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Surfing genomes - systematic analysis of cell death regulation through comparative genomics
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Transcript of Surfing genomes - systematic analysis of cell death regulation through comparative genomics
Surfing genomes - systematic analysis of cell death
regulation through comparative genomics
• Sea of sequences Sea of Genomes
• Genomic sequence comprehensive view of cell death regulation system in one organism.
• Genomes understanding the evolution of systems and fundamentals of system construction and regulation.
Cell Death Is A Fundamental Biological Process
Cancers,
Autoimmune Diseases,
Neurodegenerative Diseases
Stroke, AIDS etc.
Cell Death Regulatory Pathways
rpr/hid/grim/sickle Diap1 etc
TNFFasLTrail etc.
TNFRFasDR5etc
FADDTRADD
egl-1 ced-9 ced-4Apaf-1Bcl-2 etc.Bid etc.
Bak etc.cyto. c
mitochondria
Smac etc.
Xiap etc.
ced-3
dcp-1/ drice
Effecter Caspases
Proximal Caspases
Piecing Up Cell Death Machinery in Drosophila
BID_MOUSE SESQEEIIHN IARHLAQIGDEM DHNIQPTLVRBAD_MOUSE APPNLWAAQR YGRELRRMSDEF EGSFKGLPRPBAK_MOUSE PLEPNSILGQ VGRQLALIGDDI NRRYDTEFQNBAXB_HUMAN PVPQDASTKK LSECLKRIGDEL DSNMELQRMIBimS EPEDLRPEIR IAQELRRIGDEF NETYTRRVFAHRK_HUMAN LGLRSSAAQL TAARLKALGDEL HQRTMWRRRAEgl-1 DSEISSIGYE IGSKLAAMCDDF DAQMMSYSAH
Domain-Based Searching of Cell Death Regulatory Proteins
BH3 domain
Piecing up cell death machinery in Drosophila
Protein familiesCED-4/APAF-1CED-9/Bcl-2/EGL-1/BIDIAPsRGHDED-ProteinDD-ProteinCASPASES
P35/CrmA
Domain (No. of HMM models) NBD (5)BH4(2), BH1, BH2,BH3 (3)BIR (2)RGH_N (1)DEDDD(2)CARD(2), CasN, CasCP35CrmA(2)total 24 HMM models
Known 0 0 2 3 0 0 3
0
Identifiedthrough motif search 1 2 1 0 1 2 1
N/A
(as of august 1999)
Drosophila as a model for studying apoptosis
dBok Hac-1(Ark,dApaf)
HidIap1
dcp-1
dTNF(Eiger)
dTNFR(wengen)
dFADD
ReaperGrim
Deterin
Iap2
Buffy
drIce
Decay
Dredd
Dronc
Forward and Reverse Genetics
Bcl-2-like ?
Hac-1/Ark
Caspase activation(dcp-1, drice, ?)
Reaper (grim, hid, Sickle)
Diap-1 Apoptosis
Gas and Brake Model for Caspase Activation
Zhou et al, Mol. Cell 1999
Rodriguez et al, EMBO J. 2002
Up and downs of the Reaper/grim/hid genes?
• Reaper Identified in early 90’s, cheered as gene of the year by Science in 94. Hid and grim reported in 95 and 96, respectively.
• Revelation of functional mechanism through genetic analysis.
• Smac/Diablo identified in 2000.
• Structure of the whole protein remains unsolved.
• No orthologs identified outside of Drosophila.
How relevant is the Drosophila Reaper/Grim/Hid genes?
Two classes of IAP-antagonists
Position of IAP-binding motif (IBM)
Jfrac2fly Reaper, Grim, Hid, Sickle
Smac/Diablo, HtrA2/Omi
human --
IBM-Iap binding motif
Reaper 2 AVAFYIPDGrim 2 AIAYFIPDSickle 2 AIPFFEEEHid 2 AVPFYLPE
Class I
Jfrac2 17 AKPEDNESSmac/DIA 56 AVPIAQKSOmi/HtrA 134 AVPSPPPA
Class II
The functional mechanism of class I and II Iap-antagonists is identical/similar
Wu et al., Mol. Cell. 2001
How relevant is the Drosophila Reaper/Grim/Hid genes?
Two classes of IAP-antagonists
Position of IAP-binding motif (IBM)
RegulationTranscription / PTM
Requires cleavage to expose IBM
Jfrac2fly Reaper, Grim, Hid, Sickle
Smac/Diablo, HtrA2/Omi
human --
Are RGH proteins (and Corp) conserved?
• IAPs are highly conserved.
• Binding of RGH to IAP is identical to that of Smac.
• RGH protein kills mammalian cell when transfected.
No ortholog has been identified outside of the Drosophila genus
Does mosquito have reaper/grim -like Iap-antagonists ?
Immunity-Related Genes and Gene Families in Anopheles gambiae
George K. Christophides,1* Evgeny Zdobnov,1* Carolina Barillas-Mury,2 Ewan Birney,3 Stephanie Blandin,1 Claudia Blass,1 Paul T. Brey,4 Frank H. Collins,5 Alberto Danielli,1 George Dimopoulos,6 Charles Hetru,7 Ngo T. Hoa,8 Jules A. Hoffmann,7 Stefan M. Kanzok,8 Ivica Letunic,1 Elena A. Levashina,1 Thanasis G. Loukeris,9 Gareth Lycett,1 Stephan Meister,1 Kristin Michel,1 Luis F. Moita,1 Hans-Michael Müller,1 Mike A. Osta,1 Susan M. Paskewitz,10 Jean-Marc Reichhart,7 Andrey Rzhetsky,11 Laurent Troxler,7 Kenneth D. Vernick,12 Dina Vlachou,1 Jennifer Volz,1 Christian von Mering,1 Jiannong Xu,12 Liangbiao Zheng,8 Peer Bork,1 Fotis C. Kafatos1
Science (2002) 298:159-165
Caspases of Fly and Mosquito
Iaps for Fly, Mosquito, and Human
Does mosquito have reaper/grim -like Iap-antagonists ?
• “…The expansion of both IAPs and effector caspases in the mosquito as compared to Drosophila possibly suggests coevolution of apoptotic regulators that may fine tune cell death and/or immune responses in the mosquito, such as those in midgut cells invaded by Plasmodium.”
• “…The search for mosquito pro-apoptotic genes was hampered by the rapid sequence diversification of the main players.”
Christophides, et al. (2002) Science 298
chickmice
How far away is fly, mosquito, and mice
fly
mosquito
Based on Peterson et al. PNAS 2004
fish
235 M
550 M
445 M300 M
Differential rate of evolution sequence divergence between fruit fly and mosquito is greater than that between zebra fish and mice.
chickmice
How far away is fly, mosquito, and mice
fly
mosquito
Based on Peterson et al. PNAS 2004
fish
576 M
445 M300 M
Differential rate of evolution sequence divergence between fruit fly and mosquito is greater than that between zebra fish and mice.
Comparison of cell death machinery
Mosquito Fly Human
Caspases 12 7 12
IAP 6 4 6
IAP-antagonists (Type I)
0 ? 4 ?
Reaper, Grim, Sickle, and Hid
• Pro-apoptotic genes clustered in close vicinity of chromosome region 75C.
• Together they are required for most developmental cell death.
sicklereapergrimhid
60 million years
D. mela
D. yaku
D. pseu
D. viri
D. moja
Subgenus
Sophophora
Subgenus
Drosophila
5 species / 2 subgenera
The four genes existed before the divergence of the subgenera 60 million years ago.
Zhou, 2005 Cell Death & Differentiation
Pile up of the IAP-binding motifs
Database search strategy
Build HMM models for IBM and GH3 using MEME (SDSC).
Search for matches to IBM immediately following a Methionine in genomic sequence.
Gene prediction analysis and EST search. Conservation of sequence in different mosquito
genomes.
Results: michelob_x (mx), michelob_y (my), and michelob_z (mz)
Michelob_x is the missing IAP-antagonist in mosquito genome -- sequence
b
a
0%
20%
40%
60%
80%
100%
Cel
l Sur
viva
l
Anti-Flag
Anti-HA
MX
:HA
MX
:Fla
g
MX
(-IB
M) :
HA
MX
(-IB
M) :
Fla
g
The Iap-binding motif (IBM) is required for MX pro-apoptotic activity
Reaper Mx
b
c
0%20%40%60%80%
100%
rpr + diap1
rpr(107) + diap1
grim + diap1mx + diap1
mx + p35
Cel
l Su
rviv
al
1:1
1:2
1:5
Mx is a “pure” Iap-antagonist lacking GH3 domain
Zhou et al., EMBO Reports 2005
Michelb_x is the missing Class I IAP-antagonist in mosquito genomes
• Conserved Iap-binding motif (IBM) required for pro-apoptotic activity.
• Pro-apoptotic activity is blocked by Iap.
• Transcriptionally regulated in response to cytotoxic stimuli (UVC).
• Binding between Mx and Iap
• Induction of mx upon virus or parasite infection
Evolution of the IAP /(Corp) pathway(s)
mosquitoesflies mammals
Class II
HidReaperGrimSickle
Mx ?Class I
Jfrac2 Smac, HtrA2
?
Corp ? ?
Caspases 7 12 12
IAP 4 6 6
Significance of Identifying Pro-apoptotic Genes in Mosquitoes
• Understanding transduction of malaria and other pathogens (dengue virus, west nile virus, etc) in mosquitoes.
• Vector population control strategies.
Understanding irradiation -induced apoptosis -- HPC for the Junk sequences
The underlying mechanism for many medical applications of ionizing irradiation
50% cancer patients undergo radiation therapy at various stage of treatment
Irradiation -induced apoptosis
Irradiation
Apoptosis
Cellular damage
Signal transduction Checkpoint
Apoptosis machinery
Repair
-ray -induced lethality is dependent on developmental stage
Würgler and Ulrich, 1976 - Summarized by M. Ashburner 1989
X-ray only induces cell death at sensitive stages
Same irradiation – different outcomes
X-ray
Cell death / Tissue ablation
Resistance / Survival
0-3hr 13-16_X-Ray0-3 _X-Ray 4-7hr 4-7_X-ray 8-11hr 8-11_X-ray 13-16
Sample0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
3.60
3.80
4.00
4.20
4.40
4.60
4.80
5.00NormalizedIntensity
CS-Xray
Array -Based Assay of Genomic Response to Cytotoxic Stimuli
DNA damage stimuli
• X-ray
•UV (C & B)
• Etoposide, Camptothecin
Irradiation treatments (2-3)
RNA sample
Affy hybridization
triplicate
Data analysis
Embryos at different stage
• Undifferentiated
• Differentiating
• Post-mitotic
Data analysis (2) – probing the mechanism
Transcrip. factors
Transcrip. response
Signal Transduction
Check Point
DNA damage stimuli
Genomic sequence of co-regulated genes
Identifying shared pattern using MEME
Functional testing of “Sensitive” and
“resistant” elements Genomic
sequence comparison
What are the molecular differences between sensitive and resistant cells ?
1012 10872648
4-7P 8-11P
9263
1454 479
3115
Sensitive stage Resistant stageDetectable genes
Status of cell death regulatory genes in sensitive vs resistant stages
1012 10872648
4-7P 8-11P
9263
1454 479
3115
Sensitive stage
Resistant stageDetectable genes
What are the molecular differences between sensitive and resistant cells ?
• Higher expression level (active transcriptional regulation) of cell death regulatory genes in sensitive embryos (cells).
Immediate genomic response to irradiationX-ray induced Genes
1012 10872648
4-7P 8-11P
9263
11 23
0
Sensitive stage Resistant stageInduced genes
153035_AT CG10965
143680_AT rpr apoptosis apoptosis activator
DNA damage response
induction of apoptosis by ionic changes
154125_AT CG7590 146318_AT CG15480 152052_AT BcDNA:GH06193
152208_AT Fkbp13 peptidyl-prolyl cis-trans isomerase
FK506 binding
143411_AT W apoptosis activator
apoptosis
153091_AT CycT transcription elongation factor
transcription heat shock response
cyclin-dependent protein kinase\, regulator
154421_AT fax 143384_AT trx DNA binding positive regulation of homeotic gene (trithorax group) 147969_AT CG9184
Hid
Immediate x-ray responsive genes in sensitive embryos
Sensitive stage X-ray induced genes (15-20 mins)1.) Array Measurements
(CG10965)
Hid
Control ControlX-ray X-ray
Sensitive stage X-ray induced genes2.) Northern Hybridization
Sensitive stage X-ray induced genesTime Course with QT-PCR
Time Course
0
2
4
6
8
10
12
0 50 100 150
Time (mins)
Ra
tio
(x
-ra
y/co
ntr
ol)
S_rpr
R_rpr
S_corp
R_corp
apoptosis apoptosis
Coordinated genomic responses mediate x-ray induced cell death
X-ray
Repair / Cell cycle control
Checkpoint mechanism
reaperhidcorp
Sensitive cells
Resistant cells
Death
Same irradiation – different outcomes
X-ray
Cell death / Tissue ablation
Resistance / Survival
Regulation of cell death gene expression – Junk sequences
sicklereapergrimhid
43kb75kb
Reaper: 1 kb transcript, 195 bp ORF
Greedy Nature of “Junk” Sequence Analysis
Untranslated DNA sequence in reaper locus (120kb)
Protein seq. of HID, Reaper, Grim, Sickle
D. mela
D. yaku
D. pseu
D. viri
D. moja
1 cpu, 1-2 minutes
64 cpu, over 2 days
MEME -mode tcm, -maxmotifs 12,
Functional significance of the “junk” region
43kb75kb
Deletion lethal
reaper sickle
Motifs identified by MEME
E47 c-Myb RSRFC4
29
165bps
32246
1065bps86 25 30 30
Finding the signal pattern among noises – Binding-site level identification of shared
signal-module (Bliss)
Meng, Banerjee, & Zhou, 2006, in preparation
Computers In The Wet Lab
• Local database for data integration.
IKBAR(Integrated Knowledge Base for Apoptosis Research)
Regulatory Pathways
Interactions
Cell death regulatory genes
Gene (genomic)
Expressed Seqs
Proteins
IKBAR: A community-based system-specific knowledge base
IKBAR
ETR sever
Web severT
ech.
Man
agemen
t
Expert Users
Reg. Users
IKBAR Team
ETR: Event-Trigger-Rule Huang et al, 2003 Bioinformatics
Computers In The Wet Lab
• Local database for data integration.
• XML.
• Visualization tools, like Apollo.
• State of the art applications, MEME(SDSC), Primer3(MIT/WI), Blast(NCBI/NIH), GSEA (MIT), etc.
Acknowledgement
UF College of Medicine:
Gina Chan
Yanping Zhang
Hailong Meng
Carl P. Santos
Lei Zhou
BMS, Inc.
Dr. Pam Carroll
Bo Gua
Hong XiaoDr. Lei Xiao
Guohua Jiang
UF College of Engineering
UF HPC Grid
Dr. Charles A. Taylor
Dr. Alan D.George
Dr. Arunava Banerjee.
Dr. Stanley Su
Supported by NIH/NCI, ACS, UFSCC, HHMI
Synergism between x-ray inducible pro-apoptotic genes
Reaper
HID
Corp(CG10965)
ReaperHID Corp
+
++++ -
Future directions (II)
• Transcriptional mechanism mediating sensitivity/resistance to X-ray induced gene expression and cell death.– Decision were made within minutes of x-ray– P53 only is not sufficient to restore
sensitivity– Genetic screen for mutants affect this
response
Iap –interacting motif
GH3 motif
Reaper
Grim
Sickle
HID
GH3 (Grim Helix 3) domain
sickle 70 4.79e-16 PPSAEEQLLA W K F L A I T M C K V L K Q F YQQHKSSGKS
GRIM 86 3.03e-15 GSMTMSEFGC W D L L A Q I L C Y A L R I Y SYSSSQRQPT
RPR 32 5.27e-14 QQILRLRESQ W R F L A T V V L E T L R Q Y TSCHPKTGRK
CG15343 182 2.90e-11 YTAWKFQPQR W D F L K V G L D Q I A D R V QYRLQKDGKW
CG10965 56 1.59e-10 KALILTNGKD Y V F E A H L L E Y F L L L F PCPEITFHLR
Corp(CG10965)
Claveria et al 2002 EMBO J
Corp is expressed in cells destined to die
Excision mutants of corp
10 kb
1 kbStopATG
corp
CG1632
CG15343
E19A 100%
E1A 71%
E95B 84%
lethality
Corp is required for mediating x-ray induced cell death
Gal4 / UAS system – GOF analysis
CorpUASGal4enhancer
Gal4enhancer
CorpUAS
Dramatic Synergism Between Corp and
Hid
Coordinated genomic response mediate x-ray induced cell death
X-ray
Repair / Cell cycle control
Checkpoint mechanism
reaperhidcorp
Sensitive cells
Resistant cells
Death
Michelb_x is the missing IAP-antagonist in mosquito genome -- evidences
0%20%40%60%80%
100%120%
cont
rol
rpr
rpr +
Diap
1 mx
mx +
p35
mx +
Dia
p1
mx(
-IBM
)
mx(
-IBM
) + D
iap1
Series1
Michelb_x is the missing IAP-antagonist in mosquito genome -- implications
Michelob_x does not have the GH3 domain
GH3 domain evolved separately with the IAP-binding motif (IBM), joined together with IBM in Drosophila.
GH3 domain is not required for the function of RGH proteins, not stable during evolution, lost in mosquitoes.
Mining Knowledge From Genomic Data
1. Identifying novel cell death regulatory genes
Irradiation Responsive genes
Genes have potential cell death regulatory motif
The Tough Questions
• How significant is the collaboration between Hid (IAP antagonist) and Corp (GH3 domain protein) -- Is it relevant to human?
• Is the pathway conserved?
Status of cell death machinery in different embryonic stages
Focusing on cell death regulatory genes
Selection of cell death regulatory genes
Based on Gene Ontology
http://www.geneontology.org/
Function assignment with GO
probe set gene name E1 E2 E3 E4 Role in cell death 154947_AT CG7263(AIF) 222.4 87.3 132.7 97 144023_AT Rep1 69.5 A/P A/P A/P 144206_AT Rep4 26.3 A/P Absent Absent
DNA fragmentation
153796_AT Dcp-1 316.4 595.6 A/P A/P 152941_AT Ice 433.7 729.4 289.5 355 147043_AT damm Absent Absent Absent Absent 153776_AT dream Absent Absent Absent Absent
Effecter capases
141423_AT Nc 183.7 112 A/P A/P 144201_AT decay Absent Absent A/P A/P 152345_AT Dredd Absent Absent Absent Absent
Apical caspases
148853_AT PDCD-5 412.3 278.367 A/P A/P 151854_AT morgue 157.9 118.533 A/P A/P 149628_AT bruce 71.7 A/P A/P A/P
Ubiquitination / protein
degradation
150057_at det 284.4 290.7 A/P 68.4 152631_AT th 289.7 1,141.50 974.4 780 154697_AT Iap2 162 138.9 145.8 125.2
Inhibitor of apoptosis protein
(IAP)
143680_AT rpr Absent A/P Absent Absent 143411_AT W A/P 97.3 A/P A/P 143862_AT grim Absent Absent A/P Absent 148994_AT sickle Absent Absent Absent Absent
IAP antagonist
152705_AT Ark Absent 67.5 Absent Absent 143115_AT Cyt-C1 Absent Absent Absent Absent 141371_AT Cyt-C2 1322.833 1366.8 1211.833 1331.933
Ced4 / apaf-1
142130_AT debcl (1) 129.3 A/P Absent Absent 151475_AT debcl (2) 612.3 A/P 151.8 153.8 151003_AT Buffy Absent Absent Absent Absent
Ced9 / Bcl-2 family
143728_AT Eip93F Absent Absent Absent Absent 143073_AT aop 362.8 1,077.20 280.3 282.7 142193_AT pnt Absent A/P Absent Absent 150416_at p53 99.9 A/P A/P Absent
Transcription factors
154982_AT CG1388 (tak1)
205.3 207.7 A/P A/P
154411_AT lok 621.5 139.4 Absent Absent 144125_AT Pten 148.9 96.6 A/P Absent 141791_AT Akt1 186.4 102.9 A/P A/P
Signal transduction
154450_AT abs 34.6 64.8 A/P A/P 154197_at CG10473 244.4 297.3 123.1 130.8 146029_AT CG13393 877.2 1,184.80 1,206.40 986.7
RNA interacting proteins
143858_AT crq 68.8 42.4 A/P A/P 151493_AT dib Absent Absent Absent Absent 143414_AT wg Absent 86.4 A/P A/P 146921_AT eiger(TNF) A/P A/P A/P A/P 153667_AT Aac11 184.4 421.8 A/P A/P
Membrane / secreted proteins
143953_AT Cas 577.2 141.1 Absent Absent 146383_at mdy(cg17938) 187.6 252.4 104.1 128.3 154661_at mdy (cg1793) 139.2 129.7 231.9 225.3 146381_AT mdy(cg13273) Absent Absent Absent Absent
other cellular protein
200-500
>500
50 - 200
Absent - 50