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

http://www.ikbar.org/

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