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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

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


Acknowledgements
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


Outline
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.


Programmed cell death pcd
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


Aims

J. Mol. Recognit. 2003; 16: 337–348

  • Molecular machinery involved? Relationships?

  • Which genes are necessary & sufficient?

  • Which genes are associated with human disease?


Genome sciences centre programmed cell death group

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)


Types of programmed cell death pcd
Types of Programmed Cell Death (PCD)

I. Apoptosis

II. Autophagic PCD

(adapted from Baehrecke, 2002)



Autophagy

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


Autophagic pcd in development
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


  • Autophagic pcd in disease disease models
    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


    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


    The drosophila salivary glands
    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)


    • 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


    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


    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


    Sage velculescu et al 1995
    SAGE(Velculescu et al. 1995)

    • Potential for gene discovery


    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


    1244 transcripts are expressed differentially p 05 prior to salivary gland pcd

    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)


    Sage identifies genes associated previously with salivary gland death

    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


    Genes associated with autophagic pcd
    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


    Gene expression is reduced in a salivary gland death defective mutant
    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)


    Function based strategies for characterizing differentially expressed genes

    Mutants unavailable death-defective mutant

    • 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


    Prioritization
    Prioritization death-defective mutant

    • 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)


    Finding pcd genes by orthology and expression
    Finding PCD genes by orthology and expression death-defective mutant

    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)


    Genes upregulated in pcd
    Genes upregulated in PCD death-defective mutant

    E. Pleasance


    Cg4091

    A death-defective mutant

    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)


    Drosophila l 2 mbn cell line
    Drosophila l(2)mbn death-defective mutant 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


    Rnai screen design

    No treatment death-defective mutant

    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)


    Concept

    RNAi death-defective mutant

    Concept

    • Induction of death

    • Ecdysone

    • Diap1-RNAi

    Required genes


    Pcd pathways in drosophila
    PCD pathways in Drosophila death-defective mutant

    (Meier et al., Nature 2000)


    Cg4091 rnai partially blocks pcd induced by diap1 rnai
    CG4091-RNAi partially blocks PCD induced by Diap1-RNAi death-defective mutant

    No. of live cells

    RNAi construct


    Pcd pathways in drosophila1
    PCD pathways in Drosophila death-defective mutant

    TNF?

    CG4091

    (Meier et al., Nature 2000)


    Cg4091 work in progress

    RNAi in death-defective mutant

    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)


    Summary
    Summary death-defective mutant

    • 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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