Surfing genomes - systematic analysis of cell death regulation through comparative genomics

2. 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.

3. Cell Death Is A Fundamental Biological Process Cancers, Autoimmune Diseases, Neurodegenerative Diseases Stroke, AIDS etc.

5. egl-1 ced-9 ced-4 ced-3 Apaf-1 Bid etc. Bcl-2 etc. Bak etc. cyto. c mitochondria Smac etc. rpr/hid/grim/sickle Diap1 etc dcp-1/ drice Xiap etc. FADD TRADD TNFR Fas DR5 etc TNF FasL Trail etc. Proximal Caspases Effecter Caspases Cell Death Regulatory Pathways

6. Piecing Up Cell Death Machinery in Drosophila Domain-Based Searching of Cell Death Regulatory Proteins BID_MOUSESESQEEIIHN IARHLAQIGDEM DHNIQPTLVR BAD_MOUSE APPNLWAAQR YGRELRRMSDEF EGSFKGLPRP BAK_MOUSE PLEPNSILGQ VGRQLALIGDDI NRRYDTEFQN BAXB_HUMAN PVPQDASTKK LSECLKRIGDEL DSNMELQRMI BimS EPEDLRPEIR IAQELRRIGDEF NETYTRRVFA HRK_HUMAN LGLRSSAAQL TAARLKALGDEL HQRTMWRRRA Egl-1 DSEISSIGYE IGSKLAAMCDDFDAQMMSYSAH BH3 domain

7. Piecing up cell death machinery inDrosophila Identified through motif search 1 2 1 0 1 2 1 N/A Domain (No. of HMM models) NBD (5) BH4(2), BH1, BH2, BH3 (3) BIR (2) RGH_N (1) DED DD(2) CARD(2), CasN, CasC P35CrmA(2) total 24 HMM models Known 0 0 2 3 0 0 3 0 Protein families CED-4/APAF-1 CED-9/Bcl-2/ EGL-1/BID IAPs RGH DED-Protein DD-Protein CASPASES P35/CrmA (as of august 1999)

8. Drosophila as a model for studying apoptosis Forward and Reverse Genetics Buffy dBok Hac-1 (Ark, dApaf) Dronc Iap2 dcp-1 Grim Iap1 Reaper drIce Hid Deterin Decay Dredd dTNF (Eiger) dTNFR (wengen) dFADD

9. Gas and Brake Model for Caspase Activation Bcl-2-like ? Hac-1/Ark Reaper (grim, hid, Sickle) Apoptosis Caspase activation (dcp-1, drice, ?) Diap-1 Zhou et al, Mol. Cell 1999 Rodriguez et al, EMBO J. 2002

10. 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.

11. Reaper, Grim, Hid, Sickle Jfrac2 fly -- Smac/Diablo, HtrA2/Omi human How relevant is the Drosophila Reaper/Grim/Hid genes? Two classes of IAP-antagonists Position of IAP-binding motif (IBM)

12. Reaper 2 AVAFYIPD Grim 2 AIAYFIPD Sickle 2 AIPFFEEE Hid 2 AVPFYLPE Class I Jfrac2 17 AKPEDNES Smac/DIA 56 AVPIAQKS Omi/HtrA 134 AVPSPPPA Class II IBM-Iap binding motif

13. The functional mechanism of class I and II Iap-antagonists is identical/similar Wu et al., Mol. Cell. 2001

14. Requires cleavage to expose IBM Transcription / PTM Regulation Reaper, Grim, Hid, Sickle Jfrac2 fly -- Smac/Diablo, HtrA2/Omi human How relevant is the Drosophila Reaper/Grim/Hid genes? Two classes of IAP-antagonists Position of IAP-binding motif (IBM)

15. 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

16. Does mosquito have reaper/grim -like Iap-antagonists ?

17. 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

18. Caspases of Fly and Mosquito

19. Iaps for Fly, Mosquito, and Human

20. 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

21. How far away is fly, mosquito, and mice 300 M 445 M 550 M 235 M mice chick fish mosquito fly Differential rate of evolution sequence divergence between fruit fly and mosquito is greater than that between zebra fish and mice. Based on Peterson et al. PNAS 2004

22. How far away is fly, mosquito, and mice mice 300 M chick 445 M fish mosquito 576 M fly Differential rate of evolution sequence divergence between fruit fly and mosquito is greater than that between zebra fish and mice. Based on Peterson et al. PNAS 2004

23. Comparison of cell death machinery

24. 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. hid grim reaper sickle

25. 5 species / 2 subgenera D. mela SubgenusSophophora D. yaku D. pseu D. viri SubgenusDrosophila D. moja 60 million years

26. The four genes existed before the divergence of the subgenera 60 million years ago. Zhou, 2005 Cell Death & Differentiation

27. Pile up of the IAP-binding motifs

28. 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)

29. Michelob_x is the missing IAP-antagonist in mosquito genome -- sequence

30. MX(-IBM) :HA MX(-IBM) :Flag MX:HA MX:Flag Anti-Flag Anti-HA b The Iap-binding motif (IBM) is required for MX pro-apoptotic activity Reaper Mx

31. b Mx is a “pure” Iap-antagonist lacking GH3 domain Zhou et al., EMBO Reports 2005

32. 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

33. Mx ? Class I Hid Reaper Grim Sickle Smac, HtrA2 Jfrac2 ? Corp ? ? Evolution of the IAP /(Corp) pathway(s) flies mosquitoes mammals Class II

34. 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.

35. 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

36. Repair Cellular damage Signal transduction Checkpoint Apoptosis machinery Irradiation -induced apoptosis Irradiation Apoptosis

37. -ray -induced lethality is dependent on developmental stage Würgler and Ulrich, 1976 - Summarized by M. Ashburner 1989

38. X-ray only induces cell death at sensitive stages

39. Same irradiation – different outcomes X-ray Cell death / Tissue ablation Resistance / Survival

40. Embryos at different stage • Undifferentiated • Differentiating • Post-mitotic Irradiation treatments (2-3) RNA sample Affy hybridization Data analysis triplicate Array -Based Assay of Genomic Response to Cytotoxic Stimuli • DNA damage stimuli • X-ray • UV (C & B) • Etoposide, Camptothecin

41. Signal Transduction Check Point Transcrip. factors Identifying shared pattern using MEME DNA damage stimuli Functional testing of “Sensitive” and “resistant” elements Data analysis (2) – probing the mechanism Transcrip. response Genomic sequence of co-regulated genes Genomic sequence comparison

42. Sensitive stage Resistant stage Detectable genes 1454 479 3115 What are the molecular differences between sensitive and resistant cells ?

43. Sensitive stage Resistant stage Detectable genes 1454 479 3115 Status of cell death regulatory genes in sensitive vs resistant stages

44. 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).

45. Sensitive stage Resistant stage Induced genes 0 11 23 Immediate genomic response to irradiationX-ray induced Genes

46. Hid Immediate x-ray responsive genes in sensitive embryos

47. Sensitive stage X-ray induced genes (15-20 mins)1.)Array Measurements (CG10965) Hid Control X-ray Control X-ray

48. Sensitive stage X-ray induced genes2.) Northern Hybridization

49. Sensitive stage X-ray induced genesTime Course with QT-PCR apoptosis apoptosis

50. Coordinated genomic responses mediate x-ray induced cell death Sensitive cells reaper hid corp Death Checkpoint mechanism X-ray Repair / Cell cycle control Resistant cells