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Caenorhabditis elegans Functional Genomics Sheldon McKay January 22, 2004 Introduction • C. elegans as a model organism • Functional genomics Gene knockout project • Goals • Methods • Progress Gene expression project • Goals • GFP-promoter fusions • SAGE C. elegans as a model organism • A 1mm long nematode worm • Short generation time and large numbers of progeny. • A metazoan with differentiated tissues and comprehensively studied anatomy and developmental program • Sequenced genome C. elegans genome • • • • ‘Essentially complete’ as of December 1998 Contains ~100 million bp on 6 chromosomes Predicted to contain ~20, 000 genes. ~ 55% of these genes are similar to genes from other organisms. • ~ 20% associated with mutationally defined genetic loci Genetics with a sequenced genome • Knowing the sequence isn’t everything. • We need to identify functions for as many uncharacterized genes as possible • Identification of a mutant phenotype in a gene of previously unknown function can help in assigning a function Forward vs. Reverse Genetics • In classical genetic analysis, we start with a mutant phenotype, genetically map the gene(s) responsible to chromosomal locations then, hopefully, find the gene and study the DNA sequence • Reverse genetics: We start with a DNA sequence believed to encode a gene. We then attempt to learn about the gene’s function through expression analysis and perturbations of its normal function with tools such as RNAi and mutational analysis C. elegans Functional Genomics Projects: Examples • Large-scale EST sequencing • ORFeome • Microarrays • RNAi • Gene Knockout Project C. elegans Gene Knockout Project Objectives: • Study gene function by determining the null phenotype • Make knockouts for all genes, with an emphasis on mammalian orthologs Approach: • Use PCR to detect, isolate and sequence deletion alleles PCR Screening for deletions Problems • The approach is biased towards large deletions • Sensitivity is low in complex populations • Targeting is imprecise The Poison Primer Technique 1o PCR 2o PCR NO PRODUCT The Poison Primer Technique 1o PCR 2o PCR Deletions recovered with poison primers Exon Intron Normal Deleted Average deletion size: 486 bp Precision knockout of a gene within a gene Exon Intron Normal Deleted C. elegans Gene Expression Project C. elegans Gene Expression Project Objectives: Build on the knockout project and other large-scale functional genomics projects Study gene expression patterns, with an emphasis on human orthologs Approaches: • High resolution image analysis of gene expression with GFP/Promoter fusions • Serial Analysis of Gene Expression (SAGE) and microarray analysis of gene expression of life stages and cell types Goals: Understand patterns of gene expression through the course of development and in particular cell types and tissues Identify known and novel cis-regulatory elements and their role in transcriptional regulation at the gene and network levels Understand gene expression patterns and protein interaction networks in the context of space and time in a developing organism Green Fluorescence Protein Fusion Studies of Gene Expression Isolating Potential Promoter Regions PCR primers Gene model SOCKEYE -- BCCA Genome Sciences Centre PCR-based Promoter GFP fusion (Hobert, Biotechniques 32:728-30) Promoter GFP Promoter GFP Neurons C13F10.4 -- contains similarity to Listeria monocytogenes Probable DNA-directed RNA polymerase delta subunit (RNAP deltasfactor).; SW:RPOE_LISMO Neurons C13F10.4 -- contains similarity to Listeria monocytogenes Probable DNA-directed RNA polymerase delta subunit (RNAP deltasfactor).; SW:RPOE_LISMO Neurons C13F10.4 -- contains similarity to Listeria monocytogenes Probable DNA-directed RNA polymerase delta subunit (RNAP deltasfactor).; SW:RPOE_LISMO protein of unknown function -- expressed in neurons A case-study of tissue-specific upstream regulatory elements M03F4.3 7-pass G-coupled transmembrane recepter Expressed in head, gut, vulva, tail Use PCR-stitching technique to dissect the Tissue-specific Control Elements Vulva, Tail Head, Gut Serial Analysis of Gene Expression SAGE Objectives: • Determine temporal and spatial gene expression patterns Approaches: • Construct life-stage and tissue specific SAGE libraries • Use GFP markers to isolate tissue specific cell populations via FACS SAGE: Procedure Digest with “Anchoring enzyme” NlaIII Isolate mRNA, RT to cDNA Digest with “Tagging enzyme” BsmFI Sequence Ligate tags http://www.sagenet.org/home/Description.htm SAGE Data Analysis • Tag abundance transcript abundance • Abundant tags = Abundant transcripts • Identify interesting tags, find out which genes they belong to Statistical analysis of tag frequencies DISCOVERYspace -- BCCA Genome Sciences Centre Mapping SAGE tags to genes NlaIII AAAAAAA Mapping SAGE tags to genes Many predicted genes are not confirmed by Expressed Sequence Tags (ESTs) and do not include 3’ Untraslated 3’-most regions (UTRs) CATG Predicted: 5’ UTR 3’ UTR Actual: 3’-most CATG The virtual transcriptome Predicted Gene Models EST Data Transcripts with EST support Genomic DNA Sequence No EST Data UTR length distribution Estimate UTR length For unconfirmed gene models Transcripts without EST support Conceptual mRNAs NlaIII 5’ AAAAAAAAAA 6 5 4 3 600000 2 1 Real 3’ UTR 500000 Position Tags 1 2 3 4 5 6 7 8 9 10 400000 300000 200000 Total Real Fake 615706 514979 100727 53897 18734 35163 21028 7578 13450 16070 7974 8096 20213 8968 11245 6756 2163 4593 6068 2777 3291 6309 3259 3050 4080 1711 2369 1245 566 679 Estimated 3’ UTR 100000 0 1 2 3 4 5 6 7 Position of NlaIII site 8 9 10 3’ NlaIII 2 Predicted Gene Models EST Data Transcripts with EST support Genomic DNA Sequence Experimental SAGE tags No EST Data UTR length distribution Map tags Transcripts without EST support Digest in silico Adjust 3’ UTRs Theoretical SAGE tags Adjust gene models Conceptual mRNAs Relating Gene Expression to Development Source: http://nema.cap.ed.ac.uk/Caenorhabditis/C_elegans_genome C. elegans Life Cycle Adult Embryo L4 L1 L3 L2 ~3.5 days SAGE data: embryo Sage_summary -- BCCA Genome Sciences Centre Tags/100K Developmental Series: Top 12 collagen genes egg larval adult old Stress Response and Ageing Adult Embryo L4 L1 dauer L3 L2 ~3.5 days daf-2 • Insulin-like growth factor receptor • Constitutive dauer formation at 25C; reversible by shift to 15C. • Increased lifespan at 20C; • Increased thermotolerance, UV resistance. • Most alleles hypersensitive to dauer pheromone. Transcribed Telomeric Sequence (tts-1) • Most abundant trasncript in a SAGE comparison of dauer larvae and a normal mixed population (Jones, et al, 2001) • corresponds to a previously unknown non-coding gene • implicated in dauer larvae induction or maintenance tts-1 expression patterns 2500 2000 dauer longevity 1500 starvation 1000 500 0 daf-2 Relating gene expression to anatomy Micro-dissection of gut tissue Gonad-less mutant (worm’s insides mostly gut) Nick the worm’s outer integument and the body contents are everted due to internal positive pressure Dissect gut away from the body Construct SAGE libraries from gut and whole worm mRNA Microdissected gut tissue Whole Worm vs. Gut 2500 Aspartyl protease 2000 Vitellogenin 1500 Cytochrome C oxidase 1000 500 0 Whole Gut Relating gene expression to organogenesis and early development Isolating specific cells types Identify GFP constructs expressed in the tissue/cell of interest • Make transgenic strain • Isolate eggs with NaOCl treatment • Digest with chitinase/trypsin • Disrupt embryos with physical shearing • Keep individual cells alive in culture • Sort via fluorescence activated cell sorter • Construct SAGE libraries GFP labeled embryonic intestinal cells DIC Image GFP Fluorescence Image The elt-2 promoter linked to GFP acts a reporter for developing embryonic gut cells. Strain kindly provided by Dr. Jim McGhee. FACS sorted embryonic intestinal cells DIC Image GFP Fluorescence Image We have used an embryonic intestinal promoter linked to GFP as a reporter for developing embryonic cells. These cells have been purified after FACS sorting. They can be plated and allowed to differentiate or used immediately for SAGE studies. Embryonic Gut Cells Whole Embryo vs. Isolated Cell Types 900 800 700 600 500 400 300 200 100 0 Whole Gut Muscle SAGE libraries Summary Stage Tissue Embryo 14 bp tags whole Embryo 21 bp tags whole L1 starved whole L1 normal whole L2 whole L3 whole L4 whole Young adult whole 6 day Adult(fer-15) whole 1 day Adult(fer-15;daf-2) whole 6 day Adult (fer-15;daf-2) whole 10 day Adult(fer-15;daf-2) whole Adult (glp-4) dissected gut 138346 Adult (glp-4) whole Embryo (myo-3::GFP) FACS sorted gut Embryo (myo-3::GFP) FACS sorted muscle Mixed stage whole Dauer larvae whole Meta library (14 bp tags) Meta library (21 bp tags) Tag Total 133825 220032 116363 109994 130209 127924 141878 119222 110306 101939 100737 116336 14386 117529 81393 58147 175995 65828 1806431 359572 Genes Unique 25885 44992 19494 17532 24658 24039 25701 23128 19861 16960 14004 19183 4892 19140 19649 16967 37894 18136 130112 62893 8187 8929 6429 6705 7264 7667 8046 6302 6758 5159 4687 5594 6974 6069 4850 9222 5373 14661 9887 Ongoing projects: • SAGE/Affy Transcription profiling of: – developmental stages – ageing – tissue differentiation • SAGE-based genome annotation – gene discovery – alternative splicing – 5’ SAGE • Gene expression clustering • Cis-regulatory element discovery Research Team University of British Columbia Don Moerman Heidi Kai Adam Warner Erin Halfnight Rebecca Newbury Nicholas Dube Francis Ouellette Simon Fraser University David Baillie Robert Johnsen Lily Fang Emily Ha Allan Mah Domena Tu John Tyson Zhongying Zhao Martin Jones Kathleen Huang Robert Hollebakken Dion Li Victor Jensen BCCA Genome Sciences Centre Sheldon McKay Peter Huang Kim Wong Courtney Mills Igor Ostrovsky Peter Ruzanov Scott Zuyderduyn Richard Varhol Erin Pleasance Greg Vatcher Steven Jones Marco Marra Jaswinder Khattra Jennifer Asano Susanna Chan Shaun Coughlin Noreen Girn Helen McDonald Pawan Pandoh Rob Holt George Yang Jeff Stott University of Calgary Jim McGhee UBC/Genome BC Don Riddle