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 S. Gorski, Genome Sciences Centre, BC Cancer Agency

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

known cell death genes are regulated transcriptionally hr (APF, 18°C) RT diap2 rpr hid Drosophila salivary gland PCD (adapted from Jiang et al., 1997) autophagic stage-specific synchronous 20 hr24 hr26 hr

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

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 SAGE Library Tags analyzed Transcripts Total transcripts 16 hr34,9893,126 4, hr31,2153, hr30,8232, % 6.2% 6.5% 25.3%

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

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

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

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

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

Mutant analyses indicate that Akap200 has a PCD phenotype wild-type (41 hr APF)Akap200 EP2254 (41 hr APF) Tag16 hr23 hrP valueGeneGO Molecular Function CATGCGAATAATCC E-19Akap200Protein Kinase A anchoring

Acknowledgements BC Cancer Agency BC Cancer Foundation 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

Overview of SAGE method (Velculescu et al. 1995)

Tag-to-gene Mapping in Drosophila (E. Pleasance, M. Marra and S. Jones, submitted) AAAAA CATGAGGAGTGAAT Gene X 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)

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

Akap200 has a PCD phenoytpe Tag16 hr23 hrP valueGeneGO Molecular Function CATGCGAATAATCC E-19Akap200Protein Kinase A anchoring Akap200 wild-type