1. Genome Biology and Biotechnology 7. The phenome Prof. M. Zabeau Department of Plant Systems Biology Flanders Interuniversity Institute for Biotechnology (VIB) University of Gent International course 2005

2. Functional Maps or “-omes” proteins ORFeome Localizome Phenome Transcriptome Interactome Proteome Genes or proteins Genes Mutational phenotypes Expression profiles Protein interactions 1 2 3 4 5 n DNA InteractomeProtein-DNA interactions “Conditions” After: Vidal M., Cell, 104, 333 (2001)Vidal M., Cell, 104, 333 (2001) Cellular, tissue location

3. The phenome: genome-wide phenotypic analysis ¤Classical (forward) genetic screens –Saturated mutagenesis to identify all the genes that exhibit a specific phenotype –Draw back characterization of the gene through positional cloning is slow and laborious ¤Phenomics platforms: Reverse genetics –Systematic alteration of gene function to identify the functions of predicted genes –Advantage Identity of the gene is known beforehand ¤Phenomics platforms Transposon-based mutant libraries –Extensively used in yeast and Arabidopsis RNA interference (RNAi)-based mutant libraries –the technology of choice for gene knock-outs

4. Large-scale analysis of the yeast genome by transposon tagging and gene disruption ¤Paper presents –a transposon-tagging strategy to perform large-scale analysis of gene function in yeast to simultaneously study phenotypes gene expression protein localization –a large collection (>11,000 strains) of yeast mutants carrying a transposon inserted in genes Tagged 30% of all yeast genes Ross-Macdonald et al., Nature 402: 413 (1999)

5. Transposon-based Method for the Large-scale Functional Genomics ¤Minitransposon (mTn) –Derived from the bacterial transposable element Tn3 –LacZ reporter gene lacking an initiator methionine and upstream promoter sequence  -galactosidase (  -gal) is produced when lacz is fused in-frame to the protein-coding sequence –Haemaglutinin (3xHA) epitope tag Recombination of the lox sites produces epitope tagged proteins Reprinted from: Ross-Macdonald et al., Nature 402: 413 (1999) No ATG: gene fusions Haemaglutinin tag

6. Minitransposon mTn–3xHA/lacZ Gene-lacZ fusion protein Cre-mediated recobination Gene-3xHA fusion protein

7. High Throughput Insertion Mutagenesis ¤Yeast genomic DNA library –mutagenized with mTn –plasmids were digested with Not I –transformed into a diploid yeast strain –Integrated by homologous recombination –Transformants were assayed for  -gal activity Reprinted from: Ross-Macdonald et al., Nature 402: 413 (1999)

8. Analysis of the MTn Insertion Strains ¤Identified 11,232 strains expressing lacZ ¤Sequenced the site of insertion in 6,358 strains –5,442 in or within 200 bp of an annotated ORF Insertions affect 1,917 different ORFs (~30%) ¤Identified 328 previously non-annotated ORFs –52% overlap an ORF in the antisense direction –33% are in intergenic regions - small ORFs –15% overlap an ORF in the same orientation in a different frame –In the annotation genes are missed because of Arbitrary lower size limit of 100 amino acids Not annotating partially overlapping ORFs Reprinted from: Ross-Macdonald et al., Nature 402: 413 (1999)

9. Analysis of Mutant Phenotypes ¤Phenotypes of essential genes –14.1% of the insertions are non viable in haploid strains Represent genes that are essential for viability ¤Large scale scoring of “other” phenotypes –growth under 20 different growth conditions 'phenotypic macroarrays' (96-well format) –Insertions in 407 genes (20%) result in a phenotype different from the wild type ¤The majority (80%) of the insertions exhibit no phenotype! –Expand the range of phenotypic assays –Utilize more precise criteria for phenotypic analysis Growth rate Reprinted from: Ross-Macdonald et al., Nature 402: 413 (1999)

10. Phenotypic Macroarray Analysis of Yeast Mutants Reprinted from: Ross-Macdonald et al., Nature 402: 413 (1999) mutants deficient in oxidative phosphorylation mutants deficient in cell-wall maintenance

11. Genomic Scale Analysis of Phenotypes ¤Phenotypes observed –Expected phenotypes genes involved in microtubule functions - sensitive to benomyl –Unexpected phenotypes Genes involved in cell wall biogenesis - stress-related responses –Pleiotropic phenotypes: observed in apparently unrelated assays Sensitivity to hydroxyurea, benomyl and calcofluor ¤Pleitrophic mutants are the rule –Many mutants exhibit phenotypes in specific subsets of conditions ¤Mutants appear to ‘group' into discrete classes –“pheno-clusters” represent groups of mutants having common disruption phenotypes Reprinted from: Ross-Macdonald et al., Nature 402: 413 (1999)

12. Cluster Analysis of the Phenotypic Data Reprinted from: Ross-Macdonald et al., Nature 402: 413 (1999) Transformants sorted by increasing distance from the cluster average Growth conditions

13. Cluster Analysis of the Phenotypic Data ¤Pheno-clusters –predict the cellular functions associated with an ORF 'YPG' cluster: mutants that do not grow on glycerol –Cluster highly enriched in genes involved in cellular respiration –predict the function of uncharacterized genes “Guilt by association” ¤Assay-clusters –‘Two-dimensional cluster' analysis of the data groups phenotypic assays identifying strains exhibiting similar phenotypic profiles –Assays for growth in hydroxyurea and MMS are closely associated identify mutants defective in DNA metabolism Reprinted from: Ross-Macdonald et al., Nature 402: 413 (1999)

14. Analysis of Subcellular Localization of Proteins ¤HAT-epitope tagged proteins –sub cellular localization –Immunofluorescence with antibodies against the HAT- epitope ¤Analysis of 1,340 strains –201 proteins localized in cellular compartments nucleus, nucleolus, mitochondria, plasma membrane, cell neck and spindle pole body –214 proteins localized in the cytoplasm cytoplasm actin filaments plasma membrane Immuno fluorescence DAPI Reprinted from: Ross-Macdonald et al., Nature 402: 413 (1999)

15. Conclusions ¤Insertion strategy generates in a single mutagenic event –reporter gene fusions –epitope-tagging constructs –insertion alleles ¤Random approaches are intrinsically limited –in achieving saturation mutagenesis Small genes are less likely to be mutagenized than are large genes to mutagenize 90% of the yeast genes an additional 30,000 mTn insertions in yeast ORFs would be required –This amounts to a 5 to 10 fold redundancy –For multicellular organisms collections of 100.000 to 250.000 insertions are needed Reprinted from: Ross-Macdonald et al., Nature 402: 413 (1999)

16. RNA Interference (RNAi) ¤Phenomenon first discovered in transgenic plants –“anti-sense mediated gene silencing” Anti-sense constructs reduce the expression of the cognate gene –“co-suppresion” Enhanced gene expression constructs occasionally lead to reduced gene expression ¤“related” phenomena were later found in C. elegans –Small temporal RNAs (stRNAs) responsible for the control of gene expression during development –stRNAs contain sequences complementary to specific target mRNAs ¤Broader significance of RNA-mediated gene regulation became apparent in recent years

17. RNA-mediated Gene Regulation ¤Small regulatory RNAs are involved in two pathways for RNA- mediated gene regulation: –micro RNA pathway (miRNAs) responsible for the control of gene expression during development –miRNAs contain sequences complementary to specific target mRNAs – specific silencing of one or more target genes –Short interfering RNA pathway (siRNAs) responsible for gene silencing by RNA interference (RNAi) –dsRNA triggers destruction of a homologous mRNA that has the same sequence as one of the dsRNA strands guide DNA modifying (methylating) enzymes to corresponding genomic regions –converting these regions to heterochromatin

18. RNA-mediated Gene Regulation Pathways Reprinted from: Ambros V., Science, 293, 811 (2001) micro RNA pathway short interfering RNA pathway 21-23bp dsRNA22bp dsRNA Heterochromatin

19. RNA-mediated Gene Regulation ¤RNA-mediated gene regulation is ancient in origin –Evolved before the divergence of plants and animals –Two pathways are interconnected and share molecular components Highly conserved nuclease Dicer Small dsRNAs about 21 to 23 nucleotides in length –RNA Interference (RNAi) is thought to be a primitive genetic surveillance mechanism that protects cells from viruses ¤RNAi is well suited for large scale gene knockout –First pioneered in C. elegans –Now used in all model organisms

20. RNA Interference (RNAi) in C. Elegans ¤Injection of anti-sense or double stranded RNA into cells –can be used to interfere with the function of endogenous genes –results in silencing of the corresponding gene ¤The RNA interference process involves –a catalytic or amplification component Only a few molecules of injected dsRNA are required –injection of dsRNA into the extracellular body cavity in C. Elegans, results in silencing in the whole animal ¤Experimentally, gene silencing is achieved in nematodes –Feeding worms E. coli expressing dsRNAs

21. RNA Interference (RNAi) in C. Elegans ¤dsRNA is expressed in E. coli by –bi-directional transcription by phage T7 RNA polymerase Reprinted from: Timmons et al., Nature 395: 854 (1998) T7 promoter Open Reading Frame Feeding on wt E.coli Feeding on E.coli expressing ds GFP RNA

22. Functional Genomic Analysis of C. Elegans Chromosome I by Systematic RNAi ¤Paper reviews/presents –RNAi approach to systematically investigate loss-of-function phenotypes of predicted genes of C. Elegans chromosome I –by feeding worms with E. coli bacteria that express double-stranded RNA –Demonstrates that high-throughput genome-wide RNAi screens can be performed using a library of dsRNA-expressing bacteria The specificity of RNAi make it an ideal tool for investigating gene function Fraser et al., Nature 408: 325 (2000)

23. Functional Analysis of Chromosome I Genes ¤Constructed a library of E.coli expressing dsRNA for –the predicted genes on chromosome I 2,416 predicted genes (87.3% of the predicted genes) ¤Screened the library for detectable phenotypes –L3–L4 stage worms were were fed for 72 h at 15 °C on bacterial cultures for each targeted gene –Phenotypes of adults and progeny were scored Embryonic lethal (Emb) –10–100% embryonic lethality Sterile (Ste) –brood size of 50) Progeny sterile (Stp) –brood size of <= to 10 in the progeny of fed worms Reprinted from: Fraser et al., Nature 408: 325 (2000)

24. Functional Analysis of Chromosome I Genes ¤Assigned a phenotype to 13.9% of the genes –Confirmed 90% of the known embryonic lethal genes –number of genes with known phenotypes increased from 70 to 378 –Not all genes give a RNAi phenotype Did not find phenotypes for some previously characterized genes –genes involved in neuronal function ¤Highly conserved genes are more likely to have an RNAi phenotype than genes that show no conservation –>72% of genes with an RNAi phenotype have a Drosophila match Reprinted from: Fraser et al., Nature 408: 325 (2000)

25. Functional Analysis of Chromosome I Genes ¤Embryonic lethal (Emb) mutants: essential genes –genes involved in the basal cellular machinery: RNA-binding proteins, chromosome condensation and separation, components of signal transduction pathways –genes involved in basic metabolic processes –largest class: >60% of the mutants ¤Uncoordinated and post-embryonic mutants –High proportion (30% to 40%) of genes of unknown function genes that regulate the development are still largely unknown Reprinted from: Fraser et al., Nature 408: 325 (2000)

26. Biochemical Function and RNAi Phenotype Reprinted from: Fraser et al., Nature 408: 325 (2000)

27. Toward Improving Caenorhabditis elegans Phenome Mapping With an ORFeome-Based RNAi Library ¤Paper presents –the use of the C. elegans ORFeome as a starting point for high throughput RNAi with enhanced flexibility increasing the possibilities for phenome mapping in C. elegans –additional HT-RNAi libraries can be generated to perform gene knockdowns under various conditions Rual et. al., Genome Research 14:2162-2168(2004)

28. Generating RNAi resources from flexible Gateway ORFeome and promoterome collections Reprinted from: Rual et. al., Genome Research 14:2162-2168(2004)

29. Screening the ORFeome-RNAi v1.1 Library ¤The C. elegans ORFeome v1.1 library –contains 11,942 ORFs cloned as Gateway Entry clones –ORFs were transferred into the RNAi Destination vector (T 7 promoter vector) ¤Genome-Wide Phenotypic Analysis –RNAi-by-feeding at the first larval stage –observed phenotypes for 1066 (10%) of the ORFs tested Reprinted from: Rual et. al., Genome Research 14:2162-2168(2004)

30. Genome-Wide RNAi Analysis of Growth and Viability in Drosophila Cells ¤Paper presents –a high-throughput RNA-interference (RNAi) screen of nearly all (91%) predicted Drosophila genes –Using in Drosophila cultured cells to characterize genes in cell growth and viability Treatment of cells with dsRNA leads to detect specific phenotypes Systematic screen for loss-of-function phenotypes Genome-wide RNAi performed on two embryonic cell lines –Established a quantitative assay of cell death: z-score Boutros et. al., Science, 303, 832-835(2004)

31. Genome-wide RNAi screen for viability defects Reprinted from: Boutros et. al., Science, 303, 832-835(2004)

32. Distribution of the frequency of RNAi phenotypes ¤438 dsRNAs (3%) resulted in significantly reduced cell number –with a z score of 3 or more Reprinted from: Boutros et. al., Science, 303, 832-835(2004)

33. Pheno clusters of quantitative RNAi phenotypes Reprinted from: Boutros et. al., Science, 303, 832-835(2004)

34. Genome-wide RNAi screening in Arabidopsis ¤The Arabidopsis GST Entry clone resource was used to –Generate a library of hairpin RNA (hpRNA) expression plasmids Large scale transformation of Arabidopsis Reprinted from: Hilson et. al., Genome Research 14:2176-2189 (2004) GST hairpin RNA expression constructs

35. Phenotypes of plants carrying a GST hpRNA transgene targeting a subunit of cellulose synthase Reprinted from: Hilson et. al., Genome Research 14:2176-2189 (2004)

36. Phenotypes of plants carrying a GST hpRNA transgene targeting a H+-ATPase subunit Reprinted from: Hilson et. al., Genome Research 14:2176-2189 (2004)

37. Conclusions ¤The function of 10 to 20% of the genes is identified by insertional mutagenesis and RNAi –Expect that the detection of phenotypes for other genes will require alternative approaches different growth conditions, for example, environmental stress in other genetic backgrounds ¤Reverse and forward genetics are complementary –Reverse genetics Has the advantage of being high throughput and non-redundant Mutant phenotype is automatically connected to a known sequence –Classical forward genetics Has the disadvantage that positional cloning is slow and laborious Some genes are resistant to RNAi, while all genes are sensitive to mutagens Can also yield gain-of-function mutations

38. Recommended reading ¤RNA interference (RNAi) –The World of Small RNAs Zamore and Haley, Science 309: 1519-1524 (2005) –Functional Genomic Analysis of C. Elegans by Systematic RNAi Fraser et al., Nature 408: 325 (2000)

39. Further reading ¤Phenome analysis –Transposon-based mutant libraries Ross-Macdonald et al., Nature 402: 413 (1999) –RNAi in C.elegans Rual et. al., Genome Research 14:2162-2168(2004) –RNAi in Drosophila Boutros et. al., Science, 303, 832-835(2004)Boutros et. al., Science, 303, 832-835(2004)