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. 2002 Oct;162(2):633–645. doi: 10.1093/genetics/162.2.633

Restoration of silencing in Saccharomyces cerevisiae by tethering of a novel Sir2-interacting protein, Esc8.

Guido Cuperus 1, David Shore 1
PMCID: PMC1462306  PMID: 12399377

Abstract

We previously described two classes of SIR2 mutations specifically defective in either telomeric/HM silencing (class I) or rDNA silencing (class II) in S. cerevisiae. Here we report the identification of genes whose protein products, when either overexpressed or directly tethered to the locus in question, can establish silencing in SIR2 class I mutants. Elevated dosage of SCS2, previously implicated as a regulator of both inositol biosynthesis and telomeric silencing, suppressed the dominant-negative effect of a SIR2-143 mutation. In a genetic screen for proteins that restore silencing when tethered to a telomere, we isolated ESC2 and an uncharacterized gene, (YOL017w), which we call ESC8. Both Esc2p and Esc8p interact with Sir2p in two-hybrid assays, and the Esc8p-Sir2 interaction is detected in vitro. Interestingly, Esc8p has a single close homolog in yeast, the ISW1-complex factor Ioc3p, and has also been copurified with Isw1p, raising the possibility that Esc8p is a component of an Isw1p-containing nucleosome remodeling complex. Whereas esc2 and esc8 deletion mutants alone have only marginal silencing defects, cells lacking Isw1p show a strong silencing defect at HMR but not at telomeres. Finally, we show that Esc8p interacts with the Gal11 protein, a component of the RNA pol II mediator complex.

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Selected References

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  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Brachmann C. B., Sherman J. M., Devine S. E., Cameron E. E., Pillus L., Boeke J. D. The SIR2 gene family, conserved from bacteria to humans, functions in silencing, cell cycle progression, and chromosome stability. Genes Dev. 1995 Dec 1;9(23):2888–2902. doi: 10.1101/gad.9.23.2888. [DOI] [PubMed] [Google Scholar]
  3. Braunstein M., Rose A. B., Holmes S. G., Allis C. D., Broach J. R. Transcriptional silencing in yeast is associated with reduced nucleosome acetylation. Genes Dev. 1993 Apr;7(4):592–604. doi: 10.1101/gad.7.4.592. [DOI] [PubMed] [Google Scholar]
  4. Bryk M., Banerjee M., Murphy M., Knudsen K. E., Garfinkel D. J., Curcio M. J. Transcriptional silencing of Ty1 elements in the RDN1 locus of yeast. Genes Dev. 1997 Jan 15;11(2):255–269. doi: 10.1101/gad.11.2.255. [DOI] [PubMed] [Google Scholar]
  5. Buck S. W., Shore D. Action of a RAP1 carboxy-terminal silencing domain reveals an underlying competition between HMR and telomeres in yeast. Genes Dev. 1995 Feb 1;9(3):370–384. doi: 10.1101/gad.9.3.370. [DOI] [PubMed] [Google Scholar]
  6. Craven R. J., Petes T. D. The Saccharomyces cerevisiae suppressor of choline sensitivity (SCS2) gene is a multicopy Suppressor of mec1 telomeric silencing defects. Genetics. 2001 May;158(1):145–154. doi: 10.1093/genetics/158.1.145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cuperus G., Shafaatian R., Shore D. Locus specificity determinants in the multifunctional yeast silencing protein Sir2. EMBO J. 2000 Jun 1;19(11):2641–2651. doi: 10.1093/emboj/19.11.2641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Derbyshire M. K., Weinstock K. G., Strathern J. N. HST1, a new member of the SIR2 family of genes. Yeast. 1996 Jun 15;12(7):631–640. doi: 10.1002/(SICI)1097-0061(19960615)12:7%3C631::AID-YEA960%3E3.0.CO;2-8. [DOI] [PubMed] [Google Scholar]
  9. Deuring R., Fanti L., Armstrong J. A., Sarte M., Papoulas O., Prestel M., Daubresse G., Verardo M., Moseley S. L., Berloco M. The ISWI chromatin-remodeling protein is required for gene expression and the maintenance of higher order chromatin structure in vivo. Mol Cell. 2000 Feb;5(2):355–365. doi: 10.1016/s1097-2765(00)80430-x. [DOI] [PubMed] [Google Scholar]
  10. Dhillon N., Kamakaka R. T. A histone variant, Htz1p, and a Sir1p-like protein, Esc2p, mediate silencing at HMR. Mol Cell. 2000 Oct;6(4):769–780. doi: 10.1016/s1097-2765(00)00076-9. [DOI] [PubMed] [Google Scholar]
  11. Edmondson D. G., Smith M. M., Roth S. Y. Repression domain of the yeast global repressor Tup1 interacts directly with histones H3 and H4. Genes Dev. 1996 May 15;10(10):1247–1259. doi: 10.1101/gad.10.10.1247. [DOI] [PubMed] [Google Scholar]
  12. Fassler J. S., Winston F. The Saccharomyces cerevisiae SPT13/GAL11 gene has both positive and negative regulatory roles in transcription. Mol Cell Biol. 1989 Dec;9(12):5602–5609. doi: 10.1128/mcb.9.12.5602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fritze C. E., Verschueren K., Strich R., Easton Esposito R. Direct evidence for SIR2 modulation of chromatin structure in yeast rDNA. EMBO J. 1997 Nov 3;16(21):6495–6509. doi: 10.1093/emboj/16.21.6495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Frye R. A. Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins. Biochem Biophys Res Commun. 2000 Jul 5;273(2):793–798. doi: 10.1006/bbrc.2000.3000. [DOI] [PubMed] [Google Scholar]
  15. Gasser S. M., Cockell M. M. The molecular biology of the SIR proteins. Gene. 2001 Nov 14;279(1):1–16. doi: 10.1016/s0378-1119(01)00741-7. [DOI] [PubMed] [Google Scholar]
  16. Gavin Anne-Claude, Bösche Markus, Krause Roland, Grandi Paola, Marzioch Martina, Bauer Andreas, Schultz Jörg, Rick Jens M., Michon Anne-Marie, Cruciat Cristina-Maria. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature. 2002 Jan 10;415(6868):141–147. doi: 10.1038/415141a. [DOI] [PubMed] [Google Scholar]
  17. Ghidelli S., Donze D., Dhillon N., Kamakaka R. T. Sir2p exists in two nucleosome-binding complexes with distinct deacetylase activities. EMBO J. 2001 Aug 15;20(16):4522–4535. doi: 10.1093/emboj/20.16.4522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Goldmark J. P., Fazzio T. G., Estep P. W., Church G. M., Tsukiyama T. The Isw2 chromatin remodeling complex represses early meiotic genes upon recruitment by Ume6p. Cell. 2000 Oct 27;103(3):423–433. doi: 10.1016/s0092-8674(00)00134-3. [DOI] [PubMed] [Google Scholar]
  19. Gottlieb S., Esposito R. E. A new role for a yeast transcriptional silencer gene, SIR2, in regulation of recombination in ribosomal DNA. Cell. 1989 Mar 10;56(5):771–776. doi: 10.1016/0092-8674(89)90681-8. [DOI] [PubMed] [Google Scholar]
  20. Gottschling D. E., Aparicio O. M., Billington B. L., Zakian V. A. Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription. Cell. 1990 Nov 16;63(4):751–762. doi: 10.1016/0092-8674(90)90141-z. [DOI] [PubMed] [Google Scholar]
  21. Grunstein M. Yeast heterochromatin: regulation of its assembly and inheritance by histones. Cell. 1998 May 1;93(3):325–328. doi: 10.1016/s0092-8674(00)81160-5. [DOI] [PubMed] [Google Scholar]
  22. Güldener U., Heck S., Fielder T., Beinhauer J., Hegemann J. H. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. 1996 Jul 1;24(13):2519–2524. doi: 10.1093/nar/24.13.2519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Himmelfarb H. J., Pearlberg J., Last D. H., Ptashne M. GAL11P: a yeast mutation that potentiates the effect of weak GAL4-derived activators. Cell. 1990 Dec 21;63(6):1299–1309. doi: 10.1016/0092-8674(90)90425-e. [DOI] [PubMed] [Google Scholar]
  24. Imai S., Armstrong C. M., Kaeberlein M., Guarente L. Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature. 2000 Feb 17;403(6771):795–800. doi: 10.1038/35001622. [DOI] [PubMed] [Google Scholar]
  25. James P., Halladay J., Craig E. A. Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics. 1996 Dec;144(4):1425–1436. doi: 10.1093/genetics/144.4.1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kagiwada S., Hosaka K., Murata M., Nikawa J., Takatsuki A. The Saccharomyces cerevisiae SCS2 gene product, a homolog of a synaptobrevin-associated protein, is an integral membrane protein of the endoplasmic reticulum and is required for inositol metabolism. J Bacteriol. 1998 Apr;180(7):1700–1708. doi: 10.1128/jb.180.7.1700-1708.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kennedy B. K., Gotta M., Sinclair D. A., Mills K., McNabb D. S., Murthy M., Pak S. M., Laroche T., Gasser S. M., Guarente L. Redistribution of silencing proteins from telomeres to the nucleolus is associated with extension of life span in S. cerevisiae. Cell. 1997 May 2;89(3):381–391. doi: 10.1016/s0092-8674(00)80219-6. [DOI] [PubMed] [Google Scholar]
  28. Kent N. A., Karabetsou N., Politis P. K., Mellor J. In vivo chromatin remodeling by yeast ISWI homologs Isw1p and Isw2p. Genes Dev. 2001 Mar 1;15(5):619–626. doi: 10.1101/gad.190301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kim Y. J., Björklund S., Li Y., Sayre M. H., Kornberg R. D. A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cell. 1994 May 20;77(4):599–608. doi: 10.1016/0092-8674(94)90221-6. [DOI] [PubMed] [Google Scholar]
  30. Kirchmaier A. L., Rine J. DNA replication-independent silencing in S. cerevisiae. Science. 2001 Jan 26;291(5504):646–650. doi: 10.1126/science.291.5504.646. [DOI] [PubMed] [Google Scholar]
  31. Koleske A. J., Young R. A. The RNA polymerase II holoenzyme and its implications for gene regulation. Trends Biochem Sci. 1995 Mar;20(3):113–116. doi: 10.1016/s0968-0004(00)88977-x. [DOI] [PubMed] [Google Scholar]
  32. Landry J., Sutton A., Tafrov S. T., Heller R. C., Stebbins J., Pillus L., Sternglanz R. The silencing protein SIR2 and its homologs are NAD-dependent protein deacetylases. Proc Natl Acad Sci U S A. 2000 May 23;97(11):5807–5811. doi: 10.1073/pnas.110148297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Laroche T., Martin S. G., Gotta M., Gorham H. C., Pryde F. E., Louis E. J., Gasser S. M. Mutation of yeast Ku genes disrupts the subnuclear organization of telomeres. Curr Biol. 1998 May 21;8(11):653–656. doi: 10.1016/s0960-9822(98)70252-0. [DOI] [PubMed] [Google Scholar]
  34. Li Y. C., Cheng T. H., Gartenberg M. R. Establishment of transcriptional silencing in the absence of DNA replication. Science. 2001 Jan 26;291(5504):650–653. doi: 10.1126/science.291.5504.650. [DOI] [PubMed] [Google Scholar]
  35. Li Y., Bjorklund S., Jiang Y. W., Kim Y. J., Lane W. S., Stillman D. J., Kornberg R. D. Yeast global transcriptional regulators Sin4 and Rgr1 are components of mediator complex/RNA polymerase II holoenzyme. Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):10864–10868. doi: 10.1073/pnas.92.24.10864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Liu Y., Ranish J. A., Aebersold R., Hahn S. Yeast nuclear extract contains two major forms of RNA polymerase II mediator complexes. J Biol Chem. 2001 Mar 9;276(10):7169–7175. [PubMed] [Google Scholar]
  37. Malik S., Roeder R. G. Transcriptional regulation through Mediator-like coactivators in yeast and metazoan cells. Trends Biochem Sci. 2000 Jun;25(6):277–283. doi: 10.1016/s0968-0004(00)01596-6. [DOI] [PubMed] [Google Scholar]
  38. Marcand S., Buck S. W., Moretti P., Gilson E., Shore D. Silencing of genes at nontelomeric sites in yeast is controlled by sequestration of silencing factors at telomeres by Rap 1 protein. Genes Dev. 1996 Jun 1;10(11):1297–1309. doi: 10.1101/gad.10.11.1297. [DOI] [PubMed] [Google Scholar]
  39. Mishra K., Shore D. Yeast Ku protein plays a direct role in telomeric silencing and counteracts inhibition by rif proteins. Curr Biol. 1999 Oct 7;9(19):1123–1126. doi: 10.1016/s0960-9822(99)80483-7. [DOI] [PubMed] [Google Scholar]
  40. Moretti P., Freeman K., Coodly L., Shore D. Evidence that a complex of SIR proteins interacts with the silencer and telomere-binding protein RAP1. Genes Dev. 1994 Oct 1;8(19):2257–2269. doi: 10.1101/gad.8.19.2257. [DOI] [PubMed] [Google Scholar]
  41. Moretti P., Shore D. Multiple interactions in Sir protein recruitment by Rap1p at silencers and telomeres in yeast. Mol Cell Biol. 2001 Dec;21(23):8082–8094. doi: 10.1128/MCB.21.23.8082-8094.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Myers L. C., Kornberg R. D. Mediator of transcriptional regulation. Annu Rev Biochem. 2000;69:729–749. doi: 10.1146/annurev.biochem.69.1.729. [DOI] [PubMed] [Google Scholar]
  43. Nugent C. I., Bosco G., Ross L. O., Evans S. K., Salinger A. P., Moore J. K., Haber J. E., Lundblad V. Telomere maintenance is dependent on activities required for end repair of double-strand breaks. Curr Biol. 1998 May 21;8(11):657–660. doi: 10.1016/s0960-9822(98)70253-2. [DOI] [PubMed] [Google Scholar]
  44. Papamichos-Chronakis M., Conlan R. S., Gounalaki N., Copf T., Tzamarias D. Hrs1/Med3 is a Cyc8-Tup1 corepressor target in the RNA polymerase II holoenzyme. J Biol Chem. 2000 Mar 24;275(12):8397–8403. doi: 10.1074/jbc.275.12.8397. [DOI] [PubMed] [Google Scholar]
  45. Perrod S., Cockell M. M., Laroche T., Renauld H., Ducrest A. L., Bonnard C., Gasser S. M. A cytosolic NAD-dependent deacetylase, Hst2p, can modulate nucleolar and telomeric silencing in yeast. EMBO J. 2001 Jan 15;20(1-2):197–209. doi: 10.1093/emboj/20.1.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Piruat J. I., Chávez S., Aguilera A. The yeast HRS1 gene is involved in positive and negative regulation of transcription and shows genetic characteristics similar to SIN4 and GAL11. Genetics. 1997 Dec;147(4):1585–1594. doi: 10.1093/genetics/147.4.1585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Polotnianka R. M., Li J., Lustig A. J. The yeast Ku heterodimer is essential for protection of the telomere against nucleolytic and recombinational activities. Curr Biol. 1998 Jul 2;8(14):831–834. doi: 10.1016/s0960-9822(98)70325-2. [DOI] [PubMed] [Google Scholar]
  48. Ravindra A., Weiss K., Simpson R. T. High-resolution structural analysis of chromatin at specific loci: Saccharomyces cerevisiae silent mating-type locus HMRa. Mol Cell Biol. 1999 Dec;19(12):7944–7950. doi: 10.1128/mcb.19.12.7944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Roy N., Runge K. W. Two paralogs involved in transcriptional silencing that antagonistically control yeast life span. Curr Biol. 2000 Jan 27;10(2):111–114. doi: 10.1016/s0960-9822(00)00298-0. [DOI] [PubMed] [Google Scholar]
  50. San-Segundo P. A., Roeder G. S. Pch2 links chromatin silencing to meiotic checkpoint control. Cell. 1999 Apr 30;97(3):313–324. doi: 10.1016/s0092-8674(00)80741-2. [DOI] [PubMed] [Google Scholar]
  51. Sekinger E. A., Gross D. S. Silenced chromatin is permissive to activator binding and PIC recruitment. Cell. 2001 May 4;105(3):403–414. doi: 10.1016/s0092-8674(01)00329-4. [DOI] [PubMed] [Google Scholar]
  52. Shou W., Seol J. H., Shevchenko A., Baskerville C., Moazed D., Chen Z. W., Jang J., Shevchenko A., Charbonneau H., Deshaies R. J. Exit from mitosis is triggered by Tem1-dependent release of the protein phosphatase Cdc14 from nucleolar RENT complex. Cell. 1999 Apr 16;97(2):233–244. doi: 10.1016/s0092-8674(00)80733-3. [DOI] [PubMed] [Google Scholar]
  53. Smith J. S., Brachmann C. B., Celic I., Kenna M. A., Muhammad S., Starai V. J., Avalos J. L., Escalante-Semerena J. C., Grubmeyer C., Wolberger C. A phylogenetically conserved NAD+-dependent protein deacetylase activity in the Sir2 protein family. Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6658–6663. doi: 10.1073/pnas.97.12.6658. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Spellman P. T., Sherlock G., Zhang M. Q., Iyer V. R., Anders K., Eisen M. B., Brown P. O., Botstein D., Futcher B. Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. Mol Biol Cell. 1998 Dec;9(12):3273–3297. doi: 10.1091/mbc.9.12.3273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Steyerberg E. W., Kievit J., de Mol Van Otterloo J. C., van Bockel J. H., Eijkemans M. J., Habbema J. D. Perioperative mortality of elective abdominal aortic aneurysm surgery. A clinical prediction rule based on literature and individual patient data. Arch Intern Med. 1995 Oct 9;155(18):1998–2004. [PubMed] [Google Scholar]
  56. Straight A. F., Shou W., Dowd G. J., Turck C. W., Deshaies R. J., Johnson A. D., Moazed D. Net1, a Sir2-associated nucleolar protein required for rDNA silencing and nucleolar integrity. Cell. 1999 Apr 16;97(2):245–256. doi: 10.1016/s0092-8674(00)80734-5. [DOI] [PubMed] [Google Scholar]
  57. Sussel L., Shore D. 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. 1991 Sep 1;88(17):7749–7753. doi: 10.1073/pnas.88.17.7749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Sussel L., Vannier D., Shore D. Epigenetic switching of transcriptional states: cis- and trans-acting factors affecting establishment of silencing at the HMR locus in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Jul;13(7):3919–3928. doi: 10.1128/mcb.13.7.3919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Sussel L., Vannier D., Shore D. Suppressors of defective silencing in yeast: effects on transcriptional repression at the HMR locus, cell growth and telomere structure. Genetics. 1995 Nov;141(3):873–888. doi: 10.1093/genetics/141.3.873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Suzuki Y., Nishizawa M. The yeast GAL11 protein is involved in regulation of the structure and the position effect of telomeres. Mol Cell Biol. 1994 Jun;14(6):3791–3799. doi: 10.1128/mcb.14.6.3791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Thomas B. J., Rothstein R. Elevated recombination rates in transcriptionally active DNA. Cell. 1989 Feb 24;56(4):619–630. doi: 10.1016/0092-8674(89)90584-9. [DOI] [PubMed] [Google Scholar]
  62. Triolo T., Sternglanz R. Role of interactions between the origin recognition complex and SIR1 in transcriptional silencing. Nature. 1996 May 16;381(6579):251–253. doi: 10.1038/381251a0. [DOI] [PubMed] [Google Scholar]
  63. Tsukiyama T., Palmer J., Landel C. C., Shiloach J., Wu C. Characterization of the imitation switch subfamily of ATP-dependent chromatin-remodeling factors in Saccharomyces cerevisiae. Genes Dev. 1999 Mar 15;13(6):686–697. doi: 10.1101/gad.13.6.686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Varga-Weisz P. D., Becker P. B. Chromatin-remodeling factors: machines that regulate? Curr Opin Cell Biol. 1998 Jun;10(3):346–353. doi: 10.1016/s0955-0674(98)80010-0. [DOI] [PubMed] [Google Scholar]
  65. Watson A. D., Edmondson D. G., Bone J. R., Mukai Y., Yu Y., Du W., Stillman D. J., Roth S. Y. Ssn6-Tup1 interacts with class I histone deacetylases required for repression. Genes Dev. 2000 Nov 1;14(21):2737–2744. doi: 10.1101/gad.829100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Yu G., Fassler J. S. SPT13 (GAL11) of Saccharomyces cerevisiae negatively regulates activity of the MCM1 transcription factor in Ty1 elements. Mol Cell Biol. 1993 Jan;13(1):63–71. doi: 10.1128/mcb.13.1.63. [DOI] [PMC free article] [PubMed] [Google Scholar]

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