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A SAGE approach to discovery of genes involved in autophagic cell death

CATGGCGATATTGT CATGGCGCCAATAT CATGGCGCGCATTT CATGGCGTGGGGAT CATGGCTAATAAAT CATGGCTCAAGGAG CATGGCTGGACTCC CATGGCTGTGGCCA CATGGCTTTCGTGT CATGGCTTTTTGGC CATGGGAACCGACA CATGGGACCGCCCC CATGGGACCGCTCA CATGGGATCACAAT CATGGGCAACGATC CATGGGCAGCAAGC CATGGGCAGCAATT.

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A SAGE approach to discovery of genes involved in autophagic cell death

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  1. CATGGCGATATTGT CATGGCGCCAATAT CATGGCGCGCATTT CATGGCGTGGGGAT CATGGCTAATAAAT CATGGCTCAAGGAG CATGGCTGGACTCC CATGGCTGTGGCCA CATGGCTTTCGTGT CATGGCTTTTTGGC CATGGGAACCGACA CATGGGACCGCCCC CATGGGACCGCTCA CATGGGATCACAAT CATGGGCAACGATC CATGGGCAGCAAGC CATGGGCAGCAATT A SAGE approach to discovery of genes involved in autophagic cell death S. Gorski, Genome Sciences Centre, BC Cancer Agency

  2. I. Apoptosis II. Autophagic PCD Types of Programmed Cell Death (PCD) (adapted from Baehrecke, 2002)

  3. autophagic • stage-specific • synchronous (@18ºC) 20 hr 24 hr 26 hr (adapted from Jiang et al., 1997) • known cell death genes are highly conserved and • regulated transcriptionally RT - + - + - + - + - + - + 16 18 20 22 23 24 hr (APF, 18°C) diap2 rpr hid Drosophila salivary gland PCD

  4. Serial Analysis of Gene Expression (SAGE) (Velculescu et al., 1995) • quantitative sequence based method to generate • global gene expression profiles • yields 14 bp tags that can be compared against • transcript and genome sequences to identify genes • potential for new transcript discovery

  5. known or predicted genes genomic DNA and EST (but no annotated gene) genomic DNA only no match Salivary gland SAGE library and tag mapping summary 6.5% 25.3% 61.9% 6.2%

  6. 732 genes have unknown functions 377 of these genes were not annotated (GadFly Release 2) 48 correspond solely to salivary gland ESTs 1244 genes are expressed differentially (p<.05) prior to salivary gland PCD 512 genes have associated biological annotations (Gene Ontology in Flybase)

  7. rpr hid ark dronc crq iap2 BFTZ-F1 BR-C E74 E93 Cell Death EcR/USP E75 Tag Frequency SAGE Identifies Genes Associated Previously With Salivary Gland Death

  8. Genes associated with autophagic PCD Expression fold- difference (16 hr vs 23 hr) Trans- cription* Protein synthesis Immune response/ TNF-related Autophagy Hormone related Signal transduction Apoptosis Unknowns

  9. Mutants available • Prioritization • mammalian ortholog • data mining • Phenotype analyses • salivary glands, midguts, • retinas, embryos RNAi in Drosophila cells RNAi in mammalian cells Mutants unavailable RNAi in vivo (Gal4/UAS) RNAi in tumour models Strategy for characterizing differentially expressed genes

  10. GadFly – Swissprot Homology Table Extract entries based on keyword search Mining Expression Data in Drosophila I. Keyword-based Data Mining Q: How many differentially expressed Drosophila genes have a mammalian homolog that has been associated with cancer? A: 33 genes

  11. SCC-S2 downregulated 7-fold in human mammary gland ductal carcinoma vs normal CG4091 upregulated 102-fold in 16 vs 23 hr salivary glands Mining Expression Data in Drosophila II. Cross-species Gene Expression Comparisons Human cancer (CGAP) Drosophila autophagic cell death

  12. Mutant analyses indicate that Akap200 has a PCD phenotype wild-type (41 hr APF) Akap200EP2254 (41 hr APF)

  13. Acknowledgements Genome Sciences Centre Marco Marra Victor Ling Drosophila PCD Suganthi Chittaranjan Doug Freeman Carrie Anderson Shaun Coughlin Claire Hou Bioinformatics Steven Jones Erin Pleasance Richard Varhol Scott Zuyderduyn GSC Sequencing Group University of Maryland Biotech Institute Eric Baehrecke University of Washington Stephen Jackson www.bcgsc.ca http://sage.bcgsc.ca/tagmapping/ http://www.bcgsc.ca/lab/fg/dsage/ BC Cancer Agency BC Cancer Foundation NSERC

  14. Overview of SAGE method(Velculescu et al. 1995)

  15. CATGAGGAGTGAAT AAAAA Gene X Tag-to-gene Mapping in Drosophila (E. Pleasance, M. Marra and S. Jones, submitted) • Platform: Queryable ACEDB database • Resources: • Drosophila genomic sequence and annotation (GadFly Release 2) • predicted UTRs • 259,620 ESTs and full-length cDNAs (BDGP) • 5,181 salivary gland 3’ ESTs (GSC)

  16. Gene expression is reduced in a salivary gland death-defective mutant • E93 is an ecdysone-induced gene that encodes a DNA binding protein required for salivary gland cell death (Lee et al., 2000, 2001) Fold-difference In expression (16 hr vs 23 hr)

  17. wild-type Akap200 Akap200 has a PCD phenoytpe

  18. GadFly – Swissprot Homology Table Extract entries based on keyword search II. Cross-species Gene Expression Comparisons Drosophila autophagic cell death Human cancer SCC-S2 downregulated 7-fold in human mammary gland ductal carcinoma vs normal CG4091 upregulated 102-fold in 16 vs 23 hr salivary glands Mining Expression Data in Drosophila I. Keyword-based Data Mining e.g. Keyword cancer: 33 associations

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