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

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

  5. 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)

  6. 6.5% 25.3% 61.9% 6.2% Salivary gland SAGE Library and tag mapping summary

  7. 732 genes have unknown functions 377 of these genes were unpredicted (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)

  8. Validation of SAGE Data 0.0006 SG16 rpr hid ark dronc crq iap2 0.0005 SG20 BR-C E74 E93 BFTZ-F1 SG23 Cell Death EcR/USP 0.0004 E75 0.0003 0.0002 0.0001 0 BR-C E74 E75 E93 rpr ark dronc iap2 crq I. Quantitative RT-PCR comparison • 91/96 (95%) differentially expressed genes were concordant • with respect to direction of change (cc = 0.5) II. Identification of known genes Tag Frequency

  9. 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)

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

  11. Strategy for characterizing differentially expressed genes • Prioritization • mammalian ortholog • data mining • Mutant analysis • existing mutants RNAi in Drosophila cells RNAi in mammalian cells RNAi in vivo (Gal4/UAS) RNAi in tumour models

  12. I. Keyword-based Data Mining GadFly-SwissProt Homology (tBLASTX) Keyword search of SwissProt comments, keywords and id fields e.g. Keyword apoptosis: 20 associations; keyword cancer: 33 associations II. Cross-species Expression Profile Comparisons Differential gene expression in Drosophila autophagic cell death Differential gene expression in human cancer (e.g. CGAP) Orthologs CG4091 upregulated 102-fold in 16 hr vs 23 hr salivary glands SCC-S2 downregulated 7-fold in human mammary gland ductual carcinoma vs normal Mining Expression Data in Drosophila

  13. Mutant analyses indicate that akap200 is required for PCD wild-type (41 hr APF) akap200EP2254 (41 hr APF)

  14. Acknowledgements Genome Sciences Centre Victor Ling Marco Marra Suganthi Chittaranjan Doug Freeman Carrie Anderson Shaun Coughlin Claire Hou 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

  15. Mining Gene Expression Data in Drosophila • Keyword-based Data Mining GadFly-SwissProt Homology (tBLASTX) Keyword search of SwissProt comments, keywords and id fields e.g. dronc result “unknown” result II. Cross-species Gene Expression Data Mining Differential gene expression in Drosophila autophagic cell death Differential gene expression in human cancer (e.g. CGAP) e.g. CG4091 upregulated prior to Drosophila autophagic PCD (p < ___).and human SCC-S2 (??) SCC-S2 downregulated in human breast cancer(?tissue) compared to normal breast tissue (p < ___)

  16. Data mining by sequence similarity searches and keyword queries GadFly – Swissprot Homology (tBLASTX) tBLASTX search novel EST vs Swissprot Keyword query of Swissprot comments, keywords and identification fields

  17. Aims • Identify the genes involved in autophagic cell death in vivo. • Determine which genes are necessary and sufficient for • autophagic cell death. • Determine if genes function in mammalian autophagic • cell death. • Identify the autophagic cell death genes associated with human • disease and investigate potential as molecular markers and/or • therapeutic targets.

  18. Overview of SAGE tag abundance Total number of different transcripts in all three libraries is 4,628.

  19. Questions • What is the relationship between Autophagic Cell Death and Cancer? • common mechanism in breast and other cancers? • gene mutation • What is the therapeutic potential of autophagic cell death in cancer? • solid tumours • apoptotic-resistant tumours

  20. Quantitative RT-PCR validates SAGE data I. Direction of Change: 91/96 samples = 95% concordance II. Correlation coefficient between fold-difference values (64 samples): Correlation coefficient = 0.5 Fold-difference by QRT-PCR Fold-difference by SAGE

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

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

  23. Genes fail to be differentially expressed in E93 mutant salivary glands • E93 is an ecdysone-induced gene that encodes a chromosome binding protein required for salivary gland cell death (Lee et al., 2000, 2001) Fold difference in expression (16 hr vs 23 hr) Genes

  24. rpr hid ark dronc crq BR-C E74 E93 Cell Death BFTZ-F1 EcR/USP E75 diap2 An ecdysone induced transcriptional cascade regulates salivary gland cell death

  25. GadFly – Swissprot Homology (tBLASTX) tBLASTX search novel EST vs Swissprot Keyword query of Swissprot comments, keywords and identification fields Data mining by sequence similarity searches and keyword queries

  26. 6.5% 25.3% 61.9% 6.2% Salivary gland SAGE Library and tag mapping summary • Tag-to-gene mapping (Pleasance, Marra and Jones, submitted) • 2866 (61.9%) - known or predicted genes • 289 ( 6.2 %) - genomic DNA and EST (but no predicted gene) • (25.3%) - genomic DNA and/or reverse strand of gene • 303 ( 6.5%) - no match • 4628

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