#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

Regulating Repression: Roles for the Sir4 N-Terminus in Linker DNA Protection and Stabilization of Epigenetic States


Silent information regulator proteins Sir2, Sir3, and Sir4 form a heterotrimeric complex that represses transcription at subtelomeric regions and homothallic mating type (HM) loci in budding yeast. We have performed a detailed biochemical and genetic analysis of the largest Sir protein, Sir4. The N-terminal half of Sir4 is dispensable for SIR–mediated repression of HM loci in vivo, except in strains that lack Yku70 or have weak silencer elements. For HM silencing in these cells, the C-terminal domain (Sir4C, residues 747–1,358) must be complemented with an N-terminal domain (Sir4N; residues 1–270), expressed either independently or as a fusion with Sir4C. Nonetheless, recombinant Sir4C can form a complex with Sir2 and Sir3 in vitro, is catalytically active, and has sedimentation properties similar to a full-length Sir4-containing SIR complex. Sir4C-containing SIR complexes bind nucleosomal arrays and protect linker DNA from nucleolytic digestion, but less effectively than wild-type SIR complexes. Consistently, full-length Sir4 is required for the complete repression of subtelomeric genes. Supporting the notion that the Sir4 N-terminus is a regulatory domain, we find it extensively phosphorylated on cyclin-dependent kinase consensus sites, some being hyperphosphorylated during mitosis. Mutation of two major phosphoacceptor sites (S63 and S84) derepresses natural subtelomeric genes when combined with a serendipitous mutation (P2A), which alone can enhance the stability of either the repressed or active state. The triple mutation confers resistance to rapamycin-induced stress and a loss of subtelomeric repression. We conclude that the Sir4 N-terminus plays two roles in SIR–mediated silencing: it contributes to epigenetic repression by stabilizing the SIR–mediated protection of linker DNA; and, as a target of phosphorylation, it can destabilize silencing in a regulated manner.


Vyšlo v časopise: Regulating Repression: Roles for the Sir4 N-Terminus in Linker DNA Protection and Stabilization of Epigenetic States. PLoS Genet 8(5): e32767. doi:10.1371/journal.pgen.1002727
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1002727

Souhrn

Silent information regulator proteins Sir2, Sir3, and Sir4 form a heterotrimeric complex that represses transcription at subtelomeric regions and homothallic mating type (HM) loci in budding yeast. We have performed a detailed biochemical and genetic analysis of the largest Sir protein, Sir4. The N-terminal half of Sir4 is dispensable for SIR–mediated repression of HM loci in vivo, except in strains that lack Yku70 or have weak silencer elements. For HM silencing in these cells, the C-terminal domain (Sir4C, residues 747–1,358) must be complemented with an N-terminal domain (Sir4N; residues 1–270), expressed either independently or as a fusion with Sir4C. Nonetheless, recombinant Sir4C can form a complex with Sir2 and Sir3 in vitro, is catalytically active, and has sedimentation properties similar to a full-length Sir4-containing SIR complex. Sir4C-containing SIR complexes bind nucleosomal arrays and protect linker DNA from nucleolytic digestion, but less effectively than wild-type SIR complexes. Consistently, full-length Sir4 is required for the complete repression of subtelomeric genes. Supporting the notion that the Sir4 N-terminus is a regulatory domain, we find it extensively phosphorylated on cyclin-dependent kinase consensus sites, some being hyperphosphorylated during mitosis. Mutation of two major phosphoacceptor sites (S63 and S84) derepresses natural subtelomeric genes when combined with a serendipitous mutation (P2A), which alone can enhance the stability of either the repressed or active state. The triple mutation confers resistance to rapamycin-induced stress and a loss of subtelomeric repression. We conclude that the Sir4 N-terminus plays two roles in SIR–mediated silencing: it contributes to epigenetic repression by stabilizing the SIR–mediated protection of linker DNA; and, as a target of phosphorylation, it can destabilize silencing in a regulated manner.


Zdroje

1. BraunsteinMRoseABHolmesSGAllisCDBroachJR 1993 Transcriptional silencing in yeast is associated with reduced nucleosome acetylation. Genes Dev 7 592 604

2. SukaNSukaYCarmenAAWuJGrunsteinM 2001 Highly specific antibodies determine histone acetylation site usage in yeast heterochromatin and euchromatin. Mol Cell 8 473 479

3. LooSRineJ 1994 Silencers and domains of generalized repression. Science 264 1768 1771

4. GottschlingDE 1992 Telomere-proximal DNA in Saccharomyces cerevisiae is refractory to methyltransferase activity in vivo. Proc Natl Acad Sci U S A 89 4062 4065

5. SinghJKlarAJ 1992 Active genes in budding yeast display enhanced in vivo accessibility to foreign DNA methylases: a novel in vivo probe for chromatin structure of yeast. Genes Dev 6 186 196

6. RaghuramanMKBrewerBJFangmanWL 1997 Cell cycle-dependent establishment of a late replication program. Science 276 806 809

7. GottaMLarocheTFormentonAMailletLScherthanH 1996 The clustering of telomeres and colocalization with Rap1, Sir3, and Sir4 proteins in wild-type Saccharomyces cerevisiae. J Cell Biol 134 1349 1363

8. OttavianiAGilsonEMagdinierF 2008 Telomeric position effect: from the yeast paradigm to human pathologies? Biochimie 90 93 107

9. RuscheLNKirchmaierALRineJ 2003 The establishment, inheritance, and function of silenced chromatin in Saccharomyces cerevisiae. Annu Rev Biochem 72 481 516

10. GasserSMCockellMM 2001 The molecular biology of the SIR proteins. Gene 279 1 16

11. MoazedD 2001 Common themes in mechanisms of gene silencing. Mol Cell 8 489 498

12. BuhlerMGasserSM 2009 Silent chromatin at the middle and ends: lessons from yeasts. Embo J 28 2149 2161

13. CubizollesFMartinoFPerrodSGasserSM 2006 A homotrimer-heterotrimer switch in Sir2 structure differentiates rDNA and telomeric silencing. Mol Cell 21 825 836

14. RineJHerskowitzI 1987 Four genes responsible for a position effect on expression from HML and HMR in Saccharomyces cerevisiae. Genetics 116 9 22

15. AparicioOMBillingtonBLGottschlingDE 1991 Modifiers of position effect are shared between telomeric and silent mating-type loci in S. cerevisiae. Cell 66 1279 1287

16. ShoreDStillmanDJBrandAHNasmythKA 1987 Identification of silencer binding proteins from yeast: possible roles in SIR control and DNA replication. Embo J 6 461 467

17. BuchmanARLueNFKornbergRD 1988 Connections between transcriptional activators, silencers, and telomeres as revealed by functional analysis of a yeast DNA-binding protein. Mol Cell Biol 8 5086 5099

18. MorettiPFreemanKCoodlyLShoreD 1994 Evidence that a complex of SIR proteins interacts with the silencer and telomere-binding protein RAP1. Genes Dev 8 2257 2269

19. SusselLShoreD 1991 Separation of transcriptional activation and silencing functions of the RAP1-encoded repressor/activator protein 1: isolation of viable mutants affecting both silencing and telomere length. Proc Natl Acad Sci U S A 88 7749 7753

20. BrandAHMicklemGNasmythK 1987 A yeast silencer contains sequences that can promote autonomous plasmid replication and transcriptional activation. Cell 51 709 719

21. TrioloTSternglanzR 1996 Role of interactions between the origin recognition complex and SIR1 in transcriptional silencing. Nature 381 251 253

22. TannerKGLandryJSternglanzRDenuJM 2000 Silent information regulator 2 family of NAD- dependent histone/protein deacetylases generates a unique product, 1-O-acetyl-ADP-ribose. Proc Natl Acad Sci U S A 97 14178 14182

23. ImaiSArmstrongCMKaeberleinMGuarenteL 2000 Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 403 795 800

24. ArmstrongCMKaeberleinMImaiSIGuarenteL 2002 Mutations in Saccharomyces cerevisiae gene SIR2 can have differential effects on in vivo silencing phenotypes and in vitro histone deacetylation activity. Mol Biol Cell 13 1427 1438

25. HechtALarocheTStrahl-BolsingerSGasserSMGrunsteinM 1995 Histone H3 and H4 N-termini interact with SIR3 and SIR4 proteins: a molecular model for the formation of heterochromatin in yeast. Cell 80 583 592

26. GeorgelPTPalacios DeBeerMAPietzGFoxCAHansenJC 2001 Sir3-dependent assembly of supramolecular chromatin structures in vitro. Proc Natl Acad Sci U S A 98 8584 8589

27. JohnsonALiGSikorskiTWBuratowskiSWoodcockCL 2009 Reconstitution of heterochromatin-dependent transcriptional gene silencing. Mol Cell 35 769 781

28. OppikoferMKuengSMartinoFSoeroesSHancockS 2011 The dual role of H4K16 acetylation in the establishment of yeast silent chromatin. Embo J 30 2610 2621

29. Strahl-BolsingerSHechtALuoKGrunsteinM 1997 SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast. Genes Dev 11 83 93

30. RuscheLNKirchmaierALRineJ 2002 Ordered nucleation and spreading of silenced chromatin in Saccharomyces cerevisiae. Mol Biol Cell 13 2207 2222

31. CockellMGottaMPalladinoFMartinSGGasserSM 1998 Targeting Sir proteins to sites of action: a general mechanism for regulated repression. Cold Spring Harb Symp Quant Biol 63 401 412

32. RudnerADHallBEEllenbergerTMoazedD 2005 A nonhistone protein-protein interaction required for assembly of the SIR complex and silent chromatin. Mol Cell Biol 25 4514 4528

33. EhrentrautSHasslerMOppikoferMKuengSWeberJM 2011 Structural basis for the role of the Sir3 AAA+ domain in silencing: Interaction with Sir4 and unmethylated histone H3K79. Genes & Development 25 1835 1846

34. ZillOAScannellDTeytelmanLRineJ 2010 Co-evolution of transcriptional silencing proteins and the DNA elements specifying their assembly. PLoS Biol 8 e1000550 doi:10.1371/journal.pbio.1000550

35. TannyJCKirkpatrickDSGerberSAGygiSPMoazedD 2004 Budding yeast silencing complexes and regulation of Sir2 activity by protein-protein interactions. Mol Cell Biol 24 6931 6946

36. ChangJFHallBETannyJCMoazedDFilmanD 2003 Structure of the coiled-coil dimerization motif of Sir4 and its interaction with Sir3. Structure 11 637 649

37. MurphyGASpedaleEJPowellSTPillusLSchultzSC 2003 The Sir4 C-terminal coiled coil is required for telomeric and mating type silencing in Saccharomyces cerevisiae. J Mol Biol 334 769 780

38. MorettiPShoreD 2001 Multiple interactions in Sir protein recruitment by Rap1p at silencers and telomeres in yeast. Mol Cell Biol 21 8082 8094

39. TsukamotoYKatoJIkedaH 1997 Silencing factors participate in DNA repair and recombination in Saccharomyces cerevisiae. Nature 388 900 903

40. LarocheTMartinSGGottaMGorhamHCPrydeFE 1998 Mutation of yeast Ku genes disrupts the subnuclear organization of telomeres. Curr Biol 8 653 656

41. MishraKShoreD 1999 Yeast Ku protein plays a direct role in telomeric silencing and counteracts inhibition by rif proteins. Curr Biol 9 1123 1126

42. TaddeiAHedigerFNeumannFRBauerCGasserSM 2004 Separation of silencing from perinuclear anchoring functions in yeast Ku80, Sir4 and Esc1 proteins. Embo J 23 1301 1312

43. RoyRMeierBMcAinshADFeldmannHMJacksonSP 2004 Separation-of-function mutants of yeast Ku80 reveal a Yku80p-Sir4p interaction involved in telomeric silencing. J Biol Chem 279 86 94

44. AndrulisEDNeimanAMZappullaDCSternglanzR 1998 Perinuclear localization of chromatin facilitates transcriptional silencing. Nature 394 592 595

45. MailletLBoscheronCGottaMMarcandSGilsonE 1996 Evidence for silencing compartments within the yeast nucleus: a role for telomere proximity and Sir protein concentration in silencer-mediated repression. Genes Dev 10 1796 1811

46. HedigerFNeumannFRVan HouweGDubranaKGasserSM 2002 Live imaging of telomeres: yKu and Sir proteins define redundant telomere-anchoring pathways in yeast. Curr Biol 12 2076 2089

47. AndrulisEDZappullaDCAnsariAPerrodSLaiosaCV 2002 Esc1, a nuclear periphery protein required for Sir4-based plasmid anchoring and partitioning. Mol Cell Biol 22 8292 8301

48. TaddeiAGasserSM 2004 Multiple pathways for telomere tethering: functional implications of subnuclear position for heterochromatin formation. Biochim Biophys Acta 1677 120 128

49. GartenbergMRNeumannFRLarocheTBlaszczykMGasserSM 2004 Sir-mediated repression can occur independently of chromosomal and subnuclear contexts. Cell 119 955 967

50. TaddeiAVan HouweGNagaiSErbIvan NimwegenE 2009 The functional importance of telomere clustering: global changes in gene expression result from SIR factor dispersion. Genome Res 19 611 625

51. MarshallMMahoneyDRoseAHicksJBBroachJR 1987 Functional domains of SIR4, a gene required for position effect regulation in Saccharomyces cerevisiae. Mol Cell Biol 7 4441 4452

52. MartinoFKuengSRobinsonPTsai-PflugfelderMvan LeeuwenF 2009 Reconstitution of yeast silent chromatin: multiple contact sites and O-AADPR binding load SIR complexes onto nucleosomes in vitro. Mol Cell 33 323 334

53. CockellMRenauldHWattPGasserSM 1998 Sif2p interacts with Sir4p amino-terminal domain and antagonizes telomeric silencing in yeast. Curr Biol 8 787 790

54. PijnappelWWSchaftDRoguevAShevchenkoATekotteH 2001 The S. cerevisiae SET3 complex includes two histone deacetylases, Hos2 and Hst1, and is a meiotic-specific repressor of the sporulation gene program. Genes Dev 15 2991 3004

55. PillusLRineJ 1989 Epigenetic inheritance of transcriptional states in S. cerevisiae. Cell 59 637 647

56. PattersonEEFoxCA 2008 The Ku complex in silencing the cryptic mating-type loci of Saccharomyces cerevisiae. Genetics 180 771 783

57. VandreCLKamakakaRTRivierDH 2008 The DNA end-binding protein Ku regulates silencing at the internal HML and HMR loci in Saccharomyces cerevisiae. Genetics 180 1407 1418

58. RenauldHAparicioOMZierathPDBillingtonBLChhablaniSK 1993 Silent domains are assembled continuously from the telomere and are defined by promoter distance and strength, and by SIR3 dosage. Genes Dev 7 1133 1145

59. AiWBertramPGTsangCKChanTFZhengXF 2002 Regulation of subtelomeric silencing during stress response. Mol Cell 10 1295 1305

60. HardyCFSusselLShoreD 1992 A RAP1-interacting protein involved in transcriptional silencing and telomere length regulation. Genes Dev 6 801 814

61. LowaryPTWidomJ 1998 New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning. J Mol Biol 276 19 42

62. RuaultMDe MeyerALoiodiceITaddeiA 2011 Clustering heterochromatin: Sir3 promotes telomere clustering independently of silencing in yeast. J Cell Biol 192 417 431

63. LarocheTMartinSGTsai-PflugfelderMGasserSM 2000 The dynamics of yeast telomeres and silencing proteins through the cell cycle. J Struct Biol 129 159 174

64. SmithCDSmithDLDeRisiJLBlackburnEH 2003 Telomeric protein distributions and remodeling through the cell cycle in Saccharomyces cerevisiae. Mol Biol Cell 14 556 570

65. MartinSGLarocheTSukaNGrunsteinMGasserSM 1999 Relocalization of telomeric Ku and SIR proteins in response to DNA strand breaks in yeast. Cell 97 621 633

66. MillsKDSinclairDAGuarenteL 1999 MEC1-dependent redistribution of the Sir3 silencing protein from telomeres to DNA double-strand breaks. Cell 97 609 620

67. Radman-LivajaMRubenGWeinerAFriedmanNKamakakaR 2011 Dynamics of Sir3 spreading in budding yeast: secondary recruitment sites and euchromatic localization. Embo J 30 1012 1026

68. RayAHectorRERoyNSongJHBerknerKL 2003 Sir3p phosphorylation by the Slt2p pathway effects redistribution of silencing function and shortened lifespan. Nat Genet 33 522 526

69. HoltLJTuchBBVillenJJohnsonADGygiSP 2009 Global analysis of Cdk1 substrate phosphorylation sites provides insights into evolution. Science 325 1682 1686

70. UbersaxJAWoodburyELQuangPNParazMBlethrowJD 2003 Targets of the cyclin-dependent kinase Cdk1. Nature 425 859 864

71. MillerMLJensenLJDiellaFJorgensenCTintiM 2008 Linear motif atlas for phosphorylation-dependent signaling. Sci Signal 1 ra2

72. XueYRenJGaoXJinCWenL 2008 GPS 2.0, a tool to predict kinase-specific phosphorylation sites in hierarchy. Mol Cell Proteomics 7 1598 1608

73. DenisonCRudnerADGerberSABakalarskiCEMoazedD 2005 A proteomic strategy for gaining insights into protein sumoylation in yeast. Mol Cell Proteomics 4 246 254

74. HwangCSShemorryAVarshavskyA 2011 N-terminal acetylation of cellular proteins creates specific degradation signals. Science 327 973 977

75. LiouGGTannyJCKrugerRGWalzTMoazedD 2005 Assembly of the SIR complex and its regulation by O-acetyl-ADP-ribose, a product of NAD-dependent histone deacetylation. Cell 121 515 527

76. SchnellRD'AriLFossMGoodmanDRineJ 1989 Genetic and molecular characterization of suppressors of SIR4 mutations in Saccharomyces cerevisiae. Genetics 122 29 46

77. DasguptaARamseyKLSmithJSAubleDT 2004 Sir Antagonist 1 (San1) is a ubiquitin ligase. J Biol Chem 279 26830 26838

78. ValenzuelaLDhillonNDubeyRNGartenbergMRKamakakaRT 2008 Long-range communication between the silencers of HMR. Mol Cell Biol 28 1924 1935

79. HofmannJFLarocheTBrandAHGasserSM 1989 RAP-1 factor is necessary for DNA loop formation in vitro at the silent mating type locus HML. Cell 57 725 737

80. WeissKSimpsonRT 1998 High-resolution structural analysis of chromatin at specific loci: Saccharomyces cerevisiae silent mating type locus HMLalpha. Mol Cell Biol 18 5392 5403

81. MoazedDKistlerAAxelrodARineJJohnsonAD 1997 Silent information regulator protein complexes in Saccharomyces cerevisiae: a SIR2/SIR4 complex and evidence for a regulatory domain in SIR4 that inhibits its interaction with SIR3. Proc Natl Acad Sci U S A 94 2186 2191

82. AparicioOMGottschlingDE 1994 Overcoming telomeric silencing: a trans-activator competes to establish gene expression in a cell cycle-dependent way. Genes Dev 8 1133 1146

83. KirchmaierALRineJ 2006 Cell cycle requirements in assembling silent chromatin in Saccharomyces cerevisiae. Mol Cell Biol 26 852 862

84. MillerAMNasmythKA 1984 Role of DNA replication in the repression of silent mating type loci in yeast. Nature 312 247 251

85. Martins-TaylorKDulaMLHolmesSG 2004 Heterochromatin spreading at yeast telomeres occurs in M phase. Genetics 168 65 75

86. KellumRRaffJWAlbertsBM 1995 Heterochromatin protein 1 distribution during development and during the cell cycle in Drosophila embryos. J Cell Sci 108 Pt 4 1407 1418

87. BuchenauPHodgsonJStruttHArndt-JovinDJ 1998 The distribution of polycomb-group proteins during cell division and development in Drosophila embryos: impact on models for silencing. J Cell Biol 141 469 481

88. WeiYChenYHLiLYLangJYehSP 2011 CDK1-dependent phosphorylation of EZH2 suppresses methylation of H3K27 and promotes osteogenic differentiation of human mesenchymal stem cells. Nat Cell Biol 13 87 94

89. ChenSBohrerLRRaiANPanYGanL 2010 Cyclin-dependent kinases regulate epigenetic gene silencing through phosphorylation of EZH2. Nat Cell Biol 12 1108 1114

90. FischleWTsengBSDormannHLUeberheideBMGarciaBA 2005 Regulation of HP1-chromatin binding by histone H3 methylation and phosphorylation. Nature 438 1116 1122

91. HirotaTLippJJTohBHPetersJM 2005 Histone H3 serine 10 phosphorylation by Aurora B causes HP1 dissociation from heterochromatin. Nature 438 1176 1180

92. PerrodSCockellMMLarocheTRenauldHDucrestAL 2001 A cytosolic NAD-dependent deacetylase, Hst2p, can modulate nucleolar and telomeric silencing in yeast. Embo J 20 197 209

93. GolemisEASerebriiskiiIFinleyRLJrKoloninMGGyurisJ 2001 Interaction trap/two-hybrid system to identify interacting proteins. Curr Protoc Protein Sci Chapter 19 Unit19 12

94. BjergbaekLCobbJATsai-PflugfelderMGasserSM 2005 Mechanistically distinct roles for Sgs1p in checkpoint activation and replication fork maintenance. Embo J 24 405 417

95. GottaMPalladinoFGasserSM 1998 Functional characterization of the N terminus of Sir3p. Mol Cell Biol 18 6110 6120

96. PantazisPWestMHBonnerWM 1984 Phosphorylation of histones in cells treated with hypertonic and acidic media. Mol Cell Biol 4 1186 1188

97. HerzogFPetersJM 2005 Large-scale purification of the vertebrate anaphase-promoting complex/cyclosome. Methods Enzymol 398 175 195

98. MeisterPGehlenLRVarelaEKalckVGasserSM 2010 Visualizing yeast chromosomes and nuclear architecture. Methods Enzymol 470 535 567

99. GottschlingDEAparicioOMBillingtonBLZakianVA 1990 Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription. Cell 63 751 762

100. MondouxMAZakianVA 2007 Subtelomeric elements influence but do not determine silencing levels at Saccharomyces cerevisiae telomeres. Genetics 177 2541 2546

Štítky
Genetika Reprodukčná medicína

Článok vyšiel v časopise

PLOS Genetics


2012 Číslo 5
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Aktuální možnosti diagnostiky a léčby litiáz
nový kurz
Autori: MUDr. Tomáš Ürge, PhD.

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#