#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

Functional Dissection of the Condensin Subunit Cap-G Reveals Its Exclusive Association with Condensin I


The heteropentameric condensin complexes have been shown to participate in mitotic chromosome condensation and to be required for unperturbed chromatid segregation in nuclear divisions. Vertebrates have two condensin complexes, condensin I and condensin II, which contain the same structural maintenance of chromosomes (SMC) subunits SMC2 and SMC4, but differ in their composition of non–SMC subunits. While a clear biochemical and functional distinction between condensin I and condensin II has been established in vertebrates, the situation in Drosophila melanogaster is less defined. Since Drosophila lacks a clear homolog for the condensin II–specific subunit Cap-G2, the condensin I subunit Cap-G has been hypothesized to be part of both complexes. In vivo microscopy revealed that a functional Cap-G-EGFP variant shows a distinct nuclear enrichment during interphase, which is reminiscent of condensin II localization in vertebrates and contrasts with the cytoplasmic enrichment observed for the other EGFP-fused condensin I subunits. However, we show that this nuclear localization is dispensable for Cap-G chromatin association, for its assembly into the condensin I complex and, importantly, for development into a viable and fertile adult animal. Immunoprecipitation analyses and complex formation studies provide evidence that Cap-G does not associate with condensin II–specific subunits, while it can be readily detected in complexes with condensin I–specific proteins in vitro and in vivo. Mass-spectrometric analyses of proteins associated with the condensin II–specific subunit Cap-H2 not only fail to identify Cap-G but also the other known condensin II–specific homolog Cap-D3. As condensin II–specific subunits are also not found associated with SMC2, our results question the existence of a soluble condensin II complex in Drosophila.


Vyšlo v časopise: Functional Dissection of the Condensin Subunit Cap-G Reveals Its Exclusive Association with Condensin I. PLoS Genet 9(4): e32767. doi:10.1371/journal.pgen.1003463
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003463

Souhrn

The heteropentameric condensin complexes have been shown to participate in mitotic chromosome condensation and to be required for unperturbed chromatid segregation in nuclear divisions. Vertebrates have two condensin complexes, condensin I and condensin II, which contain the same structural maintenance of chromosomes (SMC) subunits SMC2 and SMC4, but differ in their composition of non–SMC subunits. While a clear biochemical and functional distinction between condensin I and condensin II has been established in vertebrates, the situation in Drosophila melanogaster is less defined. Since Drosophila lacks a clear homolog for the condensin II–specific subunit Cap-G2, the condensin I subunit Cap-G has been hypothesized to be part of both complexes. In vivo microscopy revealed that a functional Cap-G-EGFP variant shows a distinct nuclear enrichment during interphase, which is reminiscent of condensin II localization in vertebrates and contrasts with the cytoplasmic enrichment observed for the other EGFP-fused condensin I subunits. However, we show that this nuclear localization is dispensable for Cap-G chromatin association, for its assembly into the condensin I complex and, importantly, for development into a viable and fertile adult animal. Immunoprecipitation analyses and complex formation studies provide evidence that Cap-G does not associate with condensin II–specific subunits, while it can be readily detected in complexes with condensin I–specific proteins in vitro and in vivo. Mass-spectrometric analyses of proteins associated with the condensin II–specific subunit Cap-H2 not only fail to identify Cap-G but also the other known condensin II–specific homolog Cap-D3. As condensin II–specific subunits are also not found associated with SMC2, our results question the existence of a soluble condensin II complex in Drosophila.


Zdroje

1. WoodAJ, SeversonAF, MeyerBJ (2010) Condensin and cohesin complexity: the expanding repertoire of functions. Nat Rev Genet 11: 391–404.

2. HudsonDF, MarshallKM, EarnshawWC (2009) Condensin: Architect of mitotic chromosomes. Chromosome Res 17: 131–144.

3. HiranoT (2012) Condensins: universal organizers of chromosomes with diverse functions. Genes Dev 26: 1659–1678.

4. HiranoT, KobayashiR, HiranoM (1997) Condensins, chromosome condensation protein complexes containing XCAP-C, XCAP-E and a Xenopus homolog of the Drosophila Barren protein. Cell 89: 511–521.

5. HiranoT, MitchisonTJ (1994) A heterodimeric coiled-coil protein required for mitotic chromosome condensation in vitro. Cell 79: 449–458.

6. SchleifferA, KaitnaS, Maurer-StrohS, GlotzerM, NasmythK, et al. (2003) Kleisins: a superfamily of bacterial and eukaryotic SMC protein partners. Mol Cell 11: 571–575.

7. AndradeMA, Perez-IratxetaC, PontingCP (2001) Protein repeats: structures, functions, and evolution. J Struct Biol 134: 117–131.

8. GreenLC, KalitsisP, ChangTM, CipeticM, KimJH, et al. (2012) Contrasting roles of condensin I and condensin II in mitotic chromosome formation. J Cell Sci 125: 1591–1604.

9. GerlichD, HirotaT, KochB, PetersJM, EllenbergJ (2006) Condensin I stabilizes chromosomes mechanically through a dynamic interaction in live cells. Curr Biol 16: 333–344.

10. HirotaT, GerlichD, KochB, EllenbergJ, PetersJM (2004) Distinct functions of condensin I and II in mitotic chromosome assembly. J Cell Sci 117: 6435–6445.

11. OnoT, FangY, SpectorDL, HiranoT (2004) Spatial and temporal regulation of Condensins I and II in mitotic chromosome assembly in human cells. Mol Biol Cell 15: 3296–3308.

12. OnoT, LosadaA, HiranoM, MyersMP, NeuwaldAF, et al. (2003) Differential contributions of condensin I and condensin II to mitotic chromosome architecture in vertebrate cells. Cell 115: 109–121.

13. CsankovszkiG, ColletteK, SpahlK, CareyJ, SnyderM, et al. (2009) Three distinct condensin complexes control C. elegans chromosome dynamics. Curr Biol 19: 9–19.

14. JägerH, RauchM, HeidmannS (2005) The Drosophila melanogaster condensin subunit Cap-G interacts with the centromere-specific histone H3 variant CID. Chromosoma 113: 350–361.

15. ResnickTD, DejKJ, XiangY, HawleyRS, AhnC, et al. (2009) Mutations in the chromosomal passenger complex and the condensin complex differentially affect synaptonemal complex disassembly and metaphase I configuration in Drosophila female meiosis. Genetics 181: 875–887.

16. LongworthMS, HerrA, JiJY, DysonNJ (2008) RBF1 promotes chromatin condensation through a conserved interaction with the Condensin II protein dCAP-D3. Genes Dev 22: 1011–1024.

17. SteffensenS, CoelhoPA, CobbeN, VassS, CostaM, et al. (2001) A role for Drosophila SMC4 in the resolution of sister chromatids in mitosis. Curr Biol 11: 295–307.

18. SavvidouE, CobbeN, SteffensenS, CotterillS, HeckMM (2005) Drosophila CAP-D2 is required for condensin complex stability and resolution of sister chromatids. J Cell Sci 118: 2529–2543.

19. OliveiraRA, CoelhoPA, SunkelCE (2005) The condensin I subunit Barren/CAP-H is essential for the structural integrity of centromeric heterochromatin during mitosis. Mol Cell Biol 25: 8971–8984.

20. DejKJ, AhnC, Orr-WeaverTL (2004) Mutations in the Drosophila condensin subunit dCAP-G: defining the role of condensin for chromosome condensation in mitosis and gene expression in interphase. Genetics 168: 895–906.

21. CobbeN, SavvidouE, HeckMM (2006) Diverse mitotic and interphase functions of condensins in Drosophila. Genetics 172: 991–1008.

22. BhatMA, PhilpAV, GloverDM, BellenHJ (1996) Chromatid segregation at anaphase requires the barren product, a novel chromosome-associated protein that interacts with topoisomerase II. Cell 87: 1103–1114.

23. HartlTA, SweeneySJ, KneplerPJ, BoscoG (2008) Condensin II resolves chromosomal associations to enable anaphase I segregation in Drosophila male meiosis. PLoS Genet 4: e1000228 doi:10.1371/journal.pgen.1000228.

24. HartlTA, SmithHF, BoscoG (2008) Chromosome alignment and transvection are antagonized by condensin II. Science 322: 1384–1387.

25. LongworthMS, WalkerJA, AnderssenE, MoonNS, GladdenA, et al. (2012) A shared role for RBF1 and dCAP-D3 in the regulation of transcription with consequences for innate immunity. PLoS Genet 8: e1002618 doi:10.1371/journal.pgen.1002618.

26. GoslingKM, MakaroffLE, TheodoratosA, KimYH, WhittleB, et al. (2007) A mutation in a chromosome condensin II subunit, kleisin beta, specifically disrupts T cell development. Proc Natl Acad Sci U S A 104: 12445–12450.

27. XuY, LeungCG, LeeDC, KennedyBK, CrispinoJD (2006) MTB, the murine homolog of condensin II subunit CAP-G2, represses transcription and promotes erythroid cell differentiation. Leukemia 20: 1261–1269.

28. OliveiraRA, HeidmannS, SunkelCE (2007) Condensin I binds chromatin early in prophase and displays a highly dynamic association with Drosophila mitotic chromosomes. Chromosoma 116: 259–274.

29. JamesTC, ElginSC (1986) Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophila melanogaster and its gene. Mol Cell Biol 6: 3862–3872.

30. KellumR, RaffJW, AlbertsBM (1995) Heterochromatin protein-1 distribution during development and during the cell-cycle in drosophila embryos. J Cell Sci 108: 1407–1418.

31. ShintomiK, HiranoT (2011) The relative ratio of condensin I to II determines chromosome shapes. Genes Dev 25: 1464–1469.

32. AonoN, SutaniT, TomonagaT, MochidaS, YanagidaM (2002) Cnd2 has dual roles in mitotic condensation and interphase. Nature 417: 197–202.

33. StearJH, RothMB (2002) Characterization of HCP-6, a C. elegans protein required to prevent chromosome twisting and merotelic attachment. Genes Dev 16: 1498–1508.

34. RørthP (1998) Gal4 in the Drosophila female germline. Mech Dev 78: 113–118.

35. OnnI, AonoN, HiranoM, HiranoT (2007) Reconstitution and subunit geometry of human condensin complexes. EMBO J 26: 1024–1034.

36. BazileF, St-PierreJ, D'AmoursD (2010) Three-step model for condensin activation during mitotic chromosome condensation. Cell Cycle 9: 3243–3255.

37. TadaK, SusumuH, SakunoT, WatanabeY (2011) Condensin association with histone H2A shapes mitotic chromosomes. Nature 474: 477–483.

38. YeongFM, HombauerH, WendtKS, HirotaT, MudrakI, et al. (2003) Identification of a subunit of a novel Kleisin-beta/SMC complex as a potential substrate of protein phosphatase 2A. Curr Biol 13: 2058–2064.

39. GraveleyBR, BrooksAN, CarlsonJW, DuffMO, LandolinJM, et al. (2011) The developmental transcriptome of Drosophila melanogaster. Nature 471: 473–479.

40. BauerCR, HartlTA, BoscoG (2012) Condensin II promotes the formation of chromosome territories by inducing axial compaction of polyploid interphase chromosomes. PLoS Genet 8: e1002873 doi:10.1371/journal.pgen.1002873.

41. BrandAH, PerrimonN (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118: 401–415.

42. BischofJ, MaedaRK, HedigerM, KarchF, BaslerK (2007) An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc Natl Acad Sci U S A 104: 3312–3317.

43. HornC, WimmerEA (2000) A versatile vector set for animal transgenesis. Dev Genes Evol 210: 630–637.

44. GruberS, ArumugamP, KatouY, KuglitschD, HelmhartW, et al. (2006) Evidence that loading of cohesin onto chromosomes involves opening of its SMC hinge. Cell 127: 523–537.

45. VenkenKJ, HeY, HoskinsRA, BellenHJ (2006) P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster. Science 314: 1747–1751.

46. SchittenhelmRB, HeegerS, AlthoffF, WalterA, HeidmannS, et al. (2007) Spatial organization of a ubiquitous eukaryotic kinetochore protein network in Drosophila chromosomes. Chromosoma 116: 385–402.

47. WodarzA, HinzU, EngelbertM, KnustE (1995) Expression of crumbs confers apical character on plasma- membrane domains of ectodermal epithelia of drosophila. Cell 82: 67–76.

48. WeissA, HerzigA, JacobsH, LehnerCF (1998) Continuous Cyclin E expression inhibits progression through endoreduplication cycles in Drosophila. Curr Biol 8: 239–242.

49. MicklemDR, DasguptaR, ElliottH, GergelyF, DavidsonC, et al. (1997) The mago nashi gene is required for the polarisation of the oocyte and the formation of perpendicular axes in Drosophila. Curr Biol 7: 468–478.

50. WenH, AndrejkaL, AshtonJ, KaressR, LipsickJS (2008) Epigenetic regulation of gene expression by Drosophila Myb and E2F2-RBF via the Myb-MuvB/dREAM complex. Genes Dev 22: 601–614.

51. SchuhM, LehnerCF, HeidmannS (2007) Incorporation of Drosophila CID/CENP-A and CENP-C into centromeres during early embryonic anaphase. Curr Biol 17: 237–243.

52. EvanGI, LewisGK, RamsayG, BishopJM (1985) Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol 5: 3610–3616.

53. Harlow E, Lane D (1988) Antibodies. A Laboratory Manual: Cold Spring Harbor Laboratory Press.

54. Sullivan W, Ashburner M, Hawley RS (2000) Drosophila protocols. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. xiv, 697 p. p.

55. CandianoG, BruschiM, MusanteL, SantucciL, GhiggeriGM, et al. (2004) Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25: 1327–1333.

Štítky
Genetika Reprodukčná medicína

Článok vyšiel v časopise

PLOS Genetics


2013 Číslo 4
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Získaná hemofilie - Povědomí o nemoci a její diagnostika
nový kurz

Eozinofilní granulomatóza s polyangiitidou
Autori: doc. MUDr. Martina Doubková, Ph.D.

Všetky kurzy
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa

#ADS_BOTTOM_SCRIPTS#