Hedgehog/Gli Control by Ubiquitination/Acetylation Interplay

Vitamins and Hormones

Volumn 88, Issue , 2012, Pages 211-227

  Gulino, Alberto ^a,c   Di Marcotullio, Lucia ^a   Canettieri, Gianluca ^a   De Smaele, Enrico ^b   Screpanti, Isabella ^a  

^a SAPIENZA UNIVERSITY OF ROME   (Italy)

^b SAPIENZA UNIVERSITY OF ROME   (Italy)

^c IRCCS NEUROMED   (Italy)

Author keywords

Gli; HECT E3 ligases; Hedgehog; Histone deacetylases; RING E3 ligases; Ubiquitin

Indexed keywords

EID: 84857778358     PISSN: 00836729     EISSN: None     Source Type: Book Series    
DOI: 10.1016/B978-0-12-394622-5.00009-2     Document Type: Chapter

References (99)

1. Ahn S., Joyner A.L. In vivo analysis of quiescent adult neural stem cells responding to Sonic hedgehog. Nature 2005, 437:894-897.
2. Angers A., Ramjaun A.R., McPherson P.S. The HECT domain ligase itch ubiquitinates endophilin and localizes to the trans-Golgi network and endosomal system. J. Biol. Chem. 2004, 279:11471-11479.
3. Argenti B., Gallo R., Di Marcotullio L., Ferretti E., Napolitano M., Canterini S., et al. Hedgehog antagonist REN(KCTD11) regulates proliferation and apoptosis of developing granule cell progenitors. J. Neurosci. 2005, 25:8338-8346.
4. Aza-Blanc P., Ramirez-Weber F.A., Laget M.P., Schwartz C., Kornberg T.B. Proteolysis that is inhibited by hedgehog targets Cubitus interruptus protein to the nucleus and converts it to a repressor. Cell 1997, 89:1043-1053.
5. Bai Y., Yang C., Hu K., Elly C., Liu Y.C. Itch E3 ligase-mediated regulation of TGF-beta signaling by modulating smad2 phosphorylation. Mol. Cell 2004, 15:825-831.
6. Barakat M.T., Humke E.W., Scott M.P. Learning from Jekyll to control Hyde: Hedgehog signaling in development and cancer. Trend Mol. Med. 2010, 16:1471-4914.
7. Berdnick D., Torok T., Gonzalez-Gaitan M., Knoblich J.A. The endocytic protein alpha-Adaptin is required for numb-mediated asymmetric cell division in Drosophila. Dev. Cell 2002, 3:221-231.
8. Boname J.M., Stevenson P.G. MHC class I ubiquitination by a viral PHD/LAP finger protein. Immunity 2001, 15:627-636.
9. Canettieri G., Di Marcotullio L., Greco A., Coni S., Antonucci L., Infante P., Pietrosanti L., De Smaele E., Ferretti E., Miele E., Pelloni M., De Simone G., et al. Histone deacetylase and Cullin3-REN(KCTD11) ubiquitin ligase interplay regulates Hedgehog signalling through Gli acetylation. Nat. Cell Biol. 2010, 12:132-142.
10. Caussinus E., Gonzalez C. Induction of tumor growth by altered stem-cell asymmetric division in Drosophila melanogaster. Nat. Genet. 2005, 37:1125-1129.
11. Cayouette M., Raff M. Asymmetric segregation of Numb: A mechanism for neural specification from Drosophila to mammals. Nat. Neurosci. 2002, 5:1265-1269.
12. Chang L., Kamata H., Solinas G., Luo J.L., Maeda S., Venuprasad K., Liu Y.C., Karin M. The E3 ubiquitin ligase itch couples JNK activation to TNFalpha-induced cell death by inducing c-FLIP(L) turnover. Cell 2006, 124:601-613.
13. Chapman G., Liu L., Sahlgren C., Dahlqvist C., Lendahl U. High levels of Notch signaling down-regulate Numb and Numblike. J. Cell Biol. 2006, 175:535-540.
14. Chastagner P., Israel A., Brou C. Itch/AIP4 mediates Deltex degradation through the formation of K29-linked polyubiquitin chains. EMBO Rep. 2006, 7:1147-1153.
15. Chen C.H., von Kessler D.P., Park W., Wang B., Ma Y., Beachy P.A. Nuclear trafficking of Cubitus interruptus in the transcriptional regulation of Hedgehog target gene expression. Cell 1999, 98:305-316.
16. Chen X., Wen S., Fukuda M.N., Gavva N.R., Hsu D., Akama T.O., Yang-Feng T., Shen C.K. Human ITCH is a coregulator of the hematopoietic transcription factor NF-E2. Genomics 2001, 73:238-241.
17. Chen M.H., Wilson C.W., Li Y.J., Law K.K., Lu C.S., Gacayan R., Zhang X., Hui C.C., Chuang P.T. Cilium-independent regulation of Gli protein function by Sufu in Hedgehog signaling is evolutionarily conserved. Genes Dev. 2009, 23:1910-1928.
18. Cheng S.Y., Bishop J.M. Suppressor of Fused represses Gli-mediated transcription by recruiting the SAP18-mSin3 corepressor complex. Proc. Natl. Acad. Sci. USA 2002, 99:5442-5447.
19. Colaluca I.N., Tosoni D., Nuciforo P., Senic-Matuglia F., Galimberti V., Viale G., Pece S., Di Fiore P.P. NUMB controls p53 tumour suppressor activity. Nature 2008, 451:76-80.
20. Correale S., Pirone L., Di Marcotullio L., De Smaele E., Greco A., Mazzà D., Moretti M., Alterio V., Vitagliano L., Di Gaetano S., Gulino A., Pedone E.M. Molecular organization of the cullin E3 ligase adaptor KCTD11. Biochimie 2011, 93:715-724.
21. Dahmane N., Ruiz I Altaba A. Sonic hedgehog regulates the growth and patterning of the cerebellum. Development 1999, 126:3089-3100.
22. De Smaele E., Ferretti E., Gulino A. Vismodegib, a small-molecule inhibitor of the hedgehog pathway for the treatment of advanced cancers. Curr. Opin. Investig. Drugs 2010, 11:707-718.
23. De Smaele E., Di Marcotullio L., Moretti M., Pelloni M., Occhione M., Infante P., Cucchi D., Greco A., Pietrosanti L., Todorovic J., Coni S., Canettieri G., et al. Identification and characterization of KCASH2 and KCASH3, two novel Cullin3 adaptors suppressing histone deacetylase and Hedgehog activity in medulloblastoma. Neoplasia 2011, 13:374-385.
24. Di Marcotullio L., Ferretti E., De Smaele E., Argenti B., Mincione C., Zazzeroni F., Gallo R., Masuelli L., Napolitano M., Maroder M., Modesti A., Giangaspero F., et al. REN(KCTD11) is a suppressor of Hedgehog signaling and is deleted in human medulloblastoma. Proc. Natl. Acad. Sci. USA 2004, 101:10833-10838.
25. Di Marcotullio L., Ferretti E., Greco A., De Smaele E., Po A., Sico M.A., Alimandi M., Giannini G., Maroder M., Screpanti I., Gulino A. Numb is a suppressor of Hedgehog signalling and targets Gli1 for Itch-dependent ubiquitination. Nat. Cell Biol. 2006, 8:1415-1423.
26. Di Marcotullio L., Greco A., Mazzà D., Canettieri G., Pietrosanti L., Infante P., Coni S., Moretti M., De Smaele E., Ferretti E., Screpanti I., Gulino A. Numb activates the E3 ligase Itch to control Gli1 function through a novel degradation signal. Oncogene 2011, 30:65-76.
27. Di Marcotullio L., Canettieri G., Infante P., Greco A., Gulino A. Protected from the inside: endogenous histone deacetylase inhibitors and the road to cancer. Biochim. Biophys. Acta. 2011, 1815:241-252.
28. Ettenberg S., Yang C., Mariano J., Tiwari S., Fang S., Lipkowitz S., Weissman A.M. WW domain HECT E3s target Cbl RING finger E3s for proteasomal degradation. J. Biol. Chem. 2003, 278:43169-43177.
29. Gallagher E., Gao M., Liu Y.C., Karin M. Activation of E3 ubiquitin ligase Itch through a phosphorylation-induced conformational change. Proc. Natl. Acad. Sci. USA 2006, 103:1717-1722.
30. Gallo R., Zazzeroni F., Alesse E., Mincione C., Borello U., Buanne P., D'Eugenio R., Mackay A.R., Argenti B., Gradini R., Russo M.A., Maroder M., et al. REN: A novel, developmentally regulated gene that promotes neural cell differentiation. J. Cell Biol. 2002, 158:731-740.
31. Gao M., Labuda T., Xia Y., Gallagher E., Fang D., Liu Y.C., Karin M. Jun turnover is controlled through JNK-dependent phosphorylation of the E3 ligase Itch. Science 2004, 306:271-275.
32. Gershey E.L., Vidali G., Allfrey V.G. Chemical studies of histone acetylation. The occurrence of epsilon-N-acetyllysine in the f2a1 histone. J. Biol. Chem. 1968, 243:5018-5022.
33. Glozak M.A., Sengupta N., Zhang X., Seto E. Acetylation and deacetylation of nonhistone proteins. Gene 2005, 363:15-23.
34. Goodrich L.V., Milenkovic L., Higgins K.M., Scott M.P. Altered neural cell fates and medulloblastoma in mouse patched mutants. Science 1997, 277:1109-1113.
35. Grachtchouk M., Pero J., Yang S.H., Ermilov A.N., Michael L.E., Wang A., Wilbert D., Patel R.M., Ferris J., Diener J., Allen M., Lim S., et al. Basal cell carcinomas in mice arise from hair follicle stem cells and multiple epithelial progenitor populations. J. Clin. Invest. 2011, 121:1768-1781.
36. Gulino A., Di Marcotullio L., Screpanti I. The multiple functions of Numb. Exp. Cell Res. 2010, 316:900-906.
37. Hatakeyama S., Yada M., Matsumoto M., Ishida N., Nakayama K.I. Ubox proteins as a new family of ubiquitin-protein ligases. J. Biol. Chem. 2001, 276:33111-33120.
38. Hooper J.E., Scott M.P. Communicating with Hedgehogs. Nat. Rev. Mol. Cell Biol. 2005, 6:306-317.
39. Humke E.W., Dorn K.V., Milenkovic L., Scott M.P., Rohatgi R. The output of Hedgehog signaling is controlled by the dynamic association between Suppressor of Fused and the Gli proteins. Genes Dev. 2010, 24:670-682.
40. Huntzicker E.G., Estay I.S., Zhen H., Lokteva L.A., Jackson P.K., Oro A.E. Dual degradation signals control Gli protein stability and tumor formation. Genes Dev. 2006, 20:276-281.
41. Jenuwein T., Allis C.D. Translating the histone code. Science 2001, 293:1074-1080.
42. Jia J., Amanai K., Wang G., Tang J., Wang B., Jiang J. Shaggy/GSK3 antagonizes Hedgehog signalling by regulating Cubitus interruptus. Nature 2002, 416:548-552.
43. Jia J., Zhang L., Zhang Q., Tong C., Wang B., Hou F., Amanai K., Jiang J. Phosphorylation by double-time/CKIepsilon and CKIalpha targets cubitus interruptus for Slimb/beta-TRCP-mediated proteolytic processing. Dev. Cell 2005, 9:819-830.
44. Kelleher F.C. Hedgehog signaling and therapeutics in pancreatic cancer. Carcinogenesis 2011, 32:445-451.
45. Kent D., Bush E.W., Hooper J.E. Roadkill attenuates Hedgehog esponses through degradation of Cubitus interruptus. Development 2006, 133:2001-2010.
46. Kimura H., Stephen D., Joyner A., Curran T. Gli1 is important for medulloblastoma formation in Ptch1+/- mice. Oncogene 2005, 24:4026-4036.
47. Klein A.L., Zilian O., Suter U., Taylor V. Murine numb regulates granule cell maturation in the cerebellum. Dev. Biol. 2004, 266:161-177.
48. Kogerman P., Grimm T., Kogerman L., Krause D., Unden A.B., Sandstedt B., Toftgard R., Zaphiropoulos P.G. Mammalian suppressor-of-fused modulates nuclear-cytoplasmic shuttling of Gli-1. Nat. Cell Biol. 1999, 1:312-319.
49. Kuo C.T., Mirzadeh Z., Soriano-Navarro M., Rasin M., Wang D., Shen J., Sestan N., Garcia-Verdugo J., Alvarez-Buylla A., Jan L.Y., Jan Y.N. Postnatal deletion of numb/numblike reveals repair and remodeling capacity in the subventricular neurogenic niche. Cell 2006, 127:1253-1264.
50. Kwon J.E., La M., Oh K.H., Oh Y.M., Kim G.R., Seol J.H., Baek S.H., Chiba T., Tanaka K., Bang O.S., et al. BTB domain-containing speckle-type POZ protein (SPOP) serves as an adaptor of Daxx for ubiquitination by Cul3-based ubiquitin ligase. J. Biol. Chem. 2006, 281:12664-12672.
51. Lai K., Kaspar B.K., Gage F.H., Schaffer D.V. Sonic hedgehog regulates adult neural progenitor proliferation in vitro and in vivo. Nat. Neurosci. 2003, 6:21-27.
52. Lee J.D., Amanai K., Shearn A., Treisman J.E. The ubiquitin ligase Hyperplastic discs negatively regulates hedgehog and decapentaplegic expression by independent mechanisms. Development 2002, 129:5697-5706.
53. Lee E.Y., Ji H., Ouyangc Z., Zhoud B., Mae W., Vokesf S.A., McMahong A.P., Wongd W.H., Scott M.P. Hedgehog pathway-regulated gene networks in cerebellum development and tumorigenesis. Proc. Natl. Acad. Sci. 2010, 107:9736-9741.
54. Li H.S., Wang D., Shen Q., Schonemann M.D., Gorski J.A., Jones K.R., Temple S., Jan L.Y., Jan Y.N. Inactivation of Numb and Numblike in embryonic dorsal forebrain impairs neurogenesis and disrupts cortical morphogenesis. Neuron 2003, 40:1105-1118.
55. Lim K.J., Bisht S., Bar E.E., Maitra A., Eberhart C.G. A polymeric nanoparticle formulation of curcumin inhibits growth, clonogenicity and stem-like fraction in malignant brain tumors. Cancer Biol. Ther. 2011, 11:464-473.
56. Liu Y.C. Ubiquitin ligases and the immune response. Annu. Rev. Immunol. 2004, 22:81-127.
57. Liu S., Dontu G., Mantle I.D., Patel S., Ahn N.S., Jackson K.W., Suri P., Wicha M.S. Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells. Cancer Res. 2006, 66:6063-6071.
58. Lobo N.A., Shimono Y., Qian D., Clarke M.F. The biology of cancer stem cells. Annu. Rev. Cell Dev. Biol. 2007, 23:675-699.
59. Machold R., Hayashi S., Rutlin M., Muzumdar M.D., Nery S., Corbin J.G., Gritli-Linde A., Dellovade T., Porter J.A., Rubin L.L., Dudek H., McMahon A.P., et al. Sonic hedgehog is required for progenitor cell maintenance in telencephalic stem cell niches. Neuron 2003, 39:937-950.
60. Mancarelli M.M., Zazzeroni F., Ciccocioppo L., Capece D., Po A., Murgo S., Di Camillo R., Rinaldi C., Ferretti E., Gulino A., Alesse E. The tumor suppressor gene KCTD11 (REN) is regulated by Sp1 and methylation and its expression is reduced in tumors. Mol. Cancer 2010, 9:169-172.
61. McGill M.A., McGlade C.J. Mammalian numb proteins promote Notch1 receptor ubiquitination and degradation of the Notch1 intracellular domain. J. Biol. Chem. 2003, 278:23196-23203.
62. Morrison S.J., Kimble J. Asymmetric and symmetric stem-cell divisions in development and cancer. Nature 2006, 441:1068-1074.
63. Mund T., Pelham Hugh R.B. Conrol of the activity of WWHECT domain E3 ubiquitin ligase by NDFIP proteins. EMBO Rep. 2009, 10:501-507.
64. Nusslein-Volhard C., Wieschaus E. Mutations affecting segment number and polarity in Drosophila. Nature 1980, 287:795-801.
65. Palma V., Ruiz i Altaba A. Hedgehog-GLI signaling regulates the behavior of cells with stem cell properties in the developing neocortex. Development 2004, 131:337-345.
66. Palma V., Lim D.A., Dahmane N., Sanchez P., Brionne T.C., Herzberg C.D., Gitton Y., Carleton A., Alvarez-Buylla A., Ruiz i Altaba A. Sonic hedgehog controls stem cell behavior in the postnatal and adult brain. Development 2005, 132:335-344.
67. Peacock C.D., Wang Q., Gesell G.S., Corcoran-Schwartz I.M., Jones E., Kim J., Devereux W.L., Rhodes J.T., Huff C.A., Beachy P.A., Watkins D.N., Matsui W. Hedgehog signaling maintains a tumor stem cell compartment in multiple myeloma. Proc. Natl. Acad. Sci. USA 2007, 104:4048-4053.
68. Pece S., Serresi M., Santolini E., Capra M., Hulleman E., Galimberti V., Zurrida S., Maisonneuve P., Viale G., Di Fiore P.P. Loss of negative regulation by Numb over Notch is relevant to human breast carcinogenesis. J. Cell Biol. 2004, 167:215-221.
69. Petersen P.H., Zou K., Hwang J.K., Jan Y.N., Zhong W. Progenitor cell maintenance requires numb and numblike during mouse neurogenesis. Nature 2002, 419:929-934.
70. Pickart C.M. Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 2001, 70:503-533.
71. Po A., Ferretti E., Miele E., De Smaele E., Paganelli A., Canettieri G., Coni S., Di Marcotullio L., Biffoni M., Massimi L., Di Rocco C., Screpanti I., Gulino A. Hedgehog controls neural stem cells through p53-independent regulation of Nanog. EMBO. J. 2010, 29:2646-2658.
72. Price M.A., Kalderon D. Proteolysis of the Hedgehog signaling effector Cubitus interruptus requires phosphorylation by Glycogen Synthase Kinase 3 and Casein Kinase 1. Cell 2002, 108:823-835.
73. Qiu L., Joazeiro C., Fang N., Wang H.Y., Elly C., Altman Y., Fang D., Hunter T., Liu Y.C. Recognition and ubiquitination of Notch by Itch, a hect-type E3 ubiquitin ligase. J. Biol. Chem. 2000, 275:35734-35737.
74. Rossi M., De Laurenzi V., Munarriz E., Green D.R., Liu Y.C., Vousden K.H., Cesareni G., Melino G. The ubiquitin-protein ligase Itch regulates p73 stability. EMBO J. 2005, 24:836-848.
75. Rudin C.M., Hann C.L., Laterra J., Yauch R.L., Callahan C.A., Fu L., Holcomb T., Stinson J., Gould S.E., Coleman B., LoRusso P.M., Von Hoff D.D., et al. Treatment of medulloblastoma with hedgehog pathway inhibitor GDC-0449. N. Engl. J. Med. 2009, 361:1173-1178.
76. Ruiz i Altaba A., Sanchez P., Dahmane N. Gli and hedgehog in cancer: Tumours, embryos and stem cells. Nat. Rev. Cancer 2002, 2:361-372.
77. Ruppert J.M., Vogelstein B., Kinzler K.W. The zinc finger protein GLI transforms primary cells in cooperation with adenovirus E1A. Mol. Cell. Biol. 1991, 11:1724-1728.
78. Santolini E., Puri C., Salcini A.E., Gagliani M.C., Pelicci P.G., Tacchetti C., Di Fiore P.P. Numb is an endocytic protein. J. Cell Biol. 2000, 151:1345-1352.
79. Shahbazian M.D., Grunstein M. Functions of sitespecific histone acetylation and deacetylation. Annu. Rev. Biochem. 2007, 76:75-100.
80. Shen Q., Zhong W., Jan Y.N., Temple S. Asymmetric Numb distribution is critical for asymmetric cell division of mouse cerebral cortical stem cells and neuroblasts. Development 2002, 129:4843-4853.
81. Smelkinson M.G., Kalderon D. Processing of the Drosophila Hedgehog signaling effector Ci-155 to the repressor Ci-75 is mediated by direct binding to the SCF component Slimb. Curr. Biol. 2006, 16:110-116.
82. Song Z., Yue W., Wei B., Wang N., Li T., Guan L., Shi S., Zeng Q., Pei X., Chen L. Sonic hedgehog pathway is essential for maintenance of cancer stem-like cells in human gastric cancer. PLoS One 2011, 6:e17687.
83. Stecca B., Ruiz i Altaba A. Context-dependent regulation of the GLI code in cancer by HEDGEHOG and Non-HEDGEHOG signals. J. Mol. Cell Biol. 2010, 2:84-95.
84. Teglund S., Toftgard R. Hedgehog beyond medulloblastoma and basal cell carcinoma. Biochim. Biophys. Acta 2010, 1805:181-208.
85. Traweger A., Fang D., Liu Y.C., Stelzhammer W., Krizbai I.A., Fresser F., Bauer H.C., Bauer H. The tight junction-specific protein occludin is a functional target of the E3 ubiquitin-protein ligase itch. J. Biol. Chem. 2002, 277:10201-10208.
86. Van Sant C., Hagglund R., Lopez P., Roizman B. The infected cell protein 0 of herpes simplex virus 1 dynamically interacts with proteasomes, binds and activates the cdc34 E2 ubiquitin-conjugating enzyme, and possesses in vitro E3 ubiquitin ligase activity. Proc. Natl. Acad. Sci. USA 2001, 98:8815-8820.
87. Varnat F., Duquet A., Malerba M., Zbinden M., Mas C., Gervaz P., Ruiz i Altaba A. Human colon cancer epithelial cells harbour active HEDGEHOG-GLI signalling that is essential for tumour growth, recurrence, metastasis and stem cell survival and expansion. EMBO Mol. Med. 2009, 1:338-351.
88. Verdi J.M., Bashirullah A., Goldhawk D.E., Kubu C.J., Jamali M., Meakin S.O., Lipshitz H.D. Distinct human NUMB isoforms regulate differentiation vs. proliferation in the neuronal lineage. Proc. Natl. Acad. Sci. USA 1999, 96:10472-10476.
89. Wallace V.A. Purkinje-cell-derived Sonic hedgehog regulates granule neuron precursor cell proliferation in the developing mouse cerebellum. Curr. Biol. 1999, 9:445-448.
90. Wang Q.T., Holmgren R.A. The subcellular localization and activity of Drosophila cubitus interruptus are regulated at multiple levels. Development 1999, 126:5097-5106.
91. Wang C., Pan Y., Wang B. Suppressor of fused and Spop regulate the stability, processing and function of Gli2 and Gli3 full-length activators but not their repressors. Development 2010, 137:2001-2009.
92. Wechsler-Reya R.J., Scott M.P. Control of neuronal precursor proliferation in the cerebellum by Sonic Hedgehog. Neuron 1999, 22:103-114.
93. Welchman R.L., Gordon C., Mayer R.J. Ubiquitin and ubiquitin-like proteins as multifunctional signals. Nat. Rev. Mol. Cell Biol. 2005, 6:599-609.
94. Yang X.J., Seto E. HATs and HDACs: From structure, function and regulation to novel strategies for therapy and prevention. Oncogene 2007, 26:5310-5318.
95. Yauch R.L., Dijkgraaf G.J., Alicke B., Januario T., Ahn C.P., Holcomb T., Pujara K., Stinson J., Callahan C.A., Tang T., Bazan J.F., Kan Z., et al. Smoothened mutation confers resistance to a Hedgehog pathway inhibitor in medulloblastoma. Science 2009, 326:572-574.
96. Yoon K., Gaiano N. Notch signaling in the mammalian central nervous system: Insights from mouse mutants. Nat. Neurosci. 2005, 8:709-715.
97. Zbinden M., Duquet A., Lorente-Trigos A., Ngwabyt S.N., Borges I., Ruiz i Altaba A. NANOG regulates glioma stem cells and is essential in vivo acting in a cross-functional network with GLI1 and p53. EMBO J. 2010, 29:2659-2674.
98. Zhang Q., Zhang L., Wang B., Ou C.Y., Chien C.T., Jiang J. A hedgehog-induced BTB protein modulates hedgehog signaling by degrading Ci/Gli transcription factor. Dev. Cell 2006, 10:719-729.
99. Zhao C., Chen A., Jamieson C.H., Fereshteh M., Abrahamsson A., Blum J., Kwon H.Y., Kim J., Chute J.P., Rizzieri D., Munchhof M., VanArsdale T., et al. Hedgehog signalling is essential for maintenance of cancer stem cells in myeloid leukaemia. Nature 2009, 458:776-779.