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A Functional Genomics Approach to Autophagic Cell Death Gene Discovery

CATGGCGTGGGGAT CATGGCTAATAAAT CATGGCTCAAGGAG CATGGCTGGACTCC CATGGCTGTGGCCA CATGGCTTTCGTGT CATGGCTTTTTGGC CATGGGAACCGACA CATGGGACCGCCCC CATGGGACCGCTCA CATGGGATCACAAT CATGGGCAACGATC CATGGGCAGCAAGC CATGGGCAGCAATT. A Functional Genomics Approach to Autophagic Cell Death Gene Discovery.

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A Functional Genomics Approach to Autophagic Cell Death Gene Discovery

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  1. CATGGCGTGGGGAT CATGGCTAATAAAT CATGGCTCAAGGAG CATGGCTGGACTCC CATGGCTGTGGCCA CATGGCTTTCGTGT CATGGCTTTTTGGC CATGGGAACCGACA CATGGGACCGCCCC CATGGGACCGCTCA CATGGGATCACAAT CATGGGCAACGATC CATGGGCAGCAAGC CATGGGCAGCAATT A Functional Genomics Approach to Autophagic Cell Death Gene Discovery Genome Sciences Centre British Columbia Cancer Agency

  2. Acknowledgements GSCPCD group Sharon Gorski Suganthi Chittaranjan Doug Freeman Melissa McConechy Jennifer Kouwenberg Bioinformatics Steven Jones Erin Pleasance Richard Varhol Scott Zuyderduyn GSC Sequencing Group University of Maryland Biotech Institute Eric Baehrecke www.bcgsc.ca http://sage.bcgsc.ca/tagmapping/ http://www.bcgsc.ca/lab/fg/dsage/ BC Cancer Agency BC Cancer Foundation National Cancer Institute of Canada Michael Smith Foundation for Health Research NSERC

  3. Outline • Programmed Cell Death (PCD) • A genomic approach to gene identification in Drosophila PCD • Validation of candidate Drosophila PCD genes and an RNAi screen to assess function.

  4. Programmed cell death (PCD) • PCD isa genetically regulated type of cell death in which the cell uses specialized cellular machinery to kill itself; it is a cell suicide mechanism that enables metazoans to control cell number and eliminate cells that threaten the animal's survival • Types (Schweichel & Merker, 1973): Type I = apoptosis Type II = autophagic cell death Type III = non-lysosomal

  5. Aims J. Mol. Recognit. 2003; 16: 337–348 • Molecular machinery involved? Relationships? • Which genes are necessary & sufficient? • Which genes are associated with human disease?

  6. Genome Sciences CentreProgrammed Cell Death Group Apoptotic Cell Death Autophagic Cell Death Autophagy Gene expression profiling (SAGE) of autophagic PCD in Drosophila salivary glands Mammalian cell line transcription profiling and RNAi (M. Qadir) inxs (Doug Freeman) echinus (Ian Bosdet) Cloning and Characterization Bioinformatic analyses: associations between autophagic PCD, apoptosis, autophagy, and cancer (Erin Pleasance) RNAi screen in Drosophila cell line (Suganthi, Melissa McConechy, Jennifer Kouwenberg, Amy Leung) Novel Gene Discovery (Brent Mansfield) Role of Akap200 (Claire Hou) Role of CG4091 (Suganthi Chittaranjan) RNAi screen in mammalian cell line (M. Qadir)

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

  8. Distinctions between Type I & II PCD

  9. The Cell, A Molecular Approach, G.M. Cooper, Ed., 2000 Autophagy • Housekeeping: low level • Starvation: upregulation, provides nutrients • PCD: autophagy also upregulated. Paradox? Autophagosome (Double membrane) Autophagolysosome www.uni-marburg.de/cyto/elsaesse/auto.htm

  10. Autophagic PCD in Development • Dictyostelium sorocarp formation • insect metamorphosis • intersegmental muscle, gut, salivary glands • mammalian embryogenesis • regression of interdigital webs, sexual anlagen • mammalian adulthood • intestine, mammary gland post-weaning, ovarian atretic follicles

  11. Autophagic PCD in disease & disease models • Neurodegenerative diseases (Alzheimers, Parkinson, Huntington’s, Lurcher mouse) • cardiomyocyte degeneration • spontaneous regression of human neuroblastoma • tamoxifen-treated mammary carcinoma cells (MCF-7) • TNFα-treated T lymphoblastic leukemic cells • bcl-2 antisense treatment of human leukemic HL60 cells • Oncogenic Ras-expressing human glioma and gastric cancer cells • beclin-1 is an autophagy gene that is monoallelically deleted and expressed at reduced levels in human breast and ovarian cancers; beclin-1 knockout mouse indicated that beclin-1 is a haploinsufficient tumor suppressor gene; hets display an increased incidence of lymphoma, lung carcinoma and liver carcinoma

  12. Experimental Approach • Drosophila model system: • Known cell death genes/pathways are conserved • Genetic and molecular tools • Sequence resources • FlyBase and GadFly databases • Multiple tissues undergo PCD; well-characterized • morphologically • Gene expression profiling (SAGE) and RNAi: • Comprehensive • Gene Discovery

  13. The Drosophila Salivary Glands Cell types: duct cells & secretory cells Cell number: ≈ 100 cells/gland Size of gland: ≈ 150 x 900 µm Total RNA/pair of glands:≈ 0.6 µg (20 pairs/microSAGE library; 500 pairs /cDNA library) Development:ectodermally-derived during late embryogenesis; during metamorphosis, a pulse of ecdysone triggers larval salivary gland PCD; adult salivary glands arise from a pair of imaginal rings (from Kucharova-Mahmood et al., 2002)

  14. 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

  15. ESTs • 3’ ESTs from salivary gland specific cDNA library • 500 pairs of salivary glands from mixed stages, 16-24hrs) High quality 3’ ESTs 5181 Number of different transcripts represented 1696 Matches to BDGP* predicted genes AND BDGP ESTs 1280 Matches to BDGP ESTs only (but no predicted gene) 145 Matches to BDGP predicted genes only (no other ESTs) 75 No matches to BDGP predicted genes or ESTs 196 *Berkeley Drosophila Genome Project Release 2

  16. ESTs Cluster Size Distribution Number of clusters Cluster size 3’ ESTs Top 5 most abundant salivary gland ESTs: MT35 (mitochondrial large rRNA) 733 CG4151 (no annotation) 375 Eig71Ec (Ecdysone-induced gene 71Ec) 171 CG3132 (beta-galactosidase) 155 CG14062 (DNA/RNA non-specific endonuclease) 74

  17. SAGE(Velculescu et al. 1995) • Potential for gene discovery

  18. Salivary gland SAGE:Tag mapping summary(S. Gorski et al., Curr Biol 13: 358-363, 2003)(E. Pleasance et al., Genome Res 13: 1203-15, 2003) 6.5% known or predicted genes genomic DNA and EST (but no annotated gene) 25.3% 61.9% genomic DNA only 6.2% no match

  19. 732 genes have unknown functions 377 of these genes were not predicted (GadFly Release 2) 48 correspond solely to salivary gland ESTs 1244 transcripts are expressed differentially (p<.05) prior to salivary gland PCD 512 genes have associated biological annotations (Gene Ontology in Flybase)

  20. 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

  21. 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

  22. 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) • Genes with map locations corresponding to E93 binding sites and upregulated prior to salivary gland PCD were tested by QRT-PCR: Fold-difference in expression (16 hr vs 23 hr)

  23. Mutants unavailable • Prioritization • midgut PCD • human ortholog/cancer • l(2)mbn cells RNAi in Drosophila l(2)mbn cells Overexpression and loss-of-function in vivo siRNA in mammalian cells Function-based strategies for characterizing differentially expressed genes Mutants available • Phenotype analyses • salivary glands, midguts, • retinas, embryos

  24. Prioritization • Differentially expressed (p < 0.05) tags that unambiguously correspond to known/predicted genes and show at least 5-fold difference in expression (= 489) • similar differential expression prior to midgut PCD (Li & White, Dev Cell, 2003, & in-house QRT-PCR) (> 182; in progress) • mammalian ortholog (53%; InParanoid, Remm et al., 2001) • mammalian ortholog differentially expressed in cancer (in progress) • present in RNAi cell system (64% by Affymetrix analysis of l(2)mbn cells)

  25. Finding PCD genes by orthology and expression E. Pleasance, S. Gorski and S. Jones Human cancer and normal SAGE libraries (102 libraries from CGAP) Drosophila SAGE libraries Differentially expressed genes (16 hr vs 23 hr, p<.05 = 564 genes) Differentially expressed genes (p<.05 = 2277 genes) Human orthologues (296/564 have human RefSeq ortholog) Set of Drosophila/human orthologues perturbed in both cancer and Drosophila PCD (= 23 Drosophila genes)

  26. Genes upregulated in PCD E. Pleasance

  27. A A “ “ “ “ Double knock Double knock Double knock Double knock - - - - out” strategy out” strategy out” strategy out” strategy Add dsRNA of Add dsRNA of Add dsRNA of Add dsRNA of Add Add Add Add iap1 iap1 iap1 iap1 another gene of another gene of another gene of another gene of dsRNA dsRNA dsRNA dsRNA interest (eg. interest (eg. interest (eg. interest (eg. CG4091 CG4091 CG4091 CG4091 , , , , rpr rpr rpr rpr ) ) ) ) Mbn2 cell line Mbn2 cell line Mbn2 cell line Mbn2 cell line Tryphan blue stain Tryphan blue stain Tryphan blue stain Tryphan blue stain Apoptotic Apoptotic Apoptotic Apoptotic body body body body Cell forming Cell forming Cell forming Cell forming apoptotic apoptotic apoptotic apoptotic body body body body Dead cell Dead cell Dead cell Dead cell 1. 1. 1. 1. Gene knock Gene knock Gene knock Gene knock - - - - out of i out of i out of i out of i ap1 ap1 ap1 ap1 (anti (anti (anti (anti - - - - cell cell cell cell death gene) causes apoptosis death gene) causes apoptosis death gene) causes apoptosis death gene) causes apoptosis 1. 1. 1. 1. Knock Knock Knock Knock - - - - out of genes down out of genes down out of genes down out of genes down - - - - 2. 2. 2. 2. Knock Knock Knock Knock - - - - out of genes upstream of out of genes upstream of out of genes upstream of out of genes upstream of stream of stream of stream of stream of iap1 iap1 iap1 iap1 in the same in the same in the same in the same iap1 iap1 iap1 iap1 in the same pathway and in the same pathway and in the same pathway and in the same pathway and pathway will inhibit pathway will inhibit pathway will inhibit pathway will inhibit iap iap iap iap - - - - 1 1 1 1 the genes that do not interact the genes that do not interact the genes that do not interact the genes that do not interact induced programmed cell death. induced apoptosis induced programmed cell death. induced apoptosis with with with with iap1 iap1 iap1 iap1 will still induce will still induce will still induce will still induce programmed cell death apoptosis programmed cell death apoptosis CG4091 Met all criteria: • Upregulated prior to salivary gland PCD (X 105 in SAGE) • Upregulated prior to midgut PCD (X 9) • Expressed in mbn2 cells • Human ortholog (TNF-induced protein GG2-1/SCC-S2) • GG2-1/SCC-S2 possibly associated with human cancer: SCC-S2 amplified in a metastatic head and neck carcinoma-derived cell line compared to matched primary tumor-derived cell line (Kumar et al., JBC, 2000)

  28. Drosophila l(2)mbn cell line • established in 1978 by Gateff • consists of tumorous haemocytes isolated from a larva of the Drosophila mutant lethal (2) malignant blood neoplasm. • form vacuoles and die in response to 20-hydroxyecdysone (20HE; ecdysone) treatment • die in response to treatment with Diap1-RNAi • morphology and gene expression changes currently under investigation

  29. No treatment Ecdysone treatment Diap1-RNAi treatment RNAi screen design Prepare dsRNA using T7-tailed gene specific primers (average product size = 500 bp) Add approx 50nM dsRNA directly to Drosophila l(2)mbn cells under serum-free conditions & incubate 1 hr. Add serum. Incubate 4-5 days Cell counts/WST-1 colorimetric assay (cell viability) Microscopic observation (cell morphology)

  30. RNAi Concept • Induction of death • Ecdysone • Diap1-RNAi Required genes

  31. PCD pathways in Drosophila (Meier et al., Nature 2000)

  32. CG4091-RNAi partially blocks PCD induced by Diap1-RNAi No. of live cells RNAi construct

  33. PCD pathways in Drosophila TNF? CG4091 (Meier et al., Nature 2000)

  34. RNAi in Drosophila l(2)mbn cells Overexpression and loss-of-function in vivo siRNA in mammalian cells CG4091: work in progress P-element excision (flybase.bio.indiana.edu)

  35. Summary • 1244 / 4628 transcripts differentially expressed prior to PCD – stage • and tissue specificity of starting material represents a highly enriched • source for detection of gene expression differences. Many have candidate human orthologs differentially expressed in cancer. • There is overlap between apoptosis and autophagic PCD with • respect to the genes involved (e.g. known apoptosis genes • detected in our differentially expressed genes; a few of these • were known previously but we detected others not previously • described in the salivary gland). • There appear to be genes specific to autophagic PCD – eg. putative autophagy gene orthologs and lysosomal genes were differentially expressed prior to autophagic PCD. • An RNAi system for functional characterization of candidates has been designed. 72hr Untreated Control Cells 72hr 10µM 20-hydroxyecdysone Treated Cells 72hr Untreated Control Cells 72hr 10µM 20-hydroxyecdysone Treated Cells

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