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Functional diversification of Paramecium Ku80 paralogs safeguards genome integrity during precise programmed DNA elimination


Autoři: Arthur Abello aff001;  Vinciane Régnier aff001;  Olivier Arnaiz aff001;  Romain Le Bars aff001;  Mireille Bétermier aff001;  Julien Bischerour aff001
Působiště autorů: Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France aff001;  Université de Paris, Paris, France aff002
Vyšlo v časopise: Functional diversification of Paramecium Ku80 paralogs safeguards genome integrity during precise programmed DNA elimination. PLoS Genet 16(4): e32767. doi:10.1371/journal.pgen.1008723
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pgen.1008723

Souhrn

Gene duplication and diversification drive the emergence of novel functions during evolution. Because of whole genome duplications, ciliates from the Paramecium aurelia group constitute a remarkable system to study the evolutionary fate of duplicated genes. Paramecium species harbor two types of nuclei: a germline micronucleus (MIC) and a somatic macronucleus (MAC) that forms from the MIC at each sexual cycle. During MAC development, ~45,000 germline Internal Eliminated Sequences (IES) are excised precisely from the genome through a ‘cut-and-close’ mechanism. Here, we have studied the P. tetraurelia paralogs of KU80, which encode a key DNA double-strand break repair factor involved in non-homologous end joining. The three KU80 genes have different transcription patterns, KU80a and KU80b being constitutively expressed, while KU80c is specifically induced during MAC development. Immunofluorescence microscopy and high-throughput DNA sequencing revealed that Ku80c stably anchors the PiggyMac (Pgm) endonuclease in the developing MAC and is essential for IES excision genome-wide, providing a molecular explanation for the previously reported Ku-dependent licensing of DNA cleavage at IES ends. Expressing Ku80a under KU80c transcription signals failed to complement a depletion of endogenous Ku80c, indicating that the two paralogous proteins have distinct properties. Domain-swap experiments identified the α/β domain of Ku80c as the major determinant for its specialized function, while its C-terminal part is required for excision of only a small subset of IESs located in IES-dense regions. We conclude that Ku80c has acquired the ability to license Pgm-dependent DNA cleavage, securing precise DNA elimination during programmed rearrangements. The present study thus provides novel evidence for functional diversification of genes issued from a whole-genome duplication.

Klíčová slova:

Cell fusion – DNA cleavage – Chromatin – Immunostaining – Protein domains – RNA interference – Sequence alignment – Paramecium


Zdroje

1. Taylor JS, Raes J (2004) Duplication and divergence: the evolution of new genes and old ideas. Annu Rev Genet 38: 615–643. doi: 10.1146/annurev.genet.38.072902.092831 15568988

2. Ohno S (1970) Evolution by gene duplication. Berlin, Heidelberg: Springer-Verlag.

3. Lynch M, Force A (2000) The probability of duplicate gene preservation by subfunctionalization. Genetics 154: 459–473. 10629003

4. Force A, Lynch M, Pickett FB, Amores A, Yan YL, et al. (1999) Preservation of duplicate genes by complementary, degenerative mutations. Genetics 151: 1531–1545. 10101175

5. Aury JM, Jaillon O, Duret L, Noel B, Jubin C, et al. (2006) Global trends of whole-genome duplications revealed by the ciliate Paramecium tetraurelia. Nature 444: 171–178. doi: 10.1038/nature05230 17086204

6. McGrath CL, Gout JF, Doak TG, Yanagi A, Lynch M (2014) Insights into three whole-genome duplications gleaned from the Paramecium caudatum genome sequence. Genetics 197: 1417–1428. doi: 10.1534/genetics.114.163287 24840360

7. Shi L, Koll F, Arnaiz O, Cohen J (2018) The Ciliary Protein IFT57 in the Macronucleus of Paramecium. J Eukaryot Microbiol 65: 12–27. doi: 10.1111/jeu.12423 28474836

8. Gout JF, Kahn D, Duret L, Paramecium Post-Genomics C (2010) The relationship among gene expression, the evolution of gene dosage, and the rate of protein evolution. PLoS Genet 6: e1000944. doi: 10.1371/journal.pgen.1000944 20485561

9. Gout JF, Lynch M (2015) Maintenance and loss of duplicated genes by dosage subfunctionalization. Mol Biol Evol 32: 2141–2148. doi: 10.1093/molbev/msv095 25908670

10. Bétermier M, Duharcourt S (2014) Programmed rearrangement in ciliates: Paramecium. Microbiol Spectr 2: doi: 10.1128/microbiolspec.MDNA1123-0035-2014

11. Arnaiz O, Mathy N, Baudry C, Malinsky S, Aury JM, et al. (2012) The Paramecium germline genome provides a niche for intragenic parasitic DNA: evolutionary dynamics of internal eliminated sequences. PLoS Genet 8: e1002984. doi: 10.1371/journal.pgen.1002984 23071448

12. Guérin F, Arnaiz O, Boggetto N, Denby Wilkes C, Meyer E, et al. (2017) Flow cytometry sorting of nuclei enables the first global characterization of Paramecium germline DNA and transposable elements. BMC Genomics 18: 327. doi: 10.1186/s12864-017-3713-7 28446146

13. Baudry C, Malinsky S, Restituito M, Kapusta A, Rosa S, et al. (2009) PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements in the ciliate Paramecium tetraurelia. Genes Dev 23: 2478–2483. doi: 10.1101/gad.547309 19884254

14. Bischerour J, Bhullar S, Denby Wilkes C, Regnier V, Mathy N, et al. (2018) Six domesticated PiggyBac transposases together carry out programmed DNA elimination in Paramecium. Elife 7.

15. Dubois E, Mathy N, Regnier V, Bischerour J, Baudry C, et al. (2017) Multimerization properties of PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements. Nucleic Acids Res 45: 3204–3216. doi: 10.1093/nar/gkw1359 28104713

16. Kapusta A, Matsuda A, Marmignon A, Ku M, Silve A, et al. (2011) Highly precise and developmentally programmed genome assembly in Paramecium requires ligase IV-dependent end joining. PLoS Genet 7: e1002049. doi: 10.1371/journal.pgen.1002049 21533177

17. Bétermier M, Bertrand P, Lopez BS (2014) Is non-homologous end-joining really an inherently error-prone process? PLoS Genet 10: e1004086. doi: 10.1371/journal.pgen.1004086 24453986

18. Dubois E, Bischerour J, Marmignon A, Mathy N, Regnier V, et al. (2012) Transposon invasion of the Paramecium germline genome countered by a domesticated PiggyBac transposase and the NHEJ pathway. Int J Evol Biol 2012: 436196. doi: 10.1155/2012/436196 22888464

19. Allen SE, Hug I, Pabian S, Rzeszutek I, Hoehener C, et al. (2017) Circular concatemers of ultra-short DNA segments produce regulatory RNAs. Cell 168: 990–999 e997. doi: 10.1016/j.cell.2017.02.020 28283070

20. Bétermier M, Duharcourt S, Seitz H, Meyer E (2000) Timing of developmentally programmed excision and circularization of Paramecium internal eliminated sequences. Mol Cell Biol 20: 1553–1561. doi: 10.1128/mcb.20.5.1553-1561.2000 10669733

21. Gratias A, Bétermier M (2001) Developmentally programmed excision of internal DNA sequences in Paramecium aurelia. Biochimie 83: 1009–1022. doi: 10.1016/s0300-9084(01)01349-9 11879729

22. Grundy GJ, Moulding HA, Caldecott KW, Rulten SL (2014) One ring to bring them all—the role of Ku in mammalian non-homologous end joining. DNA Repair (Amst) 17: 30–38.

23. Aravind L, Koonin EV (2001) Prokaryotic homologs of the eukaryotic DNA-end-binding protein Ku, novel domains in the Ku protein and prediction of a prokaryotic double-strand break repair system. Genome Res 11: 1365–1374. doi: 10.1101/gr.181001 11483577

24. Dupuy P, Sauviac L, Bruand C (2019) Stress-inducible NHEJ in bacteria: function in DNA repair and acquisition of heterologous DNA. Nucleic Acids Res 47: 1335–1349. doi: 10.1093/nar/gky1212 30517704

25. Walker JR, Corpina RA, Goldberg J (2001) Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair. Nature 412: 607–614. doi: 10.1038/35088000 11493912

26. Marmignon A, Bischerour J, Silve A, Fojcik C, Dubois E, et al. (2014) Ku-mediated coupling of DNA cleavage and repair during programmed genome rearrangements in the ciliate Paramecium tetraurelia. PLoS Genet 10: e1004552. doi: 10.1371/journal.pgen.1004552 25166013

27. Frapporti A, Miro Pina C, Arnaiz O, Holoch D, Kawaguchi T, et al. (2019) The Polycomb protein Ezl1 mediates H3K9 and H3K27 methylation to repress transposable elements in Paramecium. Nat Commun 10: 2710. doi: 10.1038/s41467-019-10648-5 31221974

28. Skouri F, Cohen J (1997) Genetic approach to regulated exocytosis using functional complementation in Paramecium: identification of the ND7 gene required for membrane fusion. Mol Biol Cell 8: 1063–1071. doi: 10.1091/mbc.8.6.1063 9201716

29. Denby Wilkes C, Arnaiz O, Sperling L (2016) ParTIES: a toolbox for Paramecium interspersed DNA elimination studies. Bioinformatics 32: 599–601. doi: 10.1093/bioinformatics/btv691 26589276

30. Arnaiz O, Van Dijk E, Betermier M, Lhuillier-Akakpo M, de Vanssay A, et al. (2017) Improved methods and resources for Paramecium genomics: transcription units, gene annotation and gene expression. BMC Genomics 18: 483. doi: 10.1186/s12864-017-3887-z 28651633

31. McGrath CL, Gout JF, Johri P, Doak TG, Lynch M (2014) Differential retention and divergent resolution of duplicate genes following whole-genome duplication. Genome Res 24: 1665–1675. doi: 10.1101/gr.173740.114 25085612

32. Gout JF, Johri P, Arnaiz O, Doak TG, Bhullar S, et al. (2019) Universal trends of post-duplication evolution revealed by the genomes of 13 Paramecium species sharing an ancestral whole-genome duplication. BioRXiv https://doi.org/10.1101/573576.

33. Meyer E, Keller AM (1996) A mendelian mutation affecting mating-type determination also affects developmental genomic rearrangements in Paramecium tetraurelia. Genetics 143: 191–202. 8722774

34. Arnaiz O, Sperling L (2011) ParameciumDB in 2011: new tools and new data for functional and comparative genomics of the model ciliate Paramecium tetraurelia. Nucleic Acids Res 39: D632–636. doi: 10.1093/nar/gkq918 20952411

35. Arnaiz O, Meyer E, Sperling L (2019) ParameciumDB 2019: integrating genomic data across the genus for functional and evolutionary biology. Nucleic Acids Res 48: D499–D605.

36. Nemoz C, Ropars V, Frit P, Gontier A, Drevet P, et al. (2018) XLF and APLF bind Ku80 at two remote sites to ensure DNA repair by non-homologous end joining. Nat Struct Mol Biol 25: 971–980. doi: 10.1038/s41594-018-0133-6 30291363

37. Frit P, Ropars V, Modesti M, Charbonnier JB, Calsou P (2019) Plugged into the Ku-DNA hub: The NHEJ network. Prog Biophys Mol Biol 147: 62–76. doi: 10.1016/j.pbiomolbio.2019.03.001 30851288

38. Chen H, Xue J, Churikov D, Hass EP, Shi S, et al. (2018) Structural insights into yeast telomerase recruitment to telomeres. Cell 172: 331–343 e313. doi: 10.1016/j.cell.2017.12.008 29290466

39. Postow L, Ghenoiu C, Woo EM, Krutchinsky AN, Chait BT, et al. (2008) Ku80 removal from DNA through double strand break-induced ubiquitylation. J Cell Biol 182: 467–479. doi: 10.1083/jcb.200802146 18678709

40. Gratias A, Lepere G, Garnier O, Rosa S, Duharcourt S, et al. (2008) Developmentally programmed DNA splicing in Paramecium reveals short-distance crosstalk between DNA cleavage sites. Nucleic Acids Res 36: 3244–3251. doi: 10.1093/nar/gkn154 18420657

41. Cheng CY, Vogt A, Mochizuki K, Yao MC (2010) A domesticated piggyBac transposase plays key roles in heterochromatin dynamics and DNA cleavage during programmed DNA deletion in Tetrahymena thermophila. Mol Biol Cell 21: 1753–1762. doi: 10.1091/mbc.E09-12-1079 20357003

42. Saveliev SV, Cox MM (1995) Transient DNA breaks associated with programmed genomic deletion events in conjugating cells of Tetrahymena thermophila. Genes Dev 9: 248–255. doi: 10.1101/gad.9.2.248 7851797

43. Lin CG, Chao JL, Tsai HK, Chalker D, Yao MC (2019) Setting boundaries for genome-wide heterochromatic DNA deletions through flanking inverted repeats in Tetrahymena thermophila. Nucleic Acids Res 47: 5181–5192. doi: 10.1093/nar/gkz209 30918956

44. Lin IT, Chao JL, Yao MC (2012) An essential role for the DNA breakage-repair protein Ku80 in programmed DNA rearrangements in Tetrahymena thermophila. Mol Biol Cell 23: 2213–2225. doi: 10.1091/mbc.E11-11-0952 22513090

45. Hamilton EP, Kapusta A, Huvos PE, Bidwell SL, Zafar N, et al. (2016) Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome. Elife 5.

46. Jin Y, Chen Y, Zhao S, Guan KL, Zhuang Y, et al. (2017) DNA-PK facilitates piggyBac transposition by promoting paired-end complex formation. Proc Natl Acad Sci U S A 114: 7408–7413. doi: 10.1073/pnas.1612980114 28645898

47. Cheng CY, Young JM, Lin CG, Chao JL, Malik HS, et al. (2016) The piggyBac transposon-derived genes TPB1 and TPB6 mediate essential transposon-like excision during the developmental rearrangement of key genes in Tetrahymena thermophila. Genes Dev 30: 2724–2736. doi: 10.1101/gad.290460.116 28087716

48. Feng L, Wang G, Hamilton EP, Xiong J, Yan G, et al. (2017) A germline-limited piggyBac transposase gene is required for precise excision in Tetrahymena genome rearrangement. Nucleic Acids Res 45: 9481–9502. doi: 10.1093/nar/gkx652 28934495

49. Bétermier M, Borde V, de Villartay JP (2020) Coupling DNA damage and repair: an essential safeguard during programmed DNA double strand breaks? Trends Cell Biol 30: 87–96.

50. Gratias A, Bétermier M (2003) Processing of double-strand breaks is involved in the precise excision of Paramecium IESs. Mol Cell Biol 23: 7152–7162. doi: 10.1128/MCB.23.20.7152-7162.2003 14517286

51. Beisson J, Betermier M, Bre MH, Cohen J, Duharcourt S, et al. (2010) Paramecium tetraurelia: the renaissance of an early unicellular model. Cold Spring Harb Protoc 2010: pdb emo140. doi: 10.1101/pdb.emo140 20150105

52. Galvani A, Sperling L (2002) RNA interference by feeding in Paramecium. Trends Genet 18: 11–12. doi: 10.1016/s0168-9525(01)02548-3 11750689

53. Timmons L, Court DL, Fire A (2001) Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene 263: 103–112. doi: 10.1016/s0378-1119(00)00579-5 11223248

54. Kamath RS, Martinez-Campos M, Zipperlen P, Fraser AG, Ahringer J (2001) Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biol 2: RESEARCH0002. 0010.1186/gb-2000-0002-0001-research0002. doi: 10.1186/gb-2000-2-1-research0002 11178279

55. Garnier O, Serrano V, Duharcourt S, Meyer E (2004) RNA-mediated programming of developmental genome rearrangements in Paramecium tetraurelia. Mol Cell Biol 24: 7370–7379. doi: 10.1128/MCB.24.17.7370-7379.2004 15314149

56. Callen AM, Adoutte A, Andrew JM, Baroin-Tourancheau A, Bre MH, et al. (1994) Isolation and characterization of libraries of monoclonal antibodies directed against various forms of tubulin in Paramecium. Biol Cell 81: 95–119. doi: 10.1016/s0248-4900(94)80002-2 7531532

57. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10: 845–858. doi: 10.1038/nprot.2015.053 25950237

58. Jones DT, Taylor WR, Thornton JM (1992) The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci 8: 275–282. doi: 10.1093/bioinformatics/8.3.275 1633570

59. Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33: 1870–1874. doi: 10.1093/molbev/msw054 27004904


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