A Functional Genomics Approach to Autophagic Cell Death Gene

Discovery

Genome Sciences CentreBritish Columbia Cancer Agency

CATGGCGTGGGGAT

CATGGCTAATAAAT

CATGGCTCAAGGAG

CATGGCTGGACTCC

CATGGCTGTGGCCA

CATGGCTTTCGTGT

CATGGCTTTTTGGC

CATGGGAACCGACA

CATGGGACCGCCCC

CATGGGACCGCTCA

CATGGGATCACAAT

CATGGGCAACGATC

CATGGGCAGCAAGC

CATGGGCAGCAATT

Acknowledgements

BC Cancer AgencyBC Cancer Foundation

National Cancer Institute of CanadaMichael Smith Foundation

for Health ResearchNSERC

GSC PCD group Sharon Gorski

Suganthi ChittaranjanDoug Freeman

Melissa McConechyJennifer Kouwenberg

BioinformaticsSteven Jones

Erin PleasanceRichard Varhol

Scott Zuyderduyn

GSC Sequencing Group

University of Maryland Biotech InstituteEric Baehrecke

www.bcgsc.cahttp://sage.bcgsc.ca/tagmapping/http://www.bcgsc.ca/lab/fg/dsage/

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)

• PCD is a 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 = apoptosisType II = autophagic cell deathType III = non-lysosomal

J. Mol. Recognit. 2003; 16: 337–348

Aims

•Molecular machinery involved? Relationships?•Which genes are necessary & sufficient?•Which genes are associated with human disease?

Genome Sciences CentreProgrammed Cell Death

GroupApoptotic Cell Death Autophagic Cell Death Autophagy

inxs(Doug Freeman)

echinus(Ian Bosdet)

Cloning and Characterization

Mammalian cell linetranscription profiling

and RNAi(M. Qadir)

Gene expression profiling (SAGE)of autophagic PCD in Drosophila

salivary glands

Role of Akap200(Claire Hou)

Role of CG4091(Suganthi Chittaranjan)

Bioinformatic analyses:

associations between autophagic PCD,

apoptosis,autophagy, and cancer

(Erin Pleasance)

Novel GeneDiscovery

(Brent Mansfield)

RNAi screenin Drosophila

cell line(Suganthi, Melissa

McConechy, Jennifer Kouwenberg, Amy Leung)

RNAi screen inmammalian cell line

(M. Qadir)

Types of Programmed Cell Death (PCD)

(adapted from Baehrecke, 2002)

I. Apoptosis

II. Autophagic PCD

Distinctions between Type I & II PCD

Characteristic Type I (Apoptosis) Type II (Autophagic)

Hallmark Condensation, membrane blebbing & apoptotic body formation

Formation of autophagic vacuoles

Typical Occurrence

Isolated cells Groups of cells

Cytoskeleton Cytoskeletal collapse Cytoskeletal preservation

Cytoplasm Condensation & fragmentation

Engulfed by autophagic vacuoles

Organelles Preserved Engulfed by autophagic vacuoles

Nucleus Chromatin condensation & nuclear fragmentation early

Degradation is late; follows cytoplasmic degradation

Degradation Phagocytes or neighbouring cells (heterophagy)

Autophagic vacuoles fuse with lysosomes (autophagy); remnants by phagocytes

Caspases Caspase-dependent Caspase-independent or -dependent

Triggers DNA damage, oncogene activation, extracellular signals, etc.

Ras activation, ecdysone, constitutive autophagy, ??

Autophagy

Autophagosome(Double membrane)

Autophagolysosome

www.uni-marburg.de/cyto/elsaesse/auto.htmThe Cell, A Molecular Approach, G.M. Cooper, Ed., 2000

• Housekeeping: low level• Starvation: upregulation, provides nutrients

• PCD: autophagy also upregulated. Paradox?

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

• 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

Gene expression profiling (SAGE) and RNAi:• Comprehensive• Gene Discovery

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

The Drosophila Salivary Glands

Cell types: duct cells & secretory cells Cell number: ≈ 100 cells/glandSize of gland: ≈ 150 x 900 µmTotal 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)

Drosophila salivary gland PCD

(adapted from Jiang et al., 1997)

•autophagic •stage-specific•synchronous

20 hr 24 hr 26 hr (@18ºC)

• known cell death genes are highly conserved and regulated transcriptionally

hr (APF, 18°C)

RT - + - + - + - + - + - + 16 18 20 22 23 24

diap2

rpr

hid

ESTs

3’ ESTs from salivary gland specific cDNA library • 500 pairs of salivary glands from mixed stages, 16-24hrs)

High quality 3’ ESTs5181Number of different transcripts represented

1696Matches to BDGP* predicted genes AND BDGP ESTs

1280Matches to BDGP ESTs only (but no predicted gene)

145Matches to BDGP predicted genes only (no other ESTs) 75No matches to BDGP predicted genes or ESTs

196*Berkeley Drosophila Genome Project Release 2

ESTs C

lust

er

size

Top 5 most abundant salivary gland ESTs: MT35 (mitochondrial large rRNA) 733CG4151 (no annotation) 375Eig71Ec (Ecdysone-induced gene 71Ec) 171CG3132 (beta-galactosidase) 155CG14062 (DNA/RNA non-specific endonuclease) 74

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1400

Cluster sizeNumber of clusters

3’ ESTs

Nu

mb

er

of

clu

sters

Cluster Size Distribution

SAGE(Velculescu et al. 1995)

•Potential for gene discovery

known or predicted genes

genomic DNA and EST(but no predicted gene)

genomic DNA and/orreverse strand of gene

no match

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)

SAGELibrary

Tags analyzed Transcripts

Total transcripts

16 hr 34,989 3,126

4,62820 hr 31,215 3,034

23 hr 30,823 2,963

61.9%

6.2%

6.5%

25.3%

known or predicted genes

genomic DNA and EST(but no annotated gene)

genomic DNA only

no match

1244 transcripts 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 geneswere not predicted

(GadFly Release 2)

48 correspond solely to salivary

gland ESTs

SAGE Identifies Genes Associated Previously With Salivary Gland

Death

0

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

BR-C E74 E75 E93 rpr ark dronc iap2 crq

SG16

SG20

SG23

Tag Frequency

BFTZ-F1

EcR/USP

BR-CE74E93

rprhidarkdronccrq

iap2

CellDeath

E75

Genes associated with autophagic PCD

Expressionfold-difference(16 hr vs 23 hr)

Proteinsynthesis Hormone

related

Trans-cription*

Signal transduction Apoptosis

Immune response/TNF-related

Autophagy

0

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90

100E

f1g

amm

aC

G5

605

CG

384

5e

IF-4

EC

G9

769

CG

101

92C

G7

439

eIF

-5A

Hr7

8C

G1

5505

CG

759

2E

ip63

F-1

Eip

71C

D

bu

nE

P22

37

CG

995

4C

G3

350

Aka

p2

00D

oa

sktl

Ptp

me

gP

R2

CG

167

08C

G8

655

Th

orC

ecA

1C

ecC

Ce

cB De

fD

rsP

GR

P-L

AB

ES

T:G

H0

2 Dif

cact

Myd

88T

raf1

CG

409

1

Dcp

-1em

pC

G1

2789

CG

382

9bu

ffysi

ckle

CG

619

4C

G1

643

CG

542

9C

G1

0861

Ra

b-7

CG

111

59C

G3

132

CG

109

92ca

thD

CG

172

83C

G1

2163

Cp

1

CG

148

30C

G1

908

CG

540

2C

G1

5239

CG

109

65C

G1

8811

AE

003

826

AE

003

481

AE

003

446

AE

003

503

reve

rse

ES

Tre

vers

e E

ST

Unknowns

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*Ce

cB

*CG

40

91

*Ce

cA

1

Do

a

CG

48

59

CG

14

99

5

CG

52

54

CG

38

45

Ptp

me

g

CG

93

21

CG

12

78

9

CG

12

16

larp

CG

78

60

Cy

p1

So

x1

4

CG

13

44

8

CG

81

49

ark

OreR

E93

Gene expression is reduced in a salivary gland death-defective

mutant

Fold-differencein 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)•Genes with map locations corresponding to E93 binding sites and upregulated prior to salivary gland PCD were tested by QRT-PCR:

Function-based strategies for characterizing

differentially expressed genes

Mutants available

Phenotype analyses•salivary glands, midguts,

retinas, embryos

Overexpression andloss-of-function

in vivo

Prioritization•midgut PCD

•human ortholog/cancer•l(2)mbn cells

siRNA inmammalian cells

RNAi in Drosophila l(2)mbn cells

Mutants unavailable

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)

Finding PCD genes by orthology and expression

Human cancer and normal SAGE libraries

(102 libraries from CGAP)

Differentially expressed genes (p<.05 = 2277 genes)

Drosophila SAGE libraries

Differentially expressed genes (16 hr vs 23 hr, p<.05 = 564 genes)

Human orthologues (296/564 have human RefSeq

ortholog)

Set of Drosophila/human orthologues perturbed in both cancer and Drosophila PCD

(= 23 Drosophila genes)

E. Pleasance, S. Gorski and S. Jones

Genes upregulated in PCDE. Pleasance

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)

CG4091 expression profile

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SG16

SG20

SG23

16 A

PF

20 A

PF

23 A

PF

3rd

insta

r

0 APF

4 APF

6 APF

Fo

ld e

xpre

ssio

n

SAGEQRT-PCR in salivary glands

QRT-PCR in mid-gut

Percentage of Live l(2)mbn Cells After 10uM 20HE Treament

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100

0 24 48 72

Hours of Treatment

% L

ive

Cel

ls

Control Cells

20HE Treated Cells

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

RNAi screen design

Prepare dsRNA usingT7-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)

No treatment Ecdysone treatment

Diap1-RNAitreatment

Concept

Induction of death•Ecdysone•Diap1-RNAi

Required genes

RNAi

PCD pathways in Drosophila

(Meier et al., Nature 2000)

CG4091-RNAi partially blocks PCD induced by Diap1-RNAi

0.00E+00

2.00E+05

4.00E+05

6.00E+05

8.00E+05

1.00E+06

1.20E+06

1.40E+06

1.60E+06

0 after dsRNA treatment (AVE)

5 days after dsRNA treatment: (AVE)

RNAi construct

No

. of

live

cells

PCD pathways in Drosophila

(Meier et al., Nature 2000)

CG4091

TNF?

CG4091: work in progress

P-element excision(flybase.bio.indiana.edu)

Overexpression andloss-of-function

in vivo

siRNA inmammalian cells

RNAi in Drosophila l(2)mbn cells

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.