose) medium.sodium BacteriaorSC (0.67% were(wt/vol) yeast grownnitrogen base, in0.2% (wt/vol) 2× amino YT acid mediumsupplement (1% base (Difco), 0.2% amino acid supplement Growthminus media.Leu (Fisher), 2% (wt/vol) glucose) May 2010. 11 on SGD the from loaded considered were standard gold to the same Gene Ontology term. Only Gene Ontology terms from a published annotated were they if related functionally be to considered were interaction relatedness. functional of Analysis se the from an gene essential as the ORF.query The list of genes was essential downloaded containing pairs gene only include to filtered were data suppression Dosage on SGD the 20 from May 2010. were downloaded set data interaction protein of types other with interactions. interactions genetic suppression dosage of Overlap enrichment in a particular cluster was used to label the cluster on the network. in for Cytoscape containing >30 genes were tested for functionalThe network enrichment was clustered using using the the BiNGOMarkov plug-clustering algorithmIdentifying gene clusters in the dosage suppression METHODS ONLINE genetic interaction network. nature biotechnology nature 100 in grown were plasmid high-copy barcoded a ing then transferred to a shaking incubator at 37 °C for 2 h. Of the 100 100 the Of 2 h. for °C at 37 incubator a shaking to transferred then (wt/vol) to each well. Cells were incubated without shaking at 37 °C for 2 h and time which at h, 2 100 of volume total to diluted were average the as used was wells three the of from (donor) strains the MoBY-ORF bacterial three plate; the 96-well average 200 and cin YE +Gluc medium, containing 10 0.2% glucose, (wt/vol) ( p5476 plasmid carrying FRT quence.stanford.edu/ The final MoBY-ORF 2.0 plasmid library contains 4,547 clones (represent clones 4,547 contains MoBY-ORFThe library final 2.0 plasmid YT 2× onto colonies individual for out streaked were products Mating the measured we day, next The µ µ Q g ml g ∆ g ml g rrnB3 rrnB3 recA635:FRT recA635:FRT umuC:ParaBAD-I-SceI-FRT −1 −1 Saccharomyces kanamycin and incubated at 41 °C overnight. °C at 41 incubated and kanamycin ∆ kanamycin and incubated at 37 °C overnight. Miniprep DNA Miniprep overnight. °C 37 at incubated and kanamycin µ µ lacZ4787 hsdR514 l was plated onto YE +Glyc medium containing 0.2% glycerol glycerol 0.2% containing medium +Glyc YE onto plated was l The literature-curated dosage suppression data and suppression set protein- dosage The literature-curated µ g ml g Yeast strains were grown in SD −Leu (0.67% (wt/vol) yeast nitrogen g ml g 4 A Supplementary Table 7 Supplementary 8 . The Gene Ontology biological process term with the highest 600 −1 −1 l µ carbenicillin. Cultures were grown for ~22 h at 30 °C. at h 30 ~22 for grown were Cultures carbenicillin.

≈ -arabinose was added to a final concentration of 0.2% 0.2% of concentration final a to added was -arabinose 2 l in a fresh 96-well plate. Cells were shaken at 30 °C for for °C 30 at shaken were Cells plate. 96-well fresh a in l tetracycline, 50 50 tetracycline, 4 0.10 and mixed together for mating in a 1:1 ratio in a in ratio 1:1 a in mating for together mixed and 0.10 Supplementary Table 6 Supplementary were inoculated from frozen stocks in 96-well plates plates 96-well in stocks frozen from inoculated were group/yeast_deletion dl eoe eein rjc dtbs ( database Project Deletion Genome Supplementary Fig. 5 Fig. Supplementary -chlorophenylalanine, 200 200 -chlorophenylalanine, Individual ∆ 2 h (araBAD)567 2 t . Gene Ontology annotations were down were annotations Ontology Gene . t p A Two genes sharing a dosage suppression suppression a dosage Two sharing genes : / 600 / m Escherichia Escherichia coli µ values of the recipient strain and of of and strain recipient the of values g ml g o ), of which 91% have two usable bar have 91% ), two usable of which b A y . c 600 −1 _project c ∆ b kanamycin and 12.5 12.5 and kanamycin ). Two individuals carried out out carried Two ). individuals for the entire plate. Cultures Cultures plate. entire the for (rhaBAD)568 galU95 ), ), the MAGIC recipient strain ), was inoculated into 5 ml of 5 ml into inoculated was ), r . u µ t o ml g r / transformants contain o ) on 20 March 2010. March 20 on ) µ n µ g ml g µ t −1 o l of 2× YT medium medium YT 2× of l g g ml µ . carbenicillin and and carbenicillin c l 2× YT medium medium YT 2× l a −1 / ). 2 −1 carbenicillin, carbenicillin, 1 . Nine clusters MoBY-ORF MoBY-ORF spectinomy http://www- µ ∆ l mating l mating µ endA9: g ml g −1 - - - - -

nonbiotinylated/biotinylated PCR product. A raw microarray signal of 1,000 1,000 of signal microarray raw A product. PCR nonbiotinylated/biotinylated (vol/vol) 9:1 contained mix hybridization Each pool. the transformant from original barcodes the nonbiotinylated PCR-amplify to whereas used were pool, primers TAG4 suppressor universal dosage the from PCR-amplify to barcodes used the were primers TAG4 universal Biotinylated out. ried described carried were as out hybridization microarray TAG4 and barcodes the of analysis. fication data and hybridization microarray barcode Yeast described as kit miniprep Qiagen the of protocol miniprep a using modified were isolated and was plasmids thawed, age pool suppressor dos the of sample a plasmids, in suppressing °C To isolate −80 glycerol. at (wt/vol) stored 15% and pool) suppressor dosage d the 3 form after (to appeared pooled that were colonies temperature, particular a at strain given a mutant temperature-sensitive untransformed the for temperature restric tive observed the on depended strain each for temperatures The −Leu. incubation SD onto plated were cells trans 50,000 and the thawed, of was sample pool a formant suppressors, of identification For glycerol. (wt/vol) MoBY-ORFwith v2.0; library. 2.0 MoBY-ORF the ­temperature-sensitive query strain with ( suppressors dosage of Cloning the from prepared DNA was the form to mixed was culture of A 55 plate. total at 37 °C 96-well for kanamycin 15 h in a shallow 200 glucose, (wt/vol) 0.2% containing were carried out were as carried described loss Chromosome rate measurements. tests). suppression reciprocal (for strain or the corresponding semipermissive temperature of the temperature-sensitive suppressors) dosage candidate (for identified initially was suppressor dosage medium −Leu SD on the at which temperature same out at the incubated and methods standard using carried were dilutions Spot methods. standard ( strain temperature-sensitive corresponding reciprocal for test and suppression. suppressors dosage candidate of Confirmation candidate of suppressors. dosage identification for a.f.u. raw use 1,000 to of cutoff decided we stringent more difference, the (<10%) marginal a was there strain, and one cutoffs, for intensity raw 1,000 and 500 the between retrieved barcodes of respectively. retrieved, were sample the in barcodes the of (50%) 5/10 and (50%) 5/10 (40%) 4/10 500, and 1,000 2,000, of Using cutoffs respectively. retrieved, were sample the in barcodes the of (66%) 4/6 and (66%) 4/6 (50%), 3/6 500, and 1,000 2,000, of cutoffs Using respectively. retrieved, were sample the in 21/24 barcodes the of and (88%) (80%) 19/24 (58%), 14/24 500, and 1,000 2,000, of cutoffs Using was hybridization microarray competitive described. as out a ­carried and amplified, PCR barcodes the were colonies, pooled the from extracted were plasmids Barcoded dosage candidate for cutoff empirical an identify thereby and a.f.u.) (~30 cutoff background microarray observed the than intensity greater raw with signals barcodes of set distinct a identify would mixture this in pool plasmid the that We anticipated pooled. and picked indi temperature. were vidually temperature semipermissive semipermissive the at d 3 the after at appearing Colonies incubated and medium plated were selected strain on each of transformants the of sample pooled a cutoff suppressors. resenting dosage intensity candidate of microarray identification barcode for raw of determination Empirical 4 Fig. ( empirically determined (a.f.u.), units fluorescence average Because for two of the three strains, there was no difference in the number number in the no difference was there strains, for of three two the Because For For For ), was used as the cutoff for identifying candidate dosage suppressors. dosage candidate identifying for cutoff the as used was ), cdc48-9 stu2-11 nse3-ts5 Individual 2 Individual , 10 barcode signals , were 10 signals greater barcode than the standard background. , , 24 were barcode signals greater than the standard background. , 6 barcode signals were greater than the standard background. background. were than the standard , greater signals 6 barcode 5 0 Fr ah ra, cmeiie yrdzto ws car was hybridization competitive a array, each For . ≥ 50,000 transformants were pooled and frozen in 15% and frozen were pooled transformants 50,000 µ MoBY-ORF plasmids were transformed into the the into transformed were MoBY-ORF plasmids 32,5 E. coli E. E. coli E. 1 , except , that except pool. Supplementary TableSupplementary 8 MoBY-ORF version 2.0 pool. Plasmid Plasmid pool. 2.0 version MoBY-ORF µ Chromosome loss rate measurements g ml g −1 Supplementary Table8 Supplementary HOM3 carbenicillin and 50 50 and carbenicillin was replaced with with was replaced Cells (50,000) rep (50,000) Cells doi:10.1038/nbt.1855 4 ) ) was transformed 9 . Supplementary Supplementary

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© 2011 Nature America, Inc. All rights reserved. 48. and type wild for times independent three out carried V. were chromosome on Measurements doi:10.1038/nbt.1855

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21 ,

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