1. Caenorhabditis elegans Functional Genomics Sheldon McKay January 22, 2004

2. Introduction C. elegans as a model organism Functional genomics Gene knockout project Goals Methods Progress Gene expression project Goals GFP-promoter fusions SAGE

3. 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

5. 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

6. 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

7. 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

8. C. elegans Functional Genomics Projects: Examples Large-scale EST sequencing ORFeome Microarrays RNAi Gene Knockout Project

9. 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

10. PCR Screening for deletions

11. Problems The approach is biased towards large deletions Sensitivity is low in complex populations Targeting is imprecise

12. The Poison Primer Technique 1 o PCR 2 o PCR NO PRODUCT

13. The Poison Primer Technique 1 o PCR 2 o PCR

14. Deletions recovered with poison primers Average deletion size: 486 bp Exon Intron Normal Deleted

15. Precision knockout of a gene within a gene Exon Intron Normal Deleted

16. C. elegans Gene Expression Project

17. Objectives: Build on the knockout project and other large-scale functional genomics projects Study gene expression patterns, with an emphasis on human orthologs

18. 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

19. 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

20. Green Fluorescence Protein Fusion Studies of Gene Expression

21. Isolating Potential Promoter Regions PCR primers Gene model SOCKEYE -- BCCA Genome Sciences Centre

22. PCR-based Promoter GFP fusion (Hobert, Biotechniques 32:728-30) GFP Promoter GFP Promoter

23. Neurons C13F10.4 -- contains similarity to Listeria monocytogenes Probable DNA-directed RNA polymerase delta subunit (RNAP deltasfactor).; SW:RPOE_LISMO

24. Neurons C13F10.4 -- contains similarity to Listeria monocytogenes Probable DNA-directed RNA polymerase delta subunit (RNAP deltasfactor).; SW:RPOE_LISMO

26. 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

27. A case-study of tissue-specific upstream regulatory elements

28. M03F4.3 7-pass G-coupled transmembrane recepter Expressed in head, gut, vulva, tail Use PCR-stitching technique to dissect the putative promoter region

29. Head, GutVulva, Tail Tissue-specific Control Elements

30. Serial Analysis of Gene Expression

31. 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

32. Ligate tags SAGE: Procedure Digest with “Tagging enzyme” BsmFI http://www.sagenet.org/home/Description.htm Isolate mRNA, RT to cDNA Digest with “Anchoring enzyme” NlaIII Sequence

33. SAGE Data Analysis Tag abundance  transcript abundance Abundant tags = Abundant transcripts Identify interesting tags, find out which genes they belong to

34. Statistical analysis of tag frequencies DISCOVERYspace -- BCCA Genome Sciences Centre

35. Mapping SAGE tags to genes AAAAAAA NlaIII

36. Mapping SAGE tags to genes Many predicted genes are not confirmed by Expressed Sequence Tags (ESTs) and do not include 3’ Untraslated regions (UTRs) 5’ UTR3’ UTR Predicted: Actual: 3’-most CATG

37. The virtual transcriptome

38. EST DataNo EST Data Transcripts with EST support Transcripts without EST support Conceptual mRNAs Predicted Gene ModelsGenomic DNA Sequence UTR length distribution Estimate UTR length For unconfirmed gene models

39. Tags Position of NlaIII site NlaIII 543216 5’ AAAAAAAAAA 3’ Real 3’ UTR Estimated 3’ UTR

40. NlaIII 2

41. Experimental SAGE tags EST DataNo EST Data Transcripts with EST support Transcripts without EST support Conceptual mRNAs Map tags Predicted Gene ModelsGenomic DNA Sequence Theoretical SAGE tags Digest in silico Adjust 3’ UTRs UTR length distribution Adjust gene models

42. Source: http://nema.cap.ed.ac.uk/Caenorhabditis/C_elegans_genome Relating Gene Expression to Development

43. Embryo L1 L2L3 L4 Adult C. elegans Life Cycle ~3.5 days

44. SAGE data: embryo Sage_summary -- BCCA Genome Sciences Centre

45. Developmental Series: Top 12 collagen genes egg adult Tags/100K old larval

46. Embryo L1 L2L3 L4 Adult dauer ~3.5 days Stress Response and Ageing

47. 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.

48. 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

49. Starved L1 larvae Normal L1 larvae Dauer larvae Mixed population 1-day adult6-day adult1-day adult6-day adult 10-day adult tts-1 expression patterns daf-2 starvation dauer longevity

50. Relating gene expression to anatomy

51. 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

52. Microdissected gut tissue

53. Aspartyl protease Vitellogenin Cytochrome C oxidase Whole Worm vs. Gut

54. Relating gene expression to organogenesis and early development

55. 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

56. GFP labeled embryonic intestinal cells DIC ImageGFP Fluorescence Image The elt-2 promoter linked to GFP acts a reporter for developing embryonic gut cells. Strain kindly provided by Dr. Jim McGhee.

57. FACS sorted embryonic intestinal cells 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. DIC ImageGFP Fluorescence Image

58. Embryonic Gut Cells

59. Whole Embryo vs. Isolated Cell Types

60. Stage Tissue Tag Genes TotalUnique Embryo 14 bp tags whole13382525885 8187 Embryo 21 bp tags whole22003244992 8929 L1 starved whole11636319494 6429 L1 normal whole10999417532 6705 L2 whole13020924658 7264 L3 whole12792424039 7667 L4 whole14187825701 8046 Young adult whole11922223128 6302 6 day Adult (fer-15) whole11030619861 6758 1 day Adult (fer-15;daf-2) whole10193916960 5159 6 day Adult (fer-15;daf-2) whole10073714004 4687 10 day Adult (fer-15;daf-2) whole11633619183 5594 Adult (glp-4) dissected gut13834614386 4892 Adult (glp-4) whole11752919140 6974 Embryo (myo-3::GFP) FACS sorted gut8139319649 6069 Embryo (myo-3::GFP) FACS sorted muscle5814716967 4850 Mixed stage whole175995 37894 9222 Dauer larvae whole6582818136 5373 Meta library (14 bp tags)1806431130112 14661 Meta library (21 bp tags) 35957262893 9887 SAGE libraries Summary

61. 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

62. Sheldon McKay Peter Huang Kim Wong Courtney Mills Igor Ostrovsky Peter Ruzanov Scott Zuyderduyn Richard Varhol Erin Pleasance Greg Vatcher Steven Jones David Baillie Robert Johnsen Lily Fang Emily Ha Allan Mah Domena Tu Don Moerman Heidi Kai Adam Warner Erin Halfnight Rebecca Newbury Nicholas Dube Francis Ouellette Marco Marra Jaswinder Khattra Jennifer Asano Susanna Chan Shaun Coughlin Noreen Girn Helen McDonald Pawan Pandoh Rob Holt George Yang Jeff Stott Simon Fraser University BCCA Genome Sciences Centre University of British Columbia University of Calgary Jim McGhee UBC/Genome BC Don Riddle John Tyson Zhongying Zhao Martin Jones Kathleen Huang Robert Hollebakken Dion Li Victor Jensen Research Team