Genome Biology and Biotechnology Functional Genomics Prof M

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

Functional Genomics – the Paradigm Shift ¤ Large-scale genome sequencing generates – “parts lists” • complete inventories of genes and functional elements – A new challenge • to understand the function of the many genes predicted • In general 90% to 95% of the genes have unknown functions ¤ Genome sequencing has triggered a transition – from vertical (reductionist) approaches – to horizontal (large scale) approaches – Each approach has its own strengths and weaknesses Reprinted from: Vidal M. , Cell, 104, 333 (2001)

Vertical Versus Horizontal Approach Reprinted from: Vidal M. , Cell, 104, 333 (2001)

The vertical approach ¤ The vertical or reductionist approach – Studies one or a few proteins or genes at a time by applying different experimental tools to test hypotheses • Well proven by decades of research – The reductionist approach is based on the principle of • “understanding the whole by studying selected parts” ¤ The reductionist approach has severe limitations – lacks efficiency • In well-studied model organisms decades of hypothesis driven research has discovered only 5 to 10% of the genes – Fails to give a comprehensive picture of biology • The study of Gal 4 p provided a useful model of how transcription factors work but • gives no insight in global transcriptional responses

The horizontal Approach ¤ The horizontal or large scale approach – studies large numbers of genes or proteins in parallel using • high-throughput tools – Microarrays, systematic gene knock outs… • Instrumentation for automated and high-throughput analysis – Robots: automated liquid handlers – Automated data acquisition instruments: e. g. sequencers – Well suited for massively parallel studies ¤ Large scale approach is limited – lack of giving conclusive evidence • Noisy data with high rates of false positives or negatives • Observations must to be confirmed

Functional gene maps ¤ Functional genomics can be regarded as – functional mapping within two-dimensional matrices • One axis corresponds to all genes of an organism • The other axis represents a set of conditions to which the organism is exposed – Experimental conditions, various mutant backgrounds ¤ Each “omics” approach represents a different map Genes 1 2 3 4 5 “Conditions” Omics n

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

The basic rationale of functional genomics ¤ Functionally related genes share common properties – Are likely to be coregulated at the transcriptional level • Transcriptome maps consist of ''expression clusters'' of coregulated genes – Loss-of-function mutations should confer similar or opposite phenotypes • Phenome maps consist of sets of genes giving similar phenotypes or ''pheno-clusters'' – Their protein products are likely to interact physically • Interactome maps consist of networks of interacting proteins ''interaction clusters'' – Their protein products are likely to localize in similar cellular compartments • Have similar location in the localisome

Integration of Functional Maps ¤ Functional maps provide a rough indication of gene function – Integration of functional maps in a biological atlas overcomes this limitation by • overlaying sets of functional characteristics Reprinted from: Vidal M. , Cell, 104, 333 (2001)

Functional Genomics ¤ Functional Genomics provides the tools for – Identifying the function of “all genes” • overlaying sets of functional characteristics – Functional maps provide lists of clusters that contain both characterized and uncharacterized genes • Provides hypotheses for the function of uncharacterized genes ¤ Functional Genomics provides approaches for – the ultimate understanding of life at the molecular level based on the description of • Each protein individually and • The interactions between the proteins involved in particular biological processes

Genome Biology and Biotechnology 6. The ORFeome International course 2005

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

The ORFeome: Genes in the Genome ¤ The genome represents – the basic compendium of “all genes” that make up an organism ¤ The ORFeome represents – the basic compendium of all protein coding genes as defined by their Open Reading Frames (ORFs) – Predicted ORFs must be validated • In higher organisms gene identification is complicated by – Intron / exon structure ¤ The ORFeome platforms provide – Large scale approach for validating predicted genes • high throughput recombinational cloning technology – Resources for functional genomics projects

Recombinational Cloning ¤ One step cloning technology – Site specific recombination instead of restriction/ligation • Not dependent on availability of restriction sites – “ 100%” efficient: only one recombinant DNA product without byproducts • No cloning step needed: no need to assay independent clones – • Fully automatable – simple pipetting in microtiter plates – Very precise recombination system • allowing high fidelity DNA engineering ¤ Versatile cloning technology – Genes can be easily transferred into a range of vector systems • Expression, Gene fusion, RNAi… ¤ GATEWAY Recombinational Cloning – Based on the bacterio phage lambda integration & excision system Reprinted from: Walhout et al, Science 287: 116 (2000)

Phage lambda integration & excision system phage att. B att. P Bacterial genome Integration Excision att. B att. L att. R

Recombinational Cloning of ORFs Designer oligo start att. B 1 ORF c. DNA stop att. B 2 PCR att. B 1 att. B 2 ORF Phage lambda integration: Integrase & bacterial IHF TG Entry Vector TG - Toxic gene Reprinted from: Walhout et al, Science 287: 116 (2000)

Recombinational Cloning of ORFs att. P 1 att. B 1 att. P 2 att. B 2 Phage lambda integration: Integrase & bacterial IHF att. L 1 att. L 2 Reprinted from: Walhout et al, Science 287: 116 (2000)

Recombinational Cloning of gene Fusions Destination vector Entry clone att. L DNA binding domain Destination vector att. R Activation domain Phage lambda excision: Integrase, IHF & Exisionase Reprinted from: Walhout et al, Science 287: 116 (2000)

GATEWAY Recombinational Cloning ¤ First generation cloning technology – DNA Cloning Using In Vitro Site-Specific Recombination • Hartley et. al. , Genome Research 10, 1788 -1795 (2000) – Designed for large scale cloning of ORFs • High throughput platform for generating ORFeome libraries ¤ Second generation technology – Concerted Assembly and Cloning of Multiple DNA Segments Using In Vitro Site-Specific Recombination • Cheo et. al. , Genome Research 14: 2111 -2120 (2004) – Designed for large scale production of multi-segment expression clones

Second generation att sites and BP cloning Synthetic att. B and att. P sites Int cut site BP cloning Left arms Right arms 4 simultaneous reactions Reprinted from: Cheo et. al. , Genome Research 14: 2111 -2120 (2004)

Multi-segment recombination cloning Two-segment cloning Three-segment cloning Reprinted from: Cheo et. al. , Genome Research 14: 2111 -2120 (2004)

Multi-Segment Expression Clones ¤ The expanded repertoire of recombination sites for – Concerted cloning of multiple DNA segments in a predefined order, orientation, and reading frame • Generates collections of functional elements in a combinatorial fashion ¤ Applications – linkage of promoters to genes – generation of fusion proteins – assembly of multiple protein domains ¤ The technology has broad implications for – gene function analysis. – expression of multidomain proteins Reprinted from: Cheo et. al. , Genome Research 14: 2111 -2120 (2004)

The ORFeome of C. elegans version 1. 0 ¤ ORFeome cloning was first demonstrated in C. Elegans. – Predicted ORFs are amplified by PCR from a highly representative c. DNA lib • ORF-specific primers – Cloned by GATEWAY recombination cloning ¤ The C. elegans genome sequence predicted 18, 959 ORFs 9. 503 identified genes 9. 888 Untouched genes Reprinted from: Reboul et. al. , Nat. Genet. 27, 332 (2001)

PCR amplification of C. elegans ORFs PCR products of identified genes PCR products of Untouched genes Reprinted from: Reboul et. al. , Nat. Genet. 27, 332 (2001)

Successful PCR for the ORFs analyzed Reprinted from: Reboul et. al. , Nat. Genet. 27, 332 (2001)

Conclusions ¤ ORFeome strategy provides experimental evidence for – structure of genes in C. elegans ¤ ORFeome resource for large scale functional genomics version v 1. 1 – Attempted PCR amplification of the 19, 477 ORFs • cloned 10, 623 (55%) in-frame ORFs – ORF Sequence Tags improved C. elegans gene annotations • corrected the internal gene structure of 20% of the ORFs. Reprinted from: Reboul et. al. , Nat. Genet. 27, 332 (2001)

C. elegans ORFeome Version 3. 1 Gene prediction improvements Classification of the 4232 repredicted and new ORFs Reprinted from: Lamesch et. al. , Genome Research 14: 2064 -2069 (2004)

The C. elegans ORFeome is an evolving resource Reprinted from: Lamesch et. al. , Genome Research 14: 2064 -2069 (2004)

Conclusions ¤ Cloning of a complete ORFeome is an iterative process – requires multiple rounds of experimental validation together with gradually improving gene predictions (bioinformatics) – the ORFeome resource provides further verification of the predicted gene structures • Note that the procedure will not reveal alternatively spliced transcripts unless GATEWAY clones are cloned individually ¤ ORFeome projects now underway – Human – Arabidopsis – Drosophila Reprinted from: Lamesch et. al. , Genome Research 14: 2064 -2069 (2004)

Versatile Gene-Specific Sequence Tags for Arabidopsis Functional Genomics Hilson et. al. , Genome Research 14: 2176 -2189 (2004) ¤ Paper presents – The creation of a collection of gene-specific sequence tags (GSTs) • representing 21, 500 Arabidopsis genes ¤ Gene-specific sequence tags (GST) – Correspond to short (150 bp to 500 bp) segments of ORFs • selected to have no significant similarity with any other region in the genome – Synthesized by PCR amplification from genomic DNA – The GSTs provide a resource for large-scale gene function studies in multicellular eukaryotes • RNA interference • Microarray transcript profiling

Graphical representation of GSTs GST Predicted gene Reprinted from: Hilson et. al. , Genome Research 14: 2176 -2189 (2004)

GST production High throughput PCR High throughput verification Reprinted from: Hilson et. al. , Genome Research 14: 2176 -2189 (2004)

GST cloning in GATEWAY vectors Reprinted from: Hilson et. al. , Genome Research 14: 2176 -2189 (2004)

The Caenorhabditis elegans Promoterome Dupuy et. al. , Genome Research 14: 2169 -2175 (2004) ¤ Paper presents – The development of a genome-wide resource of C. elegans promoters • characterize the expression patterns of all predicted genes • expressing localization markers such as the green fluorescent protein (GFP). – "localizome" maps should provide information on • where (in what cells or tissues) genes are expressed • when (at what stage of development or under what conditions) genes are expressed • in what cellular compartments the corresponding proteins are localized

The C. elegans promoterome ¤ "promoters" correspond to upstream intergenic regions (IGR) – region from the ATG of the ORF to the end of the preceding ORF – PCR fragment upper size limit of 2 kb to ensure high cloning efficiency ORF Reprinted from: Dupuy et. al. , Genome Research 14: 2169 -2175 (2004)

Overview of promoterome cloning procedure analysis of PCR products large-scale cloning of the promoterome Reprinted from: Dupuy et. al. , Genome Research 14: 2169 -2175 (2004)

Conclusion ¤ Promoterome version 1. 1 – Resource of 6000 C. elegans promoters – cloned in the Multi. Site Gateway system ¤ Promoterome constitutes an equally valuable resource as the ORFeome – Promoters can be easily transferred into Gateway Destination vectors to drive expression of • markers such as GFP (promoter: : GFP constructs) • GFP fusion with ORFs available in ORFeome resources (promoter: : ORF: : GFP constructs) Reprinted from: Dupuy et. al. , Genome Research 14: 2169 -2175 (2004)

Applications of recombinational cloning Reprinted from: Dupuy et. al. , Genome Research 14: 2169 -2175 (2004)

Recommended reading ¤ Functional genomics – The concept of a biological atlas • Vidal M. , Cell, 104, 333 (2001) ¤ ORFeome resource and analysis – The ORFeome of C. elegans • Reboul et. al. , Nat. Genet. 27, 332 (2001) – The ORFeome of Arabidopsis • Hilson et. al. , Genome Research 14: 2176 -2189 (2004)

Further reading ¤ ORFeome analysis – GATEWAY Recombinational Cloning • Hartley et. al. , Genome Research 10, 1788 -1795 (2000) • Cheo et. al. , Genome Research 14: 2111 -2120 (2004) • Walhout et al, Science 287: 116 (2000) – C. elegans ORFeome • Lamesch et. al. , Genome Research 14: 2064 -2069 (2004) – C. elegans Promoterome • Dupuy et. al. , Genome Research 14: 2169 -2175 (2004)