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

S. Gorski, Genome Sciences Centre, BC Cancer Agency

Types of Programmed Cell death (PCD)

(adapted from Baehrecke, 2002)

I. Apoptosis

II. Autophagic PCD

(adapted from Jiang et al., 1997)

• autophagic • stage-specific • synchronous

• known cell death genes are regulated transcriptionally

hr (APF, 18°C)Reverse transcription - + - + - + - + - + - +

16 18 20 22 23 24

diap2

rpr

hid

RT-PCR analysis

Drosophila salivary gland PCD

16 hr 24 hr 26 hr (@18C)

Overview of SAGE method(Velculescu et al. 1995)

Tag-to-gene Mapping in Drosophila(E. Pleasance, M. Marra and S. Jones, submitted)

AAAAA

CATGAGGAGTGAAT

Gene X

•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)

Salivary gland SAGE Library and tag mapping summary

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

known or predictedgenes

genomic DNA and EST(but no predicted gene)

genomic DNA and/orreverse strand of gene

no match

61.9%

6.2%

6.5%

25.3%

1244 genes 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 unpredicted

(GadFly Release 2)

48 correspond solely to salivary

gland ESTs

Validation of SAGE Data

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

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

SG23BFTZ-F1

EcR/USP

BR-CE74E93

rprhidark

dronccrq

iap2

CellDeath

E75

0

10

20

30

40

50

60

70

*Cec

B

*CG

4091

*Cec

A1 Doa

CG

4859

CG

1499

5

CG

5254

CG

3845

Ptpm

eg

CG

9321

CG

1278

9

CG

1216 larp

CG

7860

Cyp

1

Sox1

4

CG

1344

8

CG

8149 ar

k

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 associated with autophagic PCD

0

10

20

30

40

50

60

70

80

90

100E

f1ga

mm

aC

G56

05C

G38

45eI

F-4E

CG

9769

CG

1019

2C

G74

39eI

F-5A

Hr7

8C

G15

505

CG

7592

Eip

63F-

1E

ip71

CD

bun

EP

2237

CG

9954

CG

3350

Aka

p200

Doa sk

tlP

tpm

egP

R2

CG

1670

8C

G86

55

Thor

Cec

A1

Cec

CC

ecB

Def

Drs

PG

RP

-LA

BE

ST:

GH

0 Dif

cact

Myd

88Tr

af1

CG

4091

Dcp

-1em

pC

G12

789

CG

3829

buffy

sick

le

CG

6194

CG

1643

CG

5429

CG

1086

1R

ab-7

CG

1115

9C

G31

32C

G10

992

cath

DC

G17

283

CG

1216

3C

p1

Expressionfold-difference(16 hr vs 23 hr)

Proteinsynthesis

Hormonerelated

Trans-cription*

Signal transduction

Immune response/TNF-related

Apoptosis Autophagy

Strategy for characterizing differentially expressed genes

Mutant analysis• existing mutants

RNAi in vivo (Gal4/UAS)

Prioritization• mammalian ortholog

• data mining

RNAi inmammalian cells

RNAi in tumour models

RNAi in Drosophila cells

Mining Expression Data in Drosophila

Keyword 16 hr 23 hr Gene Score SwissProt ID Database Description

death 0 15 dronc 251 ICE6_HUMAN CASPASE-6 PRECURSOR

tumor 45 1 botv 2044 EXL3_HUMAN TUMOUR SUPPRESSOR EXL3-LIKE

II. Cross-species Expression Profile Comparisons

Differential gene expressionin Drosophila autophagic cell death

Differential gene expressionin human cancer (e.g. CGAP)

CG4091 upregulated 102-foldin 16 hr vs 23 hr salivary glands

SCC-S2 downregulated 7-fold in humanmammary gland ductual carcinoma vs normal

Orthologs

GadFly-SwissProt Homology (tBLASTX)Keyword search of SwissProt comments, keywords and id fields

I. Keyword-based Data Mining

e.g. Keyword apoptosis: 20 associations; keyword cancer: 33 associations

Mutant analyses indicate that akap200 is required for PCD

wild-type (41 hr APF) akap200EP2254 (41 hr APF)

Tag 16 hr 23 hr P value Gene GO Molecular Function

CGAATAATCC 3 67 3.03E-19 Akap200 Protein kinase A anchoring

Acknowledgements

BC Cancer AgencyBC Cancer Foundation

Genome Sciences CentreVictor Ling

Marco Marra

Suganthi ChittaranjanDoug Freeman

Carrie AndersonShaun Coughlin

Claire Hou

Steven JonesErin PleasanceRichard Varhol

Scott Zuyderduyn

GSC Sequencing Group

University of Maryland Biotech InstituteEric Baehrecke

University of WashingtonStephen Jackson

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

Mining Gene Expression Data in Drosophila

I. Keyword-based Data Mining

II. Cross-species Gene Expression Data MiningDifferential gene expression

in Drosophila autophagic cell deathDifferential 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 < ___)

GadFly-SwissProt Homology (tBLASTX)Keyword search of SwissProt comments, keywords and id fields

e.g. dronc result“unknown” result

Data mining by sequence similarity searches and keyword queries

GadFly – Swissprot Homology (tBLASTX)

Keyword query of Swissprot comments, keywords and identification fields

tBLASTX search novel EST vs Swissprot

Keyword16 hr

23 hr

GeneSim

(%)Score Length SP ID Database Description

death 0 15 dronc 27 251 253 ICE6_HUMAN CASPASE-6 PRECURSOR

apoptosis 4 0 debcl 26 127 133 BCL2_HUMAN APOPTOSIS REGULATOR BCL-2

apoptosis 0 5 Traf1 37 300 179 TRA1_HUMAN TRAF1

autophagy 0 7 chrw 35 257 170 RB24_MOUSE RAS-RELATED PROTEIN RAB-24

cancer 1 15 CG13907 33 311 203 MOT1_HUMAN MONOCARBOXYLATE TRANSPORT

tumor 65 0 CG11335 43 423 182 LYOX_HUMAN PROT-LYSINE 6-OXIDASE PREC.

tumor 45 1 botv 49 2044 838 EXL3_HUMAN TUMOUR SUPPRESSOR EXL3-LIKE

Aims

1. Identify the genes involved in autophagic cell death in vivo.

2. Determine which genes are necessary and sufficient for autophagic cell death.

3. Determine if genes function in mammalian autophagic cell death.

4. Identify the autophagic cell death genes associated with human disease and investigate potential as molecular markers and/or

therapeutic targets.

Overview of SAGE tag abundance

SAGE

LibraryTotal tags analyzed

Transcripts % of transcripts seen at frequency:

1 2-10 11-100 >100

16 hr 34,989 3,126 32.7 55.7 10.4 1.2

20 hr 31,215 3,034 38.0 50.9 9.7 1.4

23 hr 30,823 2,963 33.3 54.0 11.2 1.4

Total number of different transcripts in all three libraries is 4,628.

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

Quantitative RT-PCR validates SAGE data

Fold-difference by SAGE

Fold-difference by QRT-PCR

1.0

10.0

100.0

1000.0

1.0 10.0 100.0 1000.0

II. Correlation coefficient between fold-difference values (64 samples):

I. Direction of Change:

91/96 samples = 95% concordance

Correlation coefficient = 0.5

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

SAGE Identifies Genes Associated Previously With Salivary Gland Death

Tag Frequency

BFTZ-F1

EcR/USP

BR-CE74E93

rprhidark

dronccrq

iap2

CellDeath

E75

SAGE Identifies Genes Associated Previously With Salivary Gland Death

0

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006B

R-C

E7

4

E7

5

E9

3

rpr

ark

dro

nc

iap

2

crq

Ta

g F

req

ue

nc

y (

tag

co

un

t/to

tal t

ag

s)

SG16

SG20

SG23BFTZ-F1

EcR/USP

BR-CE74E93

rprhidark

dronccrq

iap2

CellDeath

E75

Genes fail to be differentially expressed in E93 mutant salivary glands

0

10

20

30

40

50

60

70

*Cec

B

*CG

4091

*Cec

A1

Doa

CG

4859

CG

1499

5

CG

5254

CG

3845

Ptp

meg

CG

9321

CG

1278

9

CG

1216

larp

CG

7860

Cyp

1

Sox

14

CG

1344

8

CG

8149

ark

OreR

E93

Fold differencein expression(16 hr vs 23 hr)

Genes

• E93 is an ecdysone-induced gene that encodes a chromosome binding protein required for salivary gland cell death (Lee et al., 2000, 2001)

An ecdysone induced transcriptional cascade regulates salivary gland cell death

BFTZ-F1

EcR/USP

BR-C

E74

E93

rprhidarkdronccrq

diap2

Cell Death

E75

Data mining by sequence similarity searches and keyword queries

Keyword16 hr

23 hr

GeneSim

(%)Score Length SP ID Database Description

death

apoptosis

apoptosis

autophagy

cancer

tumor

tumor

0

4

0

0

1

65

45

15

0

5

7

15

0

1

dronc

debcl

Traf1

chrw

CG13907

CG11335

botv

27

26

37

35

33

43

49

251

127

300

257

311

423

2044

253

133

179

170

203

182

838

ICE6_HUMAN

BCL2_HUMAN

TRA1_HUMAN

RB24_MOUSE

MOT1_HUMAN

LYOX_HUMAN

EXL3_HUMAN

CASPASE-6 PRECURSOR

APOPTOSIS REGULATOR BCL-2

TRAF1

RAS-RELATED PROTEIN RAB-24

MONOCARBOXYLATE TRANSPORT

PROT-LYSINE 6-OXIDASE PREC.

TUMOUR SUPPRESSOR EXL3-LIKE

GadFly – Swissprot Homology (tBLASTX)

Keyword query of Swissprot comments, keywords and identification fields

tBLASTX search novel EST vs Swissprot

known or predictedgenesgenomic DNA and EST(but no predicted gene)genomic DNA and/orreverse strand of geneno match

Salivary gland SAGE Library and tag mapping summaryTag-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)1170 (25.3%) - genomic DNA and/or reverse strand of gene 303 ( 6.5%) - no match4628

known or predictedgenes

genomic DNA and EST(but no predicted gene)

genomic DNA and/orreverse strand of gene

no match

61.9%

6.2%

6.5%

25.3%