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A SAGE Approach to Discovery of Genes Involved in Autophagic Cell Death

A SAGE Approach to Discovery of Genes Involved in Autophagic Cell Death. Acknowledgements. Genome Sciences Centre Victor Ling Marco Marra Functional Genomics Suganthi Chittaranjan Doug Freeman Carrie Anderson Shaun Coughlin Sequencing Genome Sciences Centre Sequencing Team.

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A SAGE Approach to Discovery of Genes Involved in Autophagic Cell Death

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  1. A SAGE Approach to Discovery of Genes Involved in Autophagic Cell Death

  2. Acknowledgements Genome Sciences Centre Victor Ling Marco Marra Functional Genomics Suganthi Chittaranjan Doug Freeman Carrie Anderson Shaun Coughlin Sequencing Genome Sciences Centre Sequencing Team Bioinformatics Steven Jones Erin Garland Richard Varhol Scott Zuyderduyn SAGE team University of Maryland Biotech Institute Eric Baehrecke

  3. Programmed Cell Death (PCD) Dysfunction Function Cancer Deleting damaged cells Autoimmune diseases Culling cell number Neurodegenerative diseases Deletingstructures Developmental abnormalities Sculpting tissues

  4. Programmed cell death • Type I = Apoptotic • cytoskeletal collapse • condensation and fragmentation • of chromatin and cytoplasm • preservation of organelles • phagocytosis by macrophages or neighbouring cells (heterophagy) • occurs in isolated cells • Type II = Autophagic • preservation of cytoskeleton • formation of vacuoles that • engulf cytoplasm and organelles • fusion of vacuoles with lysosomes for self-degradation • late chromatin condensation and nuclear degeneration • occurs in groups of cells

  5. Autophagic cell death in normal physiology • 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

  6. Autophagic cell death in disease • human neurodegenerative diseases (Alzheimer and Parkinson) • cardiomyocyte degeneration • spontaneous regression of human neuroblastoma • tamoxifen-treated mammary carcinoma cells (MCF-7) • bcl-2 antisense treatment of human leukemic HL60 cells • beclin-1 (apg6) promotes autophagy and inhibits tumorigenesis; • expressed at decreased levels in human breast carcinoma

  7. 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 are conserved 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.

  8. (Jiang et al., 1997) • 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

  9. 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

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

  11. Tag-to-gene Mapping in Drosophila (E. Pleasance, M. Marra and S. Jones, in preparation) Resources: Drosophila genomic sequence, full-length cDNAs, ESTs, salivary gland ESTs • 4,628 tags: • 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

  12. Correlation coefficient = 0.5 Fold-difference by QRT-PCR Fold-difference by SAGE Comparison of SAGE and real-time quantitative RT-PCR I. Direction of Change: 91/96 samples = 95% concordance II. Correlation coefficient between fold-difference values (64 samples):

  13. 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

  14. 732 genes have unknown functions 377 of these were unpredicted 1244 genes are expressed differentially prior to salivary gland PCD 512 genes have associated biological annotations (Flybase Gene Ontology)

  15. Secondary screening of differentially expressed genes • I. Data Mining • II. Gene expression in salivary gland cell death-defective • mutant (E93) • III. Gene expression in other dying tissues • IV. Loss-of-function and gain-of-function mutant analyses

  16. I. Data mining by sequence similarity searches and keyword queries SAGE tag maps to Drosophila gene SAGE tag maps to novel EST Extract gene sequence from GadFly Database GadFly – Swissprot Homology (tBLASTX) tBLASTX search EST vs Swissprot Keyword query of Swissprot comments, keywords and identification fields Keyword query of FlyBase and PCD database

  17. II. Analysis of differential gene expression in E93 mutant salivary glands • E93 is a DNA binding protein required for salivary gland cell death • (Lee et al. 2000) • 65 different E93 chromosomal binding positions are known • Expression of known cell death genes is reduced in E93 mutant • salivary glands • we tested 20 differentially expressed genes with map positions • corresponding to E93 binding sites

  18. Gene expression is reduced in E93 mutant salivary glands 19/20 upregulated genes showed a reduction in relative levels of transcription.

  19. CG3132 Cp1 akap200 akap200 III. Expression of salivary gland genes in the embryo coincides with patterns of PCD 3/15 genes examined have embryonic expression patterns that at least partially coincide with patterns of PCD

  20. IV. Loss-of-function mutant analyses. • salivary glands • embryos • retinas e.g. akap200 mutants indicate a possible defect in PCD: wild-type (41 hr APF) akap200EP2254 (41 hr APF) * * * *

  21. Differentially expressed genes reveal molecular features associated with autophagic cell death • Autophagic cell death involves the induction of genes required • for protein synthesis • e.g. 6 different translation initiation factors • Novel transcription factors appear upregulated coordinately with • known transcriptional regulators • e.g. maf-S, CG3350 • Components of multiple signal transduction pathways are involved • e.g. TNF-a like pathway, akap200, Doa

  22. Differentially expressed genes and molecular features of autophagic cell death, continued. • apg-like genes can be regulated transcriptionally and this regulation • is associated with autophagic cell death • e.g. genes involved in two ubiquitin-like pathways • CG6194 (apg4;novel cysteine protease) • CG5429 (apg6/beclin-1) • Other autophagy-associated genes are likely involved • e.g. lysosomal enzymes, rab-7 • Autophagic and apoptotic cell death appear to utilize at least some • common pathways or pathway components

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