Molecular Genetics and Genomics

, Volume 277, Issue 5, pp 491–506 | Cite as

The role of histone ubiquitylation and deubiquitylation in gene expression as determined by the analysis of an HTB1 K123R Saccharomyces cerevisiae strain

  • A. Irina Mutiu
  • Stephen M. T. Hoke
  • Julie Genereaux
  • Gaoyang Liang
  • Christopher J. Brandl
Original Paper


In Saccharomyces cerevisiae histone H2B is ubiquitylated at lysine 123 in a process requiring the E2-ubiquitin conjugase, Rad6. We have analyzed gene expression in a strain containing a variant of histone H2B with lysine 123 converted to arginine to address the mechanisms by which ubiquitylation and deubiquitylation of histone H2B affect gene expression. The SAGA complex component, Ubp8, is one of two proteases that remove the ubiquitin moiety at lysine 123. We show that changes in gene expression observed upon deletion of ubp8 are suppressed by htb1 K123R , which provides genetic evidence that Ubp8 alters gene expression through deubiquitylation of histone H2B. Microarray analyses of the htb1 K123R strain show that loss of histone ubiquitylation results in a twofold or greater change in expression of ∼1.5% of the protein coding genes with ∼75% of these increasing. For genes in which ubiquitylation represses expression, ubiquitylation principally acts through its effects on histone methylation. In contrast, decreased expression of the CWP1 gene was not paralleled by deletions of methyltransferase components and is thus likely independent of methylation. Finally, by comparing gene expression changes in the htb1 K123R strain with those in a strain deleted for rad6, we conclude that lysine 123 affects transcription primarily because of it being a site of ubiquitylation.


Transcription Yeast Ubp8 Histone deubiquitylation SAGA complex 



We would like to thank Amy Tong and Drs. Brenda Andrews and Charlie Boone for providing reagents as well as Megan Davey, David Edgell, Eric Ball, Kerri Kobryn, and David Haniford for their discussions on this manuscript. Unpublished microarray data was kindly supplied by Drs. F. Devaux and WS Moye-Rowley. Special thanks to Karen Kennedy for technical assistance. This work was supported by a Canadian Institutes of Health Research operating grant to CJB (MT10845). SMTH is supported by a NSERC (Canada) Studentship. AIM and GL were supported by Western Graduate Research Scholarships.


  1. Arndt K, Winston F (2005) An unexpected role for ubiquitylation of a transcriptional activator. Cell 120:733–734PubMedCrossRefGoogle Scholar
  2. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) (1988) Current protocols in molecular biology. Greene/Wiley-Interscience, New York, NYGoogle Scholar
  3. Bedalov A, Gatbonton T, Irvine WP, Gottschling DE, Simon JA (2001) Identification of a small molecule inhibitor of Sir2p. Proc Natl Acad Sci USA 98:15113–15118PubMedCrossRefGoogle Scholar
  4. Brandl CJ, Furlanetto AM, Martens JA, Hamilton KS (1993) Characterization of NGG1, a novel yeast gene required for glucose repression of GAL4p-regulated transcription. EMBO J 12:5255–5265PubMedGoogle Scholar
  5. Briggs SD, Xiao T, Sun ZW, Caldwell JA, Shabanowitz J, Hunt DF, Allis CD, Strahl BD (2002) Gene silencing: trans-histone regulatory pathway in chromatin. Nature 418:498PubMedCrossRefGoogle Scholar
  6. Conaway RC, Brower CS, Conaway JW (2002) Emerging roles of ubiquitin in transcription regulation. Science 296:1254–1258PubMedCrossRefGoogle Scholar
  7. Daniel JA, Torok MS, Sun ZW, Schieltz D, Allis CD, Yates JR III, Grant PA (2004) Deubiquitination of histone H2B by a yeast acetyltransferase complex regulates transcription. J Biol Chem 279:1867–1871PubMedCrossRefGoogle Scholar
  8. Dehe PM, Pamblanco M, Luciano P, Lebrun R, Moinier D, Sendra R, Verreault A, Tordera V, Geli V (2005) Histone H3 lysine 4 mono-methylation does not require ubiquitination of histone H2B. J Mol Biol 353:477–484PubMedCrossRefGoogle Scholar
  9. Dover J, Schneider J, Tawiah-Boateng MA, Wood A, Dean K, Johnston M, Shilatifard A (2003) Methylation of histone H3 by COMPASS requires ubiquitination of histone H2B by Rad6. J Biol Chem 277:28358–28371Google Scholar
  10. Eisen MB, Spellman PT, Brown PO, Botstein D (1998) Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci 95:14863–14868PubMedCrossRefGoogle Scholar
  11. Emre NC, Ingvarsdottir K, Wyce A, Wood A, Krogan NJ, Henry KW, Li K, Marmorstein R, Greenblatt JF, Shilatifard A, Berger SL (2005) Maintenance of low histone ubiquitylation by Ubp10 correlates with telomere-proximal Sir2 association and gene silencing. Mol Cell 17:585–594PubMedCrossRefGoogle Scholar
  12. Fogel S, Welch JW (1982) Tandem gene amplification mediates copper resistance in yeast. Proc Natl Acad Sci USA 79:5342–5346PubMedCrossRefGoogle Scholar
  13. Gardner RG, Nelson ZW, Gottschling DE (2005) Ubp10/Dot4p regulates the persistence of ubiquitinated histone H2B: distinct roles in telomeric silencing and general chromatin. Mol Cell Biol 25:6123–6129PubMedCrossRefGoogle Scholar
  14. Giannattasio M, Lazzaro F, Plevani P, Muzi-Falconi M (2005) The DNA damage checkpoint response requires histone H2B ubiquitination by Rad6-Bre1 and H3 methylation by Dot1. J Biol Chem 280:9879–9886PubMedCrossRefGoogle Scholar
  15. Gregory PD, Schmid A, Zavari M, Lui L, Berger SL, Horz W (1998) Absence of Gcn5 HAT activity defines a novel state in the opening at the PHO5 promoter in yeast. Mol Cell 1:495–505PubMedCrossRefGoogle Scholar
  16. Han M, Kim UJ, Kayne P, Grunstein M (1988) Depletion of histone H4 and nucleosomes activates the PHO5 gene in Saccharomyces cerevisiae. EMBO J 7:2221–2228PubMedGoogle Scholar
  17. Henry KW, Wyce A, Lo WS, Duggan LJ, Emre NC, Kao CF, Pillus L, Shilatifard A, Osley MA, Berger SL (2003) Transcriptional activation via sequential histone H2B ubiquitylation and deubiquitylation, mediated by SAGA-associated Ubp8. Genes Dev 17:2648–2663PubMedCrossRefGoogle Scholar
  18. Hughes TR, Marton MJ, Jones AR, Roberts CJ, Stoughton R, Armour CD, Bennett HA, Coffey E, Dai H, He YD, Kidd MJ, King AM, Meyer MR, Slade D, Lum PY, Stepaniants SB, Shoemaker DD, Gachotte D, Chakraburtty K, Simon J, Bard M, Friend SH (2000) Functional discovery via a compendium of expression profiles. Cell 102:109–126PubMedCrossRefGoogle Scholar
  19. Hwang WW, Venkatasubrahmanyam S, Ianculescu AG, Tong A, Boone C, Madhani HD (2003) A conserved RING finger protein required for histone H2B monoubiquitination and cell size control. Mol Cell 11:261–266PubMedCrossRefGoogle Scholar
  20. Ingvarsdottir K, Krogan NJ, Tolga Emre NC, Wyce A, Thompson NJ, Emili A, Hughes TR, Greenblatt JF, Berger SL (2005) H2B ubiquitin protease Ubp8 and Sgf11 constitute a discrete functional module within the Saccharomyces cerevisiae SAGA complex. Mol Cell Biol 25:1162–1172PubMedCrossRefGoogle Scholar
  21. Jason LJ, Moore SC, Lewis JD, Lindsey G, Ausio J (2002) Histone ubiquitination: a tagging tail unfolds? Bioessays 24:166–174PubMedCrossRefGoogle Scholar
  22. Kaiser P, Flick K, Wittenberg C, Reed SI (2000) Regulation of transcription by ubiquitination without proteolysis: Cdc34/SCF (Met30)-mediated inactivation of the transcription factor Met4. Cell 102:303–314PubMedCrossRefGoogle Scholar
  23. Kao CF, Hillyer C, Tsukuda T, Henry K, Berger S, Osley MA (2004) Rad6 plays a role in transcriptional activation through ubiquitylation of histone H2B. Genes Dev 18:184–195PubMedCrossRefGoogle Scholar
  24. Karin M, Najarian R, Haslinger A, Valenzuela P, Welch J, Fogel S (1984) Primary structure and transcription of an amplified genetic locus: the CUP1 locus of yeast. Proc Natl Acad Sci USA 81:337–341PubMedCrossRefGoogle Scholar
  25. Keogh MC, Kurdistani SK, Morris SA, Ahn SH, Podolny V, Collins SR, Schuldiner M, Chin K, Punna T, Thompson NJ, Boone C, Emili A, Weissman JS, Hughes TR, Strahl BD, Grunstein M, Greenblatt JF, Buratowski S, Krogan NJ (2005) Cotranscriptional Set2 methylation of histone H3 lysine 36 recruits a repressive Rpd3 complex. Cell 123:593–605PubMedCrossRefGoogle Scholar
  26. Krogan NJ, Dover J, Khorrami S, Greenblatt JF, Schneider J, Johnston M, Shilatifard A (2002) COMPASS, a histone H3 (Lysine 4) methyltransferase required for telomeric silencing of gene expression. J Biol Chem 277:10753–10755PubMedCrossRefGoogle Scholar
  27. Krogan NJ, Dover J, Wood A, Schneider J, Heidt J, Boateng MA, Dean K, Ryan OW, Golshani A, Johnston M, Greenblatt JF, Shilatifard A (2003a) The Paf1 complex is required for histone H3 methylation by COMPASS and Dot1p: linking transcriptional elongation to histone methylation. Mol Cell 11:721–729CrossRefGoogle Scholar
  28. Krogan NJ, Kim M, Tong A, Golshani A, Cagney G, Canadien V, Richards DP, Beattie BK, Emili A, Boone C, Shilatifard A, Buratowski S, Greenblatt J (2003b) Methylation of histone H3 by Set2 in Saccharomyces cerevisiae is linked to transcriptional elongation by RNA polymerase II. Mol Cell Biol 278:8897–8903Google Scholar
  29. Krogan NJ, Baetz K, Keogh MC, Datta N, Sawa C, Kwok TC, Thompson MG, Davey MG, Pootoolal J, Hughes TR, Emili A, Buratowski S, Hieter P, Greenblatt JF (2004) Regulation of chromosome stability by the histone H2A variant Htz1, the Swr1 chromatin remodeling complex, and the histone acetyltransferase NuA4. Proc Natl Acad Sci USA 101:13513–13518PubMedCrossRefGoogle Scholar
  30. Lee KK, Florens L, Swanson SK, Washburn MP, Workman JL (2005) The deubiquitylation activity of Ubp8 is dependent upon Sgf11 and its association with the SAGA complex. Mol Cell Biol 25:1173–1182PubMedCrossRefGoogle Scholar
  31. McCutcheon JP, Eddy SR (2003) Computational identification of non-coding RNAs in Saccharomyces cerevisiae by comparative genomics. Nucleic Acids Res 31:4119–4128PubMedCrossRefGoogle Scholar
  32. Mutiu AI, Brandl CJ (2005) RNA isolation from yeast using silica matrices. J Biomol Tech 16:316–317PubMedGoogle Scholar
  33. Nagy PL, Griesenbeck J, Kornberg RD, Cleary ML (2002) A trithorax-group complex purified from Saccharomyces cerevisiae is required for methylation of histone H3. Proc Natl Acad Sci 99:90–94PubMedCrossRefGoogle Scholar
  34. Ng HH, Feng Q, Wang H, Erdjument-Bromage H, Tempst P, Zhang Y, Struhl K (2002) Lysine methylation within the globular domain of histone H3 by Dot1 is important for telomeric silencing and Sir protein association. Genes Dev 16:1518–1527PubMedCrossRefGoogle Scholar
  35. Ng HH, Dole S, Struhl K (2003) The Rtf1 component of the Paf1 transcriptional elongation complex is required for ubiquitination of histone H2B. J Biol Chem 278:33625–33628PubMedCrossRefGoogle Scholar
  36. Orlandi I, Bettiga M, Alberghina L, Vai M (2004) Transcriptional profiling of ubp10 null mutant reveals altered subtelomeric gene expression and insurgence of oxidative stress response. J Biol Chem 279:6414–6425PubMedCrossRefGoogle Scholar
  37. Ricci AR, Genereaux J, Brandl CJ (2002) Components of the SAGA histone acetylatransferase complex are required for repressed transcription of ARG1 in rich medium. Mol Cell Biol 22:4033–4042PubMedCrossRefGoogle Scholar
  38. Ridgeway AG, Skerjanc IS (2001) Pax3 is essential for skeletal myogenesis and the expression of Six1 and Eya2. J Biol Chem 276:19033–19039PubMedCrossRefGoogle Scholar
  39. Roberts CJ, Nelson B, Marton MJ, Stoughton R, Meyer MR, Bennett HA, He YD, Dai H, Walker WL, Hughes TR, Tyers M, Boone C, Friend SH (2000) Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles. Science 287:873–880PubMedCrossRefGoogle Scholar
  40. Robzyk K, Recht J, Osley MA (2000) Rad6-dependent ubiquitination of histone H2B in yeast. Science 287:501–504PubMedCrossRefGoogle Scholar
  41. Saleh A, Collart M, Martens JA, Genereaux J, Allard S, Côte J, Brandl CJ (1998) TOM1p, a yeast hect-domain protein which mediates transcriptional regulation through the ADA/SAGA coactivator complexes. J Mol Biol 282:933–946PubMedCrossRefGoogle Scholar
  42. Salghetti SE, Caudy AA, Chenoweth JG, Tansey WP (2001) Regulation of transcriptional activation domain function by ubiquitin. Science 293:1651–1653PubMedCrossRefGoogle Scholar
  43. Sanders SL, Jennings J, Canutescu A, Link AJ, Weil PA (2002) Proteomics of the eukaryotic transcription machinery: identification of proteins associated with components of yeast TFIID by multidimensional mass spectrometry. Mol Cell Biol 22:4723–4738PubMedCrossRefGoogle Scholar
  44. Shahbazian MD, Zhang K, Grunstein M (2005) Histone H2B ubiquitylation controls processive methylation but not monomethylation by Dot1 and Set1. Mol Cell 19:271–277PubMedCrossRefGoogle Scholar
  45. Singer MS, Kahana A, Wolf AJ, Meisinger LL, Peterson SE, Goggin C, Mahowald M, Gottschling DE (1998) Identification of high-copy disruptors of telomeric silencing in Saccharomyces cerevisiae. Genetics 150:613–632PubMedGoogle Scholar
  46. Shukla A, Stanojevic N, Duan Z, Sen PT, Bhaumik SR (2006a) Ubp8p, a histone deubiquitinase whose association with SAGA is mediated by Sgf11p, differentially regulates lysine 4 methylation of histone H3 in vivo. Mol Cell Biol 26:3339–3352CrossRefGoogle Scholar
  47. Shukla A, Stanojevic N, Duan Z, Shadle T, Bhaumik SR (2006b) Functional analysis of H2B-Lys-123 ubiquitination in regulation of H3-Lys-4 methylation and recruitment of RNA polymerase II at the coding sequences of several active genes in vivo. J Biol Chem 281:19045–19054CrossRefGoogle Scholar
  48. Strahl BD, Grant PA, Briggs SD, Sun ZW, Bone JR, Caldwell JA, Mollah S, Cook RG, Shabanowitz J, Hunt DF, Allis CD (2002) Set2 is a nucleosomal histone H3-selective methyltransferase that mediates transcriptional repression. Mol Cell Biol 22:1298–1306PubMedCrossRefGoogle Scholar
  49. Sun ZW, Allis CD (2002) Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast. Nature 418:104–108PubMedCrossRefGoogle Scholar
  50. Swerdlow PS, Schuster T, Finley D (1990) A conserved sequence in histone H2A which is a ubiquitination site in higher eucaryotes is not required for growth in Saccharomyces cerevisiae. Mol Cell Biol 10:4905–4911PubMedGoogle Scholar
  51. Turner GC, Du F, Varshavsky A (2000) Peptides accelerate their uptake by activating a ubiquitin-dependent proteolytic pathway. Nature 405:579–583PubMedCrossRefGoogle Scholar
  52. Turner SD, Ricci AR, Petropoulos H, Genereaux J, Skerjanc IS, Brandl CJ (2002) The E2 ubiquitin conjugase Rad6 is required for the ArgR/Mcm1 repression of ARG1 transcription. Mol Cell Biol 22:4011–4019PubMedCrossRefGoogle Scholar
  53. Van Leeuwen F, Gafken PR, Gottschling DE (2002) Dot1p modulates silencing in yeast by methylation of the nucleosome core. Cell 109:745–756PubMedCrossRefGoogle Scholar
  54. Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, Andre B, Bangham R, Benito R, Boeke JD, Bussey H, Chu AM, Connelly C, Davis K, Dietrich F, Dow SW, El Bakkoury M, Foury F, Friend SH, Gentalen E, Giaever G, Hegemann JH, Jones T, Laub M, Liao H, Liebundguth N, Lockhart DJ, Lucau-Danila A, Lussier M, M’Rabet N, Menard P, Mittmann M, Pai C, Rebischung C, Revuelta JL, Riles L, Roberts CJ, Ross-MacDonald P, Scherens B, Snyder M, Sookhai-Mahadeo S, Storms RK, Veronneau S, Voet M, Volckaert G, Ward TR, Wysocki R, Yen GS, Yu K, Zimmermann K, Philippsen P, Johnston M, Davis RW (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285:901–906PubMedCrossRefGoogle Scholar
  55. Wolffe AP, Hayes JJ (1999) Chromatin disruption and modification. Nucleic Acids Res 12:711–720CrossRefGoogle Scholar
  56. Wood A, Krogan NJ, Dover J, Schneider J, Heidt J, Boateng MA, Dean K, Golshani A, Zhang Y, Greenblatt JF, Johnston M, Shilatifard A (2003) Bre1, an E3 ubiquitin ligase required for recruitment and substrate selection of Rad6 at a promoter. Mol Cell 11:267–274PubMedCrossRefGoogle Scholar
  57. Xiao T, Kao CF, Krogan NJ, Sun ZW, Greenblatt JF, Osley MA, Strahl BD (2005) Histone H2B ubiquitylation is associated with elongating RNA polymerase II. Mol Cell Biol 25:637–651PubMedCrossRefGoogle Scholar
  58. Yu J, Donoviel MS, Young ET (1989) Adjacent upstream activation sequence elements synergistically regulate transcription of ADH2 in Saccharomyces cerevisiae. Mol Cell Biol 9:34–42PubMedGoogle Scholar
  59. Zhang X, Kolaczkowska A, Devaux F, Panwar SL, Hallstrom TC, Jacq C, Moye-Rowley WS (2005) Transcriptional regulation by Lge1p requires a function independent of its role in histone H2B ubiquitination. J Biol Chem 280:2759–2770PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • A. Irina Mutiu
    • 1
  • Stephen M. T. Hoke
    • 1
  • Julie Genereaux
    • 1
  • Gaoyang Liang
    • 1
    • 2
  • Christopher J. Brandl
    • 1
  1. 1.Department of Biochemistry, Schulich School of Medicine and DentistryUniversity of Western OntarioLondonCanada
  2. 2.Department of Biochemistry and BiophysicsUniversity of North Carolina at Chapel HillChapel HillUSA

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