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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%