A Functional Genomics Approach to
Autophagic Cell Death Gene Discovery
CATGGCGTGGGGAT
CATGGCTAATAAAT
CATGGCTCAAGGAG
CATGGCTGGACTCC
CATGGCTGTGGCCA
CATGGCTTTCGTGT
CATGGCTTTTTGGC
CATGGGAACCGACA
CATGGGACCGCCCC
CATGGGACCGCTCA
CATGGGATCACAAT
CATGGGCAACGATC
CATGGGCAGCAAGC
CATGGGCAGCAATT
Genome Sciences Centre
British Columbia Cancer Agency
Acknowledgements
GSC PCD 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
• 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 = 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
Apoptotic Cell Death
inxs
echinus
(Doug Freeman)
(Ian Bosdet)
Autophagic Cell Death
Gene expression profiling (SAGE)
of autophagic PCD in Drosophila
salivary glands
Bioinformatic
analyses:
(Claire Hou)
Role of CG4091
Mammalian cell line
transcription profiling
and RNAi
(M. Qadir)
Cloning and Characterization
Role of Akap200
Autophagy
associations between
autophagic PCD,
apoptosis,
autophagy, and cancer
RNAi screen
in Drosophila
cell line
(Suganthi, Melissa
McConechy, Jennifer
Kouwenberg, Amy Leung)
(Erin Pleasance)
(Suganthi Chittaranjan)
RNAi screen in
mammalian cell line
(M. Qadir)
Novel Gene
Discovery
(Brent Mansfield)
Types of Programmed Cell Death (PCD)
I. Apoptosis
II. Autophagic PCD
(adapted from Baehrecke, 2002)
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
• Housekeeping: low level
• Starvation: upregulation, provides nutrients
• PCD: autophagy also upregulated. Paradox?
Autophagosome
(Double membrane)
Autophagolysosome
www.uni-marburg.de/cyto/elsaesse/auto.htm
The Cell, A Molecular Approach, G.M. Cooper, Ed., 2000
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
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
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
(from Kucharova-Mahmood et al., 2002)
embryogenesis; during metamorphosis, a pulse of
ecdysone triggers larval salivary gland PCD; adult
salivary glands arise from a pair of imaginal rings
Drosophila salivary gland PCD
•stage-specific •synchronous
•autophagic
20 hr
24 hr
26 hr
(@18ºC)
(adapted from Jiang et al., 1997)
• known cell death genes are highly conserved and
regulated transcriptionally
RT
hr (APF, 18°C)
diap2
rpr
hid
- + - + - + -+ - + - +
16 18 20 22 23 24
ESTs
3’ ESTs from salivary gland specific cDNA library
•
500 pairs of salivary glands from mixed stages, 16-24hrs)
High quality 3’ ESTs
Number of different transcripts represented
Matches to BDGP* predicted genes AND BDGP ESTs
Matches to BDGP ESTs only (but no predicted gene)
Matches to BDGP predicted genes only (no other ESTs)
No matches to BDGP predicted genes or ESTs
*Berkeley Drosophila Genome Project Release 2
5181
1696
1280
145
75
196
800
700
1400
Cluster Size Distribution
1200
1000
500
800
400
Cluster size
Number of clusters
300
600
400
200
200
100
0
0
3’ ESTs
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
Cluster size
600
Top 5 most abundant salivary gland ESTs:
MT35 (mitochondrial large rRNA)
CG4151 (no annotation)
Eig71Ec (Ecdysone-induced gene 71Ec)
CG3132 (beta-galactosidase)
CG14062 (DNA/RNA non-specific endonuclease)
733
375
171
155
74
Number of clusters
ESTs
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)
SAGE
Library
Tags
analyzed
Transcripts
16 hr
34,989
3,126
20 hr
31,215
3,034
23 hr
30,823
2,963
6.5%
4,628
known
or predicted
genes
known
or predicted
genes
25.3%
61.9%
6.2%
Total
transcripts
genomic
DNA
EST
genomic
DNA
andand
EST
(but no annotated gene)
(but
no predicted gene)
genomic
DNA
only
genomic
DNA
and/or
reverse strand of gene
match
nono
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
512 genes have
associated
biological annotations
(Gene Ontology
in Flybase)
SAGE Identifies Genes Associated
Previously With Salivary Gland Death
BFTZ-F1
0.0006
EcR/USP
BR-C
E74
E93
rpr
hid
ark
dronc
crq
Cell
Death
SG16
SG20
SG23
0.0005
E75
iap2
0.0004
Tag
Frequency
0.0003
0.0002
0.0001
0
BR-C
E74
E75
E93
rpr
ark
dronc
iap2
crq
0
TransProtein
Immune response/
Autophagy
cription*
synthesis Hormone
TNF-related
Signal
Apoptosis
related
transduction
CG14830
CG1908
CG5402
CG15239
CG10965
CG18811
AE003826
AE003481
AE003446
AE003503
reverse EST
reverse EST
CG6194
CG1643
CG5429
CG10861
Rab-7
CG11159
CG3132
CG10992
cathD
CG17283
CG12163
Cp1
Dcp-1
emp
CG12789
CG3829
buffy
sickle
Thor
CecA1
CecC
CecB
Def
Drs
PGRP-LA
BEST:GH02
Dif
cact
Myd88
Traf1
CG4091
Akap200
Doa
sktl
Ptpmeg
PR2
CG16708
CG8655
bun
EP2237
CG9954
CG3350
Hr78
CG15505
CG7592
Eip63F-1
Eip71CD
Ef1gamma
CG5605
CG3845
eIF-4E
CG9769
CG10192
CG7439
eIF-5A
Genes associated with autophagic PCD
100
90
80
Expression
fold60
difference
(16 hr
50
vs
23 hr)
70
40
30
20
10
Unknowns
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:
70
60
OreR
E93
Fold-difference 50
in expression
(16 hr vs 23 hr) 40
30
20
10
ark
CG8149
CG13448
Sox14
Cyp1
CG7860
larp
CG1216
CG12789
CG9321
Ptpmeg
CG3845
CG5254
CG14995
CG4859
Doa
*CecA1
*CG4091
*CecB
0
Function-based strategies for characterizing
differentially expressed genes
Mutants available
Phenotype analyses
•salivary glands, midguts,
retinas, embryos
Mutants unavailable
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
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
E. Pleasance, S. Gorski and S. Jones
Drosophila SAGE libraries
Differentially expressed genes
(16 hr vs 23 hr, p<.05 = 564 genes)
Human cancer and
normal SAGE libraries
(102 libraries from CGAP)
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
E. 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
CG4091 expression profile
Fold expression
30
25
20
15
10
5
SAGE
QRT-PCR in
salivary glands
in
st
a
0 r
AP
F
4
AP
F
6
AP
F
3r
d
AP
F
AP
23 F
AP
F
20
16
23
20
SG
SG
SG
16
0
QRT-PCR in midgut
• 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
•established in 1978 by Gateff
Percentage of Live l(2)mbn Cells After
10uM 20HE Treament
Control Cells
• 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
20HE Treated Cells
100
% Live Cells
• consists of tumorous
haemocytes isolated from a larva
of the Drosophila mutant lethal
(2) malignant blood neoplasm.
80
60
40
20
0
0
24
48
Hours of Treatment
72
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.
No treatment
Ecdysone
treatment
Diap1-RNAi
treatment
Incubate 4-5 days
Cell counts/WST-1 colorimetric assay (cell viability)
Microscopic observation (cell morphology)
Concept
RNAi
Induction of death
•Ecdysone
•Diap1-RNAi
Required genes
PCD pathways in Drosophila
(Meier et al., Nature 2000)
CG4091-RNAi partially blocks PCD
induced by Diap1-RNAi
0 after dsRNA treatment (AVE)
5 days after dsRNA treatment: (AVE)
1.60E+06
1.20E+06
1.00E+06
8.00E+05
6.00E+05
4.00E+05
2.00E+05
RNAi construct
40
91
Ia
p+
CG
Ia
p+
Dr
on
c
s+
IA
P
H
Ia
p
C
G
40
91
ro
nc
D
H
s
ds
RN
co
nt
ro
l
A
0.00E+00
no
No. of live cells
1.40E+06
PCD pathways in Drosophila
TNF?
CG4091
(Meier et al., Nature 2000)
CG4091: work in progress
RNAi in
Drosophila l(2)mbn cells
Overexpression and
loss-of-function
in vivo
(flybase.bio.indiana.edu)
siRNA in
mammalian cells
P-element excision
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.