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1600 1620 siwanon jirawatnotai

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1600 1620 siwanon jirawatnotai

  1. 1. Siwanon Jirawatnotai, Ph.D. Siriraj Laboratory of Systems Pharmacology, Dept of Pharmacology, Siriraj Medical School A Direct Role of Cyclin D1 in DNA Repair
  2. 2. Cyclin D1 is a cell cycle protein that drives mammalian cell division gallus.reactome.org
  3. 3. A function of cyclin D1 in DNA repair uncovered by proteomic screenings TAP Mass-spec MCF-7, ZR-75-1 UMSCC-2 GRANTA 519 HT29 Jirawatnotai, Nature 2011
  4. 4. Cyclin D1 is required for cell survival after DNA damage 0 h 16 h sicont siD1-B 0 20 40 60 80 Percentofcells Tail length (pixels) sicont siD1-B 0 20 40 60 80 Percentofcells Tail length (pixels) *** * *** 0 h 16 h
  5. 5. Pierce A .,et al. 1999 0 20 40 60 80 100 120 Percent *** 0 20 40 60 80 100 120 Percent *** Interaction between cyclin D1 and RAD51 facilitates efficient homologous recombination Valerie et al., 2003 Oncogene
  6. 6. Cyclin D1 directly interacts with RAD51 SP49 Z138C Granta 519 H2009 ZR-75-1 HT-29 IP RAD IgG WCL WB: cyclin D1 MCF7 In vivo bindingIn vitro binding
  7. 7. N-terminus domain of cyclin D1 and C-terminus domain of RAD51 are require for the interaction ***
  8. 8. Interaction between cyclin D1 and RAD51 facilitates RAD51 loading to damage foci 0 20 40 60 80 100 0-2 3-9 10-19 Percentofcells Foci/nucleus 4 h *** *** *** *** * * 0 20 40 60 80 100 0-2 3-9 10-19 Percentofcells Foci/nucleus 0 h 0 20 40 60 80 100 0-2 3-9 10-19 Percentofcells Foci/nucleus 2 h * *** *** *** *** * 0 20 40 60 80 100 0-2 3-9 10-19 Percentofcells Foci/nucleus 8 h sicont siD1-A siD1-C *** *** *** *** ** ** 0 20 40 60 80 100 0-2 3-9 10-19 Percentofcells Foci/nucleus 6 h *** *** *** *** *** *** sicont siD1-A siD1-C DAPI RAD51 -H2AX Merge sicont siD1-A siD1-C + DNA damage
  9. 9. Cyclin D1 localizes at damaged DNA 0 2 4 6 8 Foldenrichment Before DNA break After DNA break 0 2 4 6 8 Foldenrichment siD1 0 5 10 15 20 25 IgG Cyclin D1 E2F4 Foldenrichment Before DNA break After DNA break 0 5 10 15 20 25 Foldenrichment siD1 Pierce A .,et al. 1999 Targeted ChIP DAPI - H2AX cyclin D1 Merge Immunofluorescence
  10. 10. Interaction between cyclin D1 and BRCA2 B2-1 B2-3 B2-4 B2-5 B2-6 B2-7 B2-8 B2-9 B2-2 GST Cyclin D1 binding to BRCA2 in vitro (GST-pull-down) BRCA2+/-H2AX+ siD1-A sicont Esashi, et al. 2005 Input GST RAD51 binding to BRCA2 in vitro (GST-pull-down) Esashi, et al. 2005 Cyclin A/CDK2 phosphorylation of BRCA2
  11. 11. Nocodazole D1 GST-B2-9 Roscovitin +--- -+-- ++++ Cyclin A GST-B2-9 RAD51 Cyclin D1 GST B2-9 pull-down 293 cells Phospho BRCA2 S3291 Kinase assay Kinase assay Expression of cyclin D1 helps recruit RAD51 to C-terminus domain of BRCA2 by preventing the inhibitory S3291 phosphorylation by cyclin A/CDK2 Cyclin D1 expression helps recruiting RAD51 to C-ter of BRCA2 Phospho BRCA2 S3291 BRCA2 Cyclin D1 Cyclin B1 Cyclin A2 RAD51 CDK4 b-Actin Async. G2/M (nocodazole) - D1 Ro - D1 Ro Cyclin B1 long exp 293 cells Cyclin D1 expression prevents B2-9 phosphorylation by CDK2 Lysate +++-
  12. 12. G2/M - Phospho BRCA2 S3291 Cyclin D1 Cyclin A2 CDK4 Cyclin B1 RAD51 HeLa cells Expression of cyclin D1 helps recruit RAD51 to C-terminus domain of BRCA2 by preventing the inhibitory S3291 phosphorylation by cyclin A/CDK2 Depletion of cyclin D1 by siRNA increases S3291 phosphorylation
  13. 13. IP: B2-9 G2 IR (5 Gy) - + - + Cyclin A2 Cyclin D1 BRCA2S3291 Asynchronous GST B2-9 Cyclin D1 is recruited to C-terminus of BRCA2 upon DNA damage WB
  14. 14. In vitro Competition assay rHA-Cyclin D1 GST-BRCA2 C-Ter (B2-9) - + + + + + - + + + + + rCyclin - - - - + + + + + + + + + + + + DA WB: HA GST Cyclin D1 controls phosphorylation of C-terminus BRCA2 by competing with cyclin A for BRCA2 binding
  15. 15. Cyclin D1 cooperates with DNA damage to inhibit BRCA2 phosphorylation at Ser3291 Nocodazole - + - + - + - + - + IR - - - - - - + + + + D1 - - - - + + - - + + Ros - - + + - - - - - - 1 2 3 4 5 6 7 8 9 10 S3291 Ph cyclin D1 BRCA2 CDK4 actin IB: γH2AX cyclin D1 IB: GST GST pulldown 0.5 1 2 4 6 Hour after IR actin IB: S3291 Ph BRCA2 cdc25a NoIR
  16. 16. aa 20-90 of cyclin D1 is required for BRCA2-C-terminal binding full-length 20-295 91-295 full-length 20-295 91-295 B2-5 B2-9 inp IP WB: Cyclin D1 full-length WB:GST B2-9 binding domain of cyclin D1 (aa 20-91) is required to prevent CDK2/Cyclin A2 phosphorylation on B2-9 D1 1-295 1-90 IB: GST cyclin D1 B2-9 kinase assay cyclin A-CDK2 0 20 40 60 80 100 120 0 nM 10 nM 20 nM 40 nM Relativephosphorylationlevel Concentration of recombinant cyclin D1 D1 del.91- 295 cyclin D1 Δ1-90 BSAB S A BSA BSA
  17. 17. Cyclin box domain of cyclin D1 is required for RAD51 and BRCA2 binding Cyclin box
  18. 18. Shot exp. Long exp. Shot exp. Long exp. Shot exp. Long exp. GST-B2-5 GST-B2-9 GST-pRb GST-B2-9 GST-pRb GST-B2-5 WB: GST Cyclin D1 that is in complex with CDK4 does not compete with CDK2/cyclin A Shot exp. Long exp. Kinase assay CDK4/Cyclin D1 CDK2/Cyclin A2 CDK2/Cyclin A2 CDK4/Cyclin D1 GST-B2-9 GST-Cyclin D1 GST-Cyclin A WB: GST 1 10 100 0 Gy 2 Gy 4 Gy 6 GyPercentsurvival 0 µM 1 µM 3 µM
  19. 19. Cyclin D1 depletion hypersensitizes Rb-negative cancers to IR treatment Jirawatnotai, Nature 2011
  20. 20. Cyclin D1 promotes RAD51 localization on damage DNA, and HR Jirawatnotai, Oncogene, in press
  21. 21. No DNA damage Severe DNA damage Jirawatnotai, Cancer Research, 2012
  22. 22. Thanks • Chonvara Chalermrujinanant • Gunya Sittithumcharee • Peter Sicinski, DFCI – Wojtek Michowski – Lisa Becks • Fumiko Esashi (University of Oxford) • David Livingston lab, DFCI – Yiduo Hu • Steve Gygi lab, HMS – Joshua Elias • John Quackenbush, CCCB, DFCI – Yaoyu Wang • Bing Xia (University of Medicine and Dentistry • of New Jersey)

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